Lilium lancifolium is an important economic crop in Huoshan county of Anhui province, China. Continuous cropping obstacles serious affect the yield and quality of L. