In this study, UV-B radiation of 96 kJ center dot m-2 center dot d-1 exposed to the 'Tainong 1' mango tree, and trees as the control group under natural lighting. The relative conductivity and the contents of MDA, superoxide anion, H2O2 were lower in the treatment group than in the control group before 60 DAF, and the opposite occurred after 60 DAF. The total flavonoid content in the treatment group was higher than that in the control group, but the opposite occurred later. The 60 DAF serves as the critical point. Before 60 DAF, the treatment enhanced the activity of each enzyme by inducing the upregulated expression of genes such as CHS, R02446, and CYP98A so that gallochol, leucoside, kaempferoside, quercetin, isoquercetin and luteolin, and then removed ROS through the reduction of flavonoids. After 60 DAF, the treatment enhanced the activity of each enzyme by inducing the upregulated expression of HCT and R02446, but the synthesized flavonoids are consumed by ROS. Meanwhile, ROS also accumulate continuously because of the enhanced continuous exposure of UV-B radiation. The downregulated expression of CYP75A led to a decline in enzyme activity. The accumulation rate of flavonoid compounds was lower than that of ROS, which led to ROS damage in pulp.
[Objective]To clarify the climatic similarity between cigar tobacco leaf planting areas in Hunan and fo-reign high-quality cigar tobacco leaf planting areas,so as to provide reference for climate evaluation on different cigar to-bacco planting areas in Hunan and select suitable cigar tobacco leaf types.[Method]Eight cigar tobacco leaf planting areas in Hunan(Ningxiang of Changsha,Changning of Hengyang,Shuangpai of Yongzhou,Yongding of Zhangjiajie,Cili of Zhangjiajie,Sangzhi of Zhangjiajie,Guidong of Chenzhou and Mayang of Huaihua)were selected.The monthly temperature,rainfall,relativehumidity,short-wave radiation net intensity and sunshine hours from the first month to the fifth month after transplanting were usedas climatic factors,and the climatic similarity among the planting areas and foreign high-quality cigar tobacco leaf planting areas(wrapper planting area in Saint Luis,Cuba;fillerplanting area in San Juan Martínez,Cuba;Non-Cuba wrapper planting area Ecuador;Non-Cuba filler planting area Dominica)was analyzed.Cluster analysis and orthogonal partial least squares discriminant analysis(OPLS-DA)were combined for classification of the planting areas and further explication of their differences in climatic conditions.[Result]The results showed that the climatic conditions of the eight cigar tobacco leaf planting areas in Hunan were moderately similar to those in Cuba wrapper planting area,and the climatic conditions of Ningxiang of Changsha,Sangzhi of Zhangjiajie and Guidong of Chenzhou were also moderately similar to those in Non-Cuba filler planting area Dominica.Based on climatic conditions,the above areas could be divided into two classes.Class I was the areas that had the potential to produce cigar wrapper,with five areas included Cili of Zhangjiajie,Yongding of Zhangjiajie,Mayang of Huaihua,Changning of Hengyang and Shuangpai of Yongzhou,while class II was the areas that had the potential to produce both cigar wrapper and cigar filler,including Ningxiang of Changsha,Sangzhi of Zhangjiajie and Guidong of Chenzhou.The OPLS-DA model could successfully distinguish between class I and class II areas,indicating that climatic conditions of the two classes of areas were greatly different.The analysis results of variable importance in projection(VIP)predicting value showed that seven climatic indexes including short-wave radiation net intensity in the first,second,third and fifth months and sun-shine hours in the first,second and third months after transplanting were the difference indexes between the two classes of areas(VIP>1.2).The higher short-wave radiation net intensity and sunshine hours of the class II areas were the main reasons for the production of cigar filler.[Conclusion]Cigar tobacco leaf planting areas in Hunan can be divided into wrapper production potential area and wrapper&filler production potential area.The most influential climatic factors of this classification are short-wave radiation net intensity and sunshine hours.
[Objective]This paper screened out the amino acid region with good antigenicity of non-structural protein 4(nsp4)of avian infectious bronchitis virus(IBV)and prepared polyclonal antibody,so as to provide a material basis for further research on the function of nsp4 protein in the process of IBV replication and lay a foundation for the research and development of new diagnostic kits and vaccines for IBV.[Method]In this study,nsp4 protein of IBV Beaudette strain was prokaryotically expressed.Rabbit and chicken polyclonal antibodies were prepared by immunizing Japanese white rabbit and healthy negative chicken with the fusion protein nsp4 as immunogen.And the biological function of the prepared polyclonal antibodies were investigated by indirect enzyme linked immunosorbent assay(ELISA),Western blot-ting and indirect immunofluorescence assay(IFA).[Result]The nsp4 protein prokaryotic expression vector pCZN-1-HIS-nsp4 and nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA were successfully constructed by selecting amino acids at positions 408-514 of nsp4 protein as prokaryotic expression sequences.The prokaryotic expression of fusion pro-tein nsp4 was 13.6 kD,which was consistent with its predicted size,and the fusion protein nsp4 was able to react with positive whole virus serum against IBV GX-YL5 and M41 strains.The serum titers of prepared rabbit and chicken poly-clonal antibodies were 1∶128000 and 1∶6400 respectively.Additionally,both polyclonal antibodies could react with the fusion protein nsp4 and IBV whole virus.Moreover,they could react with nsp4 protein expressed in chicken embryonic kidney(CEK)cells infected with IBV GX-YL5,Beaudette and M41 strains,and could react with nsp4 protein expressed in the transfected or IBV infected African green monkey kidney cells(Vero)Beaudette strain.The results of real-time fluo-rescence quantitative PCR showed that the viral loads of Vero cells increased gradually after transfection of nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA.In other words,in vitro overexpressed nsp4 protein promoted IBV replica-tion.[Conclusion]Two polyclonal antibodies against nsp4 of IBV strain with high titer,good reactivity and strong speci-ficity are successfully prepared,which can be used as tools for the study of protein characteristics during cell localization and expression phase analysis.In addition,they can be used to analyze the action mechanism of nsp4 protein in the proli-feration process of IBV,and also provide reference for the study of other coronavirus nsp4 protein.
[Objective]The purpose of this study was to detect the efficacy of RNA interference(RNAi)of chicken myostatin gene(MSTN)by Split-GFP bimolecular fluorescence complementary technique and to compare it with com-monly used detection methods in order to validate the effectiveness and availability of the Split-GFP bimolecular fluores-cence complementary technique in the assessment of RNAi efficacy.[Method]The three synthesized shRNA lentiviral cloning vectors(shRNA-a,shRNA-b and shRNA-c)were respectively transfected to HEK 293TGFP11-MSTN cells which sta-bly expressed the GFP11-MSTN fusion protein.The optimal shRNA lentiviral cloning vector was screened by real-time fluorescence quantitative PCR and packaged as lentivirus.Subsequently,the HEK 293TGFP11-MSTN cells were infected with the lentivirus,and mCherry positive(mCherry+)cells were screened using hygromycin B,and the RNAi efficiency was detected by real-time fluorescence quantitative PCR and Western Blotting.The obtained mCherry+ cells were trans-fected with a pcDNA3.1(+)-GFP1-10 plasmid,and the RNAi efficiency was assessed by fluorescence microscopy obser-vation and flow cytometry.[Result]All of three shRNA lentiviral vectors had extremely significant inhibitory effects on MSTN gene expression(P<0.01,the same below),among which Anti-MSTN shRNA-a lentiviral vector had the best inter-fering efficacy.Anti-MSTN shRNA-a lentivirus infected HEK 293TGFP11-MSTN cells at the optimal MOI=3,and the relative expression of the MSTN gene and the expression of the GFP11-MSTN fusion protein were inhibited under this condition.The obtained mCherry+ cells were re-transfected with pcDNA3.1(+)-GFP1-10 plasmid,and both fluorescence microsco-py observation and flow cytometry assay results showed that the number of GFP+ cells was decreased greatly,and the per-centage of GFP+cells was reduced from 31.1%to 11.5%.The results of Split-GFP assay were consistent with those of real-time fluorescence quantitative PCR and Western Blotting detections,indicating that Anti-MSTN shRNA-a lentivirus could inhibit GFP11-MSTN fusion protein expression,thus playing a RNAi role.[Conclusion]Anti-MSTN shRNA-a lentivirus has the highest interfering efficacy on MSTN gene,which extremely significantly down-regulates the expression of MSTN gene after infection of HEK 293TGFP11-MSTN cells,and the percentage of GFP+ cells in the cells is greatly decreased after re-transfection of pcDNA3.1(+)-GFP1-10 plasmid,which is consistent with the results of real-time fluorescence quantita-tive PCR and Western blotting detection results,confirming that Slipt-GFP bimolecular fluorescence complementary tech-nique is a dependable and visualized method for RNAi detection.
[Objective]The antioxidant activity and the contents of total phenolics and flavonoids of eight kinds of tropical fruits in Hainan were determined,and the differences of antioxidant activity and antioxidant substances in eight kinds of fruits were compared,so as to provide certain nutritional reference value for people to eat tropical fruits,it also provided reference for the development and utilization of tropical fruit related products with strong antioxidant activity.[Method]Eight tropical fruits including Manilkara zapota,Syzygium samarangense,Garcinia mangostana,Durio zibet-hinus,Lucuma nervosa,Passiflora edulis,Annona squamosa and Artocarpus heterophyllus were studied in this experi-ment.The antioxidant activity of the samples was determined by 1,1-dipheny1-2-picrylhydrazyl(DPPH)free radical method,ferric reducing antioxidant power(FRAP)and diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)cation free radical scavenging activity method.The total phenolics content was determined by the Folin-Ciocalteu method,and the total flavonoids content was determined by AlCl3 chromotest method.The correlation analysis of three different determination methods and two antioxidant substances were carried out,and the results of antioxidant activity were analyzed by technique for order preference by similarity to ideal solution(TOPSIS)method.[Result]The most effective antioxidant activity measured by DPPH method and FRAP method was L.nervosa(178.62 and 140.83 μmol Trolox/g),and that of M.zapota was the weakest.The highest antioxidant activity was found in G.mangostana(157.06 μmol Trolox/g)and that of M.zapota was the weakest by ABTS method.At the same time,the content of total phenolics(3.80%)and total flavonoids(7.60%)in L.nervosa was the highest among the eight kinds of fruits,the con-tent of total phenolics in M.zapota was the lowest(0.09%),and the content of total flavonoids in D.zibethinus was the lowest(1.07%).Correlation analysis of three different determination methods and two antioxidant substances was found,the correlation coefficient between DPPH method and total phenolics and total flavonoids contents reached more than 0.81,the correlation between FRAP method and total phenolics and total flavonoids contents was more than 0.90.The re-sults were analyzed by the TOPSIS method,and the antioxidant activity was ranked as:L.nervosa>G.mangostana>S.sa-marangense>P.edulis>A.squamosa>A.heterophyllus.>D.zibethinus>M.zapota.[Conclusion]All the eight kinds of tropical fruits have antioxidant activity,the antioxidant activity of L.nervosa is strong,and that of M.zapota is weak.
[Objective]The aim of this study was to investigate the effects of adding Clostridium butyricumand selenium-enriched yeast to the diet on the production performance and fecal flora structure of laying hens,so as to provide references for improving the production performance and economic value of layer hens.[Method]Hy-line brown laying hens were used as experimental animals.The control group(CK)was fed a basal diet,while the experimental groups received the basal diet supplemented with C.butyricum and selenium-enriched yeast.The supplementation levels were as follows:T1 group added 1.0 g/kg of C.butyricum and 0.2 mg/kg of selenium-enriched yeast,T2 group added 2.0 g/kg of C.butyricum and 0.2 mg/kg of selenium-enriched yeast,and T3 group added 3.0 g/kg of C.butyricum and 0.2 mg/kg of selenium-en-riched yeast.The pre-feeding period lasted for 7 d,followed by a 28 d experimental period,laying rate,average daily feed intake,average egg weight,the ratio of feed to egg and mortality rate of laying hens were measured during the period.At the end of the experiment,egg shape index,eggshell thickness,egg yolk color,albumen height,Haugh unit and egg selenium content were determined,and feces of laying hens in each group were collected and their flora structures were analyzed by bioinformatics.[Result]In terms of production performance,compared to the CK,T1 group showed a significantly increased egg production rate(P<0.05,the same below),no significant difference in average egg weight(P>0.05,the same below),and significantly decreased average daily feed intake and the ratio of feed to egg.Other groups showed slight decreases inaverage daily feed intake and the ratio of feed to egg.T1 and T3 groups had reduced mortality rates.There were no significant differences in albumen height,Haugh unit,and egg shape index among all experimental groups.The eggshell thickness and egg selenium content in T1 and T2 groups increased significantly,with the highest in T1 group.In terms of fecal flora structure,compared to the CK,T1 and T3 groups showed increases in ACE index and Chao1 index,but the differences were not significant.T1 group exhibited a decrease in Shannon index and an increase in Simpson index,but the differences were not significant.There was an upward trend in microbial richness and diversity in T1 group.At the phylum level,the dominant bacterial phyla in the CK were Firmicutes,Proteobacteria and Bacteroidetes,accounting for 74.02%,9.14%and 7.03%,respectively.The dominant bacterial phyla in all experimental groups were Fir-micutes and Proteobacteria.In T1 group,the abundance of Proteobacteria was significantly higher than in the CK,while the proportion of Firmicutes showed no significant differences with that in the CK.At the genus level,the dominant bacte-rial genus in both CK and the experimental groups was Lactobacillus,with no significant differences in proportion.The proportion of Lysinibacillus was significantly higher in T1 and T2 group compared to CK,while other genera showed no significant differences with that in the CK.[Conclusion]Adding C.butyricum and selenium-enriched yeast to basal diet can improve partial production performance of laying hens,while also promote the formation of a favorable intestinal flo-ra structure in laying hens.The best feeding effect can be reached when 1.0 g/kg of C.butyricumand 0.2 mg/kg of selenium-enriched yeast are provided.
[Objective]By using BSA-Seq sequencing analysis to determine the resistance SNP markers and resistance genes of the resistant parent NC99R of the nuclear polyhedrosis resistant variety Guican N2,lay a foundation for the iden-tification of NC99R resistance genes and provide marker resources for silkworm nuclear polyhedrosis resistant molecular marker assisted breeding.[Method]A hybrid F1,F2,and BC1 genetic population were prepared using the disease resistant variety NC99R and the susceptible variety Furong as parents,and their genetic patterns were analyzed by feeding nuclear polyhedrosis virus(BmNPV).In the F2 population of NC99R near allelic gene line NC99R-NIL hybridized with Furong,high and low concentration feeding groups with virus concentration differences of 4000 times were set up.Non-diseased individuals were selected from the high concentration feeding group to construct an extreme resistance material DNA mixing pool.Individuals with typical disease symptoms were selected from the low concentration feeding group to con-struct an extreme susceptibility material DNA mixing pool.Then the resistance DNA mixing pool,susceptible DNA mixed pool and two parental genomes were completed sequencing using second-generation sequencing,and locating resistance linked regions with population segregation analysis(BSA).[Result]The results showed that NC99R exhibited stable high resistance to BmNPV,and the genome carried anti BmNPV genes,which were jointly regulated by significant resistance major genes or closely linked genes.A total of 19276670 SNP loci and 4789615 InDel loci were obtained through whole genome sequencing analysis of extreme resistance DNA mixing pool,extreme susceptibility DNA mixing pool and paren-tal DNA.Based on the LOESS regression fitting analysis of ΔSNP-index,two regions associated with resistance to nu-clear polyhedrosis were identified,Chr.25:150-11069kb and Chr.27:8700-11009kb respectively.When the ΔSNP-index was set to≥0.8,a total of 3865 SNP markers significantly related to resistance were identified.Among them,78 SNP markers were screened in the association interval of chromosome 25 and 2694 SNP markers were screened in the associa-tion region of chromosome 27.After screening for non-synonymous mutations at the amino acid level,the Zinc carboxy-peptidase gene on chromosome 25 and the Facilitated trehalose transporter Tret1 gene on chromosome 27 were identified.[Conclusion]Using BSA-Seq,3865 SNP marker resources related to resistance against nuclear polyhedrosis are found in silkworm NC99R,and two genes related to resistance against BmNPV are preliminarily identified(Zinc carboxypepti-dase gene on chromosome 25 and Facilitated trehalose transporter Tret1 gene on chromosome 27),providing marker re-sources for molecular marker assisted breeding of silkworms resistant to nuclear polyhedrosis.
[Objective]Strains with high antagonistic activity against the pathogen of tobacco black shank were isola-ted from tobacco rhizosphere soil in the high incidence area of tobacco black shank disease in Yunnan.The taxonomic sta-tus of strains was clarified and the in vitro antagonism mechanism was preliminary studied to provide biocontrol resources and reference basis for the prevention and control of tobacco black shank disease.[Method]Strains antagonistic to tobacco black shank pathogen were isolated from tobacco rhizosphere soil using the plate standoff method.The strains with strong antagonistic activity were identified by physiological and biochemical characteristics and 16S rDNA sequence analysis.The biocontrol activity and mode of action of biocontrol bacteria against Phytophthora nicotianae in vitro were deter-mined.[Result]Six bacterial strains with antagonistic activity against P.nicotianae were isolated from the collected tobac-co rhizosphere soil samples,among which strain CD-1 showed the strongest antagonistic activity,with a radius of inhibi-tion zone of 10.12 mm.According to physiological and biochemical characteristics and 16S rDNA sequence analysis,the strain was identified as Bacillus licheniformis.Strain CD-1 had broad-spectrum antifungal properties,and both intracellu-lar and extracellular antifungal substances existed.Among them,the antifungal substance extracted using dichloromethane as bacterial fermentation liquid extractant had the best inhibitory effect.When pH was in the range of 6-8 and the tempera-ture was between 40-80℃,it showed relatively stable inhibitory activity.Strain CD-1 cultured in NB medium showed strong inhibitory activity against P.nicotianae in both culture supernatant extracts and cell extracts,with inhibition rates of 76.91%and 67.23%,respectively.The antifungal substances secreted by strain CD-1 could destroy the spore plasma membrane of P.nicotianae and inhibit spore germination.Among them,the spore membrane loss rate after treating P.ni-cotianae spores with the antifungal crude extract 0.4 mg/mL for 6 h reached 53.62%,and the spore germination was com-pletely inhibited,while obvious ion and nucleic acid leakage was detected.[Conclusion]The metabolite produced by B.li-cheniformis CD-1 has a broad antimicrobial spectrum and strong bacteriostatic activity,which has the potential to be de-veloped into antifungal agents.
[Objective]The objective of this study was to screen out the best flavonoid compound elicitors,clarify the synergistic mechanism of them on controlling tobacco bacterial wilt,and provide scientific basis for improving the con-trolling effect against tobacco bacterial wilt and developing new plant compound elicitors.[Method]Flavonoids(flavo-noid and hesperidin)were studied.Eight additives(concentrations of 200 mg/L)and 1 mmol/L flavonoid,1 mmol/L hes-peridin were mixed together,to screen the additives that could compound with flavonoid and hesperidin.The effects of them on controlling tobacco bacterial wilt were determined,and the best compound elicitors were screened out by indoor pot experiment.To further evaluate the controlling effect of the best compound elicitors against tobacco bacterial wilt,the mechanism of compound elicitors on controlling tobacco bacterial wilt was clarified by measuring the activities of enzymes such as oxidase(POD),polyphenol oxidase(PPO)and phenylalanine ammonolyase(PAL)in tobacco and the expression levels of some resistance-related genes(NtPR1a,NtPAL,NtNPR1,NtHIN1 and NtEFE26)in tobacco using fluorescence quantitative real-time PCR.[Result]Among 8 additives,silica and lecithin could enhance the effects of fla-vonoids and hesperidin on controlling tobacco bacterial wilt,and the suitable concentrations were 50 and 100 mg/L respec-tively.Compared with the single agent of flavonoids,the effect of flavonoid compound elicitors(1 mmol/L flavonoid+ 50 mg/L silica,1 mmol/L flavonoid+100 mg/L lecithin,1 mmol/L hesperidin+50 mg/L silica,1 mmol/L hesperidin+ 100 mg/L lecithin)on controlling tobacco bacterial wilt increased by 36.88%to 67.21%.The treatment of flavonoid com-pound elicitors significantly improved the activities of POD,PPO and PAL in tobacco leaves.Compared with a single agent of flavonoid,the activities of POD,PPO and PAL in tobacco leaves increased by 19.47%-37.95%,25.89%-66.91%,22.98%-254.83%in flavonoid+lecithin treatment respectively;compared with a single agent of hesperidin,the activities of POD,PPO and PAL in tobacco leave sincreased by 3.69%-114.37%,12.27%-58.04%,28.12%-104.39%in hesperidin+lecithin treatment respectively.Compared with a single agent of flavonoid or hesperidin or the water control group,the application of flavonoid compound elicitors also significantly increased the relative expression levels of resistance-related genes NtNPR1,NtPR1a and NtHIN1(P<0.05).[Conclusion]50 mg/L silica and 100 mg/L lecithin can promote the effects of 1 mmol/L flavonoids and 1 mmol/L hesperidin on controlling tobacco bacterial wilt,and strengthen the activities of defense enzymes and the expression levels of resistance-related genes.This study lays the foundation for the development of new plant elicitors.
[Objective]The purpose of the study was to investigate the effects of nano-potassium fertilizer on the growth of peach seedlings and the physiological characteristics of adult peach tree leaves,and to provide a theoretical ba-sis for the application of nano-fertilizers in peachtree cultivation.[Method]Using Prunus persica Batsch seedlings and Chunmei adult peach trees as test materials,the effects of nano-potassium fertilizers and three traditional fertilizers(potas-sium sulfate,soluble fertilizer and compound fertilizer)on seedling biomass,root morphology,photosynthetic perfor-mance,and physiological characteristics of adult tree leaves were compared.[Result]On peach seedlings,except that there was no significant difference(P>0.05,the same below)between nano-potassium fertilizer and potassium sulfate fer-tilizer in plant height,underground fresh weight and root morphology indicators,nano-potassium fertilizer could signifi-cantly increase plant height,stem diameter,the number of leaves,leaf surface area,dry(fresh)weight of aboveground and underground parts,and root system morphological indicators compared with other fertilizer treatments(P<0.05,the same below).Meanwhile,nano-potassium fertilizer also significantly increased the contents of chlorophyll a,chlorophyll b,total chlorophyll and the maximum quantum yield of photosystem Ⅱ(Fv/Fm)in the leaves of peach seedlings.On adult peach trees,compared with potassium sulfate fertilizer,nano-potassium fertilizer significantly increased the contents of total chlorophyll,soluble sugar,homogenate protein and proline in leaves by 30.2%,48.6%,40.4%and 33.6%respec-tively.The single fruit weight of peach fruits was significantly increased by 17.5%.The effects of soluble fertilizer and compound fertilizer on the leaves and fruits of adult peach trees were similar to those of nano-potassium fertilizer without reaching significant difference.[Conclusion]Comprehensive considering morphological and physiological indicators,compared with traditional potassium fertilizer,nano-potassium fertilizer can promote the growth of aboveground and un-derground parts of peach seedlings to varing degrees,and increase the chlorophyll content and photosynthetic perfor-mance of young and adult peach tree leaves,to improve tree nutrient and fruit growth,which is expected to be popularized as a new type of fertilizer in practical production of peach in the future.
[Objective]This paper explored the impact of deep tillage cultivation on the yield and production value of flue-cured tobacco,as well as the characteristics of rhizosphere soil and the micro-ecosystem.The aim was to provide scien-tific evidence for the construction of a reasonable cultivation layer and the establishment of a high-quality and efficient cultivation system for flue-cured tobacco in the tobacco-growing areas of Fujian.[Method]The experiment included a con-trol of natural tillage depth of 15 cm + mid-tillage soil cultivation(CK),tillage depth of 20 cm + mid-tillage soil cultiva-tion(D1),tillage depth of 25 cm + mid-tillage soil cultivation(D2)and tillage depth of 25 cm + no mid-tillage soil culti-vation(D3)in the tobacco-growing areas of Fujian.The study compared and analyzed the differences in yield and produc-tion value of flue-cured tobacco and the physicochemical properties of the soil under different treatments.Additionally,the high-throughput sequencing technology Illumina MiSeq was used to systematically analyze the differences in microbial structure and functional diversity in the rhizosphere under the deep tillage treatments.Spearman correlation coefficient and the random forest model were utilized to evaluate the correlation between the rhizosphere microbial community,soil nutrients and flue-cured tobacco yield.[Result]Deep tillage could significantly increase the yield and production value of flue-cured tobacco(P<0.05,the same below).Among the treatments,the D2 treatment had the best effect in terms of yield and efficiency improvement,but the improvement between the D2 and D3 treatments was not significantly different(P>0.05,the same below).In terms of soil properties,all deep tillage treatments could significantly improve soil total po-rosity and capillary porosity compared with CK.The content of alkali-hydrolyzed nitrogen,urease,sucrase and catalase activities increased with the deepening of tillage depth.Appropriate deep tillage treatment could significantly increase the bacterial α diversity.The relative abundance of Chloroflexi and Burkholderia increased significantly under deep tillage,while the relative abundance of Rhodanobacter decreased significantly.The results of Bugbase phenotypic prediction showed that deep tillage treatment could significantly increase the abundance of aerobic bacteria and gram-negative bacte-ria in rhizosphere soil.D2 and D3 treatments could significantly increase the relative abundance of oxidative stress tolerant bacteria compared with D1 treatment.The results of FAPROTAX function prediction showed that each deep tillage treat-ment could significantly improve the relative abundance of nutrient cycling functions such as nitrogen fixation and cellu-lolysis compared with CK.The random forest model predicted that nitrogen fixation was the main factor affecting the yield formation of flue-cured tobacco.[Conclusion]Under the current trend of shallow cultivation layer in the tobacco-growing areas of Fujian,moderate deep tillage(25 cm)has a positive effect on improving the rhizosphere micro-ecologi-cal environment of flue-cured tobacco.However,the mid-tillage soil cultivation before the vigorous growth stage of flue-cured tobacco under moderate deep tillage has no significant impact on the yield and production value of flue-cured tobacco.
[Objective]Based on the differences of chemical composition and sensory quality of tobacco leaves at dif-ferent oil levels,a comprehensive weight evaluation system was established,and the quality evaluation methods of tobac-co leaves with different oil levels were established to evaluate the availability of tobacco leaves with different oil levels,which provided reference for the quality evaluation and utilization of high-oil and high-quality tobacco leaves.[Method]The chemical composition and sensory quality indicators of 132 samples of Yunyan 87 tobacco leaves at different oil le-vels(a little,less oily,oily and rich)covered by the full leaf position in Lingbao,Sanmenxia,Henan tobacco area were used as the data set,and the chemical and sensory quality indicators were assigned values using the affiliation function,and then the indicators were re-assigned weights through the principal component weighting method,coefficient of varia-tion weighting method,subjective weighting method and comprehensive weighting method,so as to evaluate the availabili-ty of tobacco leaves at different oil levels.[Result]With the leaf oil level from a little to rich,with an overall decreasing trend of total nitrogen,chloride and nitrogen to alkali ratio,the total nitrogen content of a little oil level tobacco leaves was 5.15%-46.41%higher than that of tobacco leaves at other oil levels;and total sugar,reducing sugar,total alkaloids and po-tassium content showed an increasing trend,the contents of total sugar and reducing sugar in rich oil level tobacco leaves were 12.20%-28.96%and 10.09%-30.64%higher than those in tobacco leaves at other oil levels respectively.Except for total plant alkaloids and nitrogen alkali ratio indicators,the oil level of tobacco leaves was significantly(P<0.05)or ex-tremely significantly(P<0.01,the same below)correlated with the other seven chemical indicators.With the leaf oil level from a little to rich,the scores of sensory quality indicators and total scores showed an increasing trend,and the oil level of the tobacco leaves was extremely significantly and positively correlated with all the sensory indicators scores.Compared with the single weighting method,the comprehensive weighting method improved the discrimination of the usability index of tobacco leaves with different oil levels.With the oil level of tobacco decreased,the availability index greatly decreased,and the availability index of rich oil level tobacco leaves was the highest(0.644-0.749).The results of correlation analysis showed that the comprehensive weighting method was strongly correlated with the results of each single weighting me-thod.[Conclusion]The comprehensive weighting method based on the chemical composition index and sensory quality in-dex of tobacco leaves can be used as a scientific and effective method to evaluate the availability of tobacco leaves at dif-ferent oil levels,and provide a quality evaluation method for the subsequent production evaluation of high-oil tobacco leaves.
[Objective]In this study,the effects of Bacillus aryabhattai R60 strain on drought tolerance of maize un-der drought stress were analyzed,which provided theoretical reference for drought tolerant biologic fertilizer exploration and improvement of maize stress tolerance.[Method]Transcriptome sequencing technology was used to analyze the changes in transcription levels of maize root inoculated with B.aryabhattai R60 strain under drought stress.Key differen-tially expressed genes(DEGs)in maize were explored,with sterilized distilled water injected into the root as the control,and GO function annotation and KEGG signaling pathway enrichment analysis were performed.[Result]There were 1524 DEGs between the treatment group and the control group.Compared with control group,there were 421 up-regulated DEGs and 1103 down-regulated DEGs in the treatment group.The 421 up-regulated DEG swere annotated to 26 GO en-tries,including 13 entries of biological processes(cellular pathways,metabolic pathways,stimulus response,and biolo-gical regulation,etc),3 entries of cell components(cellular structures,intracellular and protein complexes),and 10 entries of molecular functions(binding,catalytic activities and transcriptional regulatory activities,etc).The top of 20 DEGs with down-regulated expression were mainly annotated in 5 entries of biological processes(cellular pathways,bio-logical regulation and stimulus response,etc),2 entries of cell components(cell structures and intracellular),and 4 entries of molecular functions(binding,transcriptional regulatory activities,etc).DEGs were mainly enriched in five signaling pathways of RNA transport,messenger mRNA pathway,plant hormone signal transduction,mitogen-activated protein kinase(MAPK)signaling pathway and plant-pathogen interaction.Compared with the control group,the expres-sions of key genes were down-regulated in the plant hormone signal transduction pathways of the treatment group,such as the negative regulation jasmonate ZIM domain-containing protein(JAZ)and downstream MYC2 family transcription factors in the jasmonic acid(JA)signaling pathway,the gibberellin(GA)receptor 1(GID1)and the negative regulation inhibitory protein(DELLA)in the GA signaling pathway,the protein phosphatase(PP2C)in the abscisic acid(ABA)signaling pathway,and the ethylene receptor transcription factor 1(ERF1)in ethylene signaling pathway,etc.In the MAPK signaling pathway,the genes expressions of MAP kinase substrate 1(MKS1)and mitogen-activated protein kinases(MPK3,MKK9 and MAPKKK18)were down-regulated.In addition,calmodulin protein(CALM)and calcium-binding protein(CML)genes expressions were down-regulated,while E3 ubiquitin-protein ligase(RHF,HOS1,and BRE1),ribose-phosphate pyrophosphokinase(PRPS),and UDP-glucuronate 4-epimerase(GAE4)encoded genes expres-sions were up-regulated.The results of real-time fluorescence quantitative PCR analysis were basically consistent with the transcriptome data.[Conclusion]The growth-promoting bacteria B.aryabhattai R60 may up-regulate the expressions of RHF,HOS1,BRE1,GAE4 and PRPS genes,and down-regulate the expressions of ERF1,DELLA and PP2C genes in the roots of maize plants,thereby activating multiple signal transduction pathways to enhance the tolerance of plants to drought stress.
[Objective]The purpose of the study was to clone the molecular chaperone gene ShDnaJ in sugarcane,and analyze its expression pattern under the stress of sugarcane streak mosaic virus(SCSMV),so as to provide a theoreti-cal reference for exploring the function and mechanism of action of ShDnaJ gene in sugarcane in response to SCSMV in-fection.[Method]ShDnaJ gene complete open reading frame(ORF)sequence was cloned by homologous cloning techno-logy and was analyzed by bioinformatics software.The subcellular localization of ShDnaJ protein was analyzed by fusion expression with green fluorescent protein(GFP).The expression levels of ShDnaJ gene in different tissues of sugarcane and under SCSMV stress were detected by real-time fluorescence quantitative PCR.[Result]The open reading frame(ORF)of ShDnaJ gene was1260 bp in length,and encoded 419 amino acid residues.ShDnaJ protein,with molecular weight of 45682.47 Da and theoretical isoelectric point(pI)of 8.78,was a stable hydrophilic protein,which contained a DnaJ structural domain and belonged to the member of DnaJ superfamily.ShDnaJ protein was located in the endoplasmic reticulum.The secondary structure of ShDnaJ protein consisted of 113 α-helixes,31 β-turns,78 extended strands and 197 random coils.The amino acid sequence of ShDnaJ protein had the highest amino acid sequence similarity with that of SbDnaJ in sorghum,which was 96.93%,indicating that the two proteins were most closely related.ShDnaJ gene was ex-pressed in roots,stems and leaves of sugarcane,and the expression levels in roots and stems were significantly higher than that in leaves(P<0.05,the same below).However,the expression level of ShDnaJ gene in immature stems was higher than that in mature stems.The expression level of ShDnaJ gene in mature leaves was higher than that in immature leaves.With the pass of treatment time,the relative expression of ShDnaJ gene in the SCSMV stress group and the control group(treated with phosphate buffer)showed an overall downward trend,but the expression levels of ShDnaJ genes were sig-nificantly higher than those in the control group at different time points after SCSMV inoculation.[Conclusion]ShDnaJ gene has tissue expression specificity,and the maturity of the tissue affects its expression level.The expression of this gene is up-regulated under SCSMV stress,suggesting that this gene is involved in regulating sugarcane plant resistance to SCSMV.
[Objective]To investigate the immune efficacy of the surface protein SclE of Streptococcus equi ssp.zooepidemicus(SEZ),to provide theoretical basis for prevention of streptococcal disease caused by SEZ.[Method]The SclE gene of SEZ was amplified by PCR and cloned into prokaryotic expression vector pCold I.The vector was trans-formed into the competent cells of Escherichia coli BL21(DE3),and finally induced by isopropyl-beta-D-thiogalactopy-ranoside(IPTG),after SDS-PAGE analysis,BALB/c mice were immunized with recombinant protein SclE(rSclE)(100 μg/mL,0.5 mL)by intraperitoneal injection(n=10,the cycle of first and second immunization was 14 d).Serum was collected 14 d after the first and second immunization,and antibody levels and specificity were detected by ELISA and Western blotting.After secondary immunization,the mice were tested for immune protection,and the bacterial load of liver,spleen,lung and kidney after challenge was detected and histopathology was observed.[Result]SDS-PAGE identi-fication showed that rSclE was successfully expressed,and the size of rSclE was about 93 kD,which was consistent with the theoretical value.Western blotting showed that the recombinant protein could bind to SEZ positive control group mice serum specifically(intraperitoneal injection of 0.5 mL of SEZ inactivated vaccine),indicating that rSclE had good immu-nogenicity.ELISA showed that the specific antibody levels in the serum increased significantly after the secondary immu-nization(P<0.05,the same below),and the mice could produce high IgG titers,the antibody level of IgG1(0.436±0.031)was significantly higher than that of IgG2a(0.161±0.009).These results indicated that IgG1 was the main subtype of rSclE induced antibody.rSclE immunized mice could significantly inhibit the colonization ability of SEZ in each organ,liver and spleen hemorrhage and inflammatory cell infiltration were alleviated,lung and kidney bleeding and other patho-logical injuries were alleviated;in the mouse immune challenge protection experiment,70%mice were protected from lethal dose of SEZ strain.[Conclusion]The successfully expressed rSclE can induce mice to produce SCLEspecific anti-bodies,and has a good protective effect,and can be used as a potential vaccine candidate antigen for SEZ.
[Objective]This paper analyzed the codon usage pattern of chloroplast genome in high production Coffea arabica L.and the influencing factors of its codon usage bias,to determine the appropriate heterologous expression hostand provide reference basis for phylogenetic analysis and gene function verification of coffee,especially heterologous expression.[Method]The complete chloroplast genome sequence of high production C.arabica(MK353209)was re-trieved and downloaded from the GenBank database.A total of 51 coding sequences(CDS)were selected,which had a length greater than 300 bp,started with ATG,ended with TAG,TAA and TGA,and had no stop codon or repetitive se-quence.And this study systematically analyzed the characteristics of codon usage in the genome of high production C.ara-bica and its influencing factors,and compared the codon usage frequency with model organismsby using EMBOSS online website and CodonW 1.4.2 software.[Result]The results showed that the GC content of the first codon positions(GC1),second codon positions(GC2),third codon positions(GC3)and the average GC content of three positions(GC)in the genome of high production C.arabica were less than 50.00%.Among the 30 codons with RSCU>1.00,96.67%codons ended with A/T and 3.33%codons ended with G/C,indicating that the chloroplast genome of high production C.arabica tended to end with A/T codons.The average values of effective number of codons(ENC),codon adaptation index(CAI),and optimal codon usage frequency(Fop)were 46.97,0.167 and 0.353 respectively,indicated that the codon usa-ge bias of chloroplast gene in high production C.arabica was weak.The 20 high-frequency codons were GCT,TGT,GAT,GAA,TTT,GGA,CAT,ATT,AAA,TTA,AAT,CCT,CAA,AGA,TCT,ACT,GTA,GTT,TAT and TAA.Neutral plot,ENC-plot,PR2-plot and correspondence analysis showed that codon usage bias was affected by natu-ral selection,mutation and other factors,among which natural selection played adecisive role.The comparison of codon usage frequency found that there were 23.44%,15.63%,40.63%and 15.63%codon usage patterns of high production C.arabica varied considerably compared with those of Arabidopsis thaliana,Nicotiana tabacum,Escherichia coli and Sac-charomyces cerevisiae respectively.Finally,21 optimal codons were determined,among which 57.14%codons ended with T,38.10%ended with A,and 4.76%ended with G.[Conclusion]The codon usage bias of the chloroplast genome of high production C.arabica is relatively weak,and tend to end with A/T codon.Codon usage bias is affected by many fac-tors,such as natural selection,mutation,gene expression level,gene length and so on.S.cerevisiae and N.tabacum are more suitable for heterologous expression receptor systems of high production C.arabicagenes.
[Objective]The purpose of the study was to conduct aphenotypic classification study on 68 macadamia germplasm resources with planting value,and to provide a scientific basis for systematic classification and evaluation of macadamia germplasm resources.[Method]The 64 phenotypic traits(39 descriptive traits and 25 numerical traits)of 68 macadamia germplasm resources originating from domestic and foreign countries were observed and described according to the identification and evaluation methods in Description Specification and Data Standard for Macadamia Germplasm Resources and Technical Specification of Identification for Macadamia Germplasm Resources,and cluster analysis and principal component analysis were performed based on them.[Result]Based on cluster analysis of flower phenotypic traits,68 germplasms were divided into 3 groups:type with long inflorescence and multiple flowers,hybrid type with me-dium length inflorescence and multiple flowers,and type with short inflorescence and few flowers.Based on cluster analy-sis of leaf phenotypic traits,68 germplasms were divided into 4 groups:Macadamia integrifolia with big leaf,M.integri-folia with medium leaf,M.integrifolia with small leaf,and M.integrifolia/M.tetraphylla hybrid with big leaf.Based on cluster analysis of fruit phenotypic traits,68 germplasms were divided into 4 groups:M.integrifolia with big fruit,M.in-tegrifolia with medium size of fruit,M.integrifolia with small fruit,and M.integrifolia/M.tetraphylla hybrid with big fruit.Based on cluster analysis of other phenotypic traits,68 germplasm were divided into 3 groups:type of high yield and mid-late ripening,type of medium yield and early to medium ripening,and type of low yield and early to medium ripe-ning.Based on comprehensive cluster analysis,68 germplasm were divided into 4 groups:type with big leaf,big fruit and multiple flowers,type with medium size leaf,small fruit and few flowers,type with small leaf,medium size fruit and few flowers,and hybrid with big leaf,big fruit and multiple flowers.Based on principal component analysis and two-di-mension scatter plot analysis of 64 phenotypic traits,15 main influencing factors that could systematically evaluate classi-fication of 68 germplasm phenotypic traits were selected.The 15 main influencing factors included fresh weight of single fruit with pericarp,fresh weight/dry weight of single shellfruit,leaf shape/leaf shape index,fruiting ability,flowering ability,flower quantity,flower color,maturity characteristics,leaf edge thorn density,surface texture of shellfruit,young leaf color,weight of single kernel,leaf area,leaf petiole length,and leaf inflorescence.[Conclusion]The cluste-ring analysis based on phenotypic traits such as flowers,leaves,and fruits supports the existing classification system of macadamia.The representative main influencing factors can scientifically and systematically evaluate the phenotypic clas-sification of macadamia germplasm resources.
[Objective]This paper cloned the heat shock transcription factor(Hsf)gene family members in mulberry,analyzed expression pattern of the MnHsfA1 genes to provide theoretical reference for in-depth research on the regulatory mechanism of this family in mulberry heat stress response and mulberry breeding.[Method]Using seedlings of Morus notabilis as experimental materials,the MnHsfs genes were cloned.And the physicochemical properties,conserved domains,signal peptides,transmembrane domains,subcellular localization,and promoter cis acting elements of MnHsfs proteins were predicted by multiple sequence alignment,phylogenetic tree construction,and MEME conserved motif.The expression pattern of MnHsfA1 genes under high temperature and drought stress were analyzed using real-time fluo-rescence quantitative PCR.[Result]Fourteen full-length coding sequences of MnHsfs genes were cloned from mulberry trees,including MnHsfA4a,MnHsfA4b,MnHsfA2,MnHsfA5,MnHsfA6b-1,MnHsfA6b-2,MnHsfB4-2,MnHsfA8,MnHsfB3,MnHsfB2b,MnHsfC1,MnHsfB2a,MnHsfB4-1 and MnHsfA1.Their open reading frame lengths were 1239,1278,1404,1431,1101,1140,1185,1197,711,996,1029,936,900 and 1437 bp and encoded 412,425,467,476,366,379,394,398,236,331,342,312,299 and 478 amino acids residues respectively.And most of MnHsfs proteins were hydrophilic and unstable acidic proteins.MnHsfs protein included domains such as function domain(DBD),oligomeriza-tion function domain(OD),AHA,and KLFGV,and were divided into class A,class B,and class C.The similarity between MnHsfA1 and HsfA1 of apple,spinach,and Arabidopsis were the highest.Compared with the control,the expression of MnHsfA1 gene and mRNA transcription level were significantly up regulated under high temperature and drought stress treatments(P<0.05).[Conclusion]MnHsfs genes have relatively high genetic conservatism,and the expression level of MnHsfA1 gene is up regulated under high temperature and drought stress.The cis acting element of MnHsfA1 gene pro-moter contains hormone responsive elements such as abscisic acid,methyl jasmonate,gibberellin,as well as transcrip-tion factors related to various abiotic stresses.It is speculated that it may regulate multiple abiotic stresses simultaneously.
[Objective]This paper explored the effect of different pH and cadmium(Cd)concentrations on physiology of seed germination and seedling growth of cigar,in order to provide reference for obtaining high-quality and safe cigar raw material.[Method]Hydroponics experiment was carried out and different pH(3,5,7,9 and 11)and Cd concentra-tions(0,10,50 and 250µmol/L)were set.The seed germination indexes(germination rate and germination energy),seedling growth physiological indexes[radicle length,embryo length,dry matter weight,chlorophyll content,nitrate re-ductase activity,malondialdehyde(MDA)content,root activity]and Cd content in various parts of seedlings were mea-sured.[Result]Under the same Cd concentration,the germination rate of cigar seeds first increased and then decreased with the increase of pH.Under the same pH,the germination rate of cigar seeds treated with Cd decreased gradually with the increase of Cd concentration.The germination energy and germination rate of seeds were the highest when pH was 7 and the Cd concentration was 10µmol/L.Under the same pH,the radicle length of cigar seedlings first increased and then decreased with the increase of Cd concentration.The embryo length of seedlings decreased with the increase of Cd concen-tration when pH was 3 and 7.Under other pH conditions,the embryo length of seedlings increased at first and then de-creased with the increase of Cd concentration.The radicle length and embryo length reached the longest when pH was 9 and Cd concentration was 10 umol/L.Except for the treatment of 0µmol/L Cd,the dry matter weight of cigar seedlings decreased with the increase of Cd concentration,and increased at first and then decreased with the increase of pH.Under the treatment of 10-250µmol/L Cd,the Cd content in roots,stems and leaves of cigar seedlings decreased gradually with the increase of pH,and increased with the increase of Cd concentration.The chlorophyll content,nitrate reductase activi-ty and root activity of cigar seedlings increased at first and then decreased with the increase of pH,but it decreased with the increase of Cd concentration.The content of MDA in cigar seedlings increased with the increase of Cd concentration when pH was 5-11.Under the treatment of 10-250µmol/L Cd,the content of MDA increased at first,then decreased and then increased with the increase of pH,and reached the minimum when pH was 7.[Conclusion]Low Cd concentration promotes the germination of cigar seeds,but the growth physiology of cigar seedlings is inhibited with the increase of Cd concentration.The change of pH will affect the absorption and accumulation of heavy metal Cd by cigar seedlings,and then affect the normal growth and dry matter accumulation of cigar seedlings.When pH is 7,10µmol/L Cd treatment in-creases the chlorophyll content,nitrate reductase activity and root activity of cigar seedlings,being beneficial to the growth of cigar seedlings.
[Objective]This paper explored the effects of microclimate characteristics of wrapper tobacco fields on the growth and development of tobacco plants and the quality of fermented wrapper tobacco leaf under shading cultivation conditions,in order to provide theoretical basis for domestic shading cultivation of wrapper tobacco leaf.[Method]Using the cigar variety Yunxue-1 as the experimental material,three treatments with 100%(CK),75%(T1),and 65%(T2)light transmittance were set up to analyze the microclimate characteristics of tobacco fields under different treatments and their effects on plant agronomic traits,biomass accumulation and distribution,and main quality characteristics of fermented tobacco leaves.[Result]Shading treatment could reduce the temperature,light intensity,diurnal temperature difference,and effective accumulated temperature≥10℃in tobacco fields,and increase relative humidity.Shading had significant impact on growth and development indicators such as tobacco plant height,stem circumference,maximum leaf area and biomass accumulation(P<0.05,the same below).Among them,tobacco under T2 treatment had the highest plant height,biggest maximum leaf area,and largest leaf biomass,with the highest increase at 40 d after shading,while tobacco under CK treatment had the longest stem circumference.Tobacco leaf under T2 treatment had the highest scores for appearance quality indicators such as leaf structure,oil content,color uniformity and branch thickness,and its tensile strength was also significantly higher than other treatment groups.The yield and yield rate of wrapper tobacco leaf under shading treatment were significantly higher than those under CK.Compared with CK,the yield of wrapper tobacco leaf under T1 and T2 treatments increased significantly by 5.87%and 13.78%respectively,and the yield rate of wrapper tobacco leaf increased significantly by 8.56%and 18.56%respectively.The correlation analysis results indicated that the decrease in temperature(accumulated temperature,effective accumulated temperature≥10℃and average temperature)under shading conditions was the main reason for the decreases in leaf thickness,stem circumference,and rhizome biomass accumulation of tobacco plants.The decrease in average light intensity and increase in average relative humidity under shading conditions were key factors in improving the appearance quality and physical properties of wrapper tobacco leaf.[Conclusion]Shading can reduce the environmental temperature and light intensity of wrapper tobacco leaf fields,in-crease relative humidity,and alter the morphological development and mass accumulation of tobacco plants,thereby in-creasing the yield and the yield rate of wrapper tobacco leaf.It also makes the appearance and physical indicators of fer-mented tobacco leaves more suitable for industrial use.Overall,65%light transmittance is more suitable for shading culti-vation of wrapper tobacco leaf in southwestern Henan.
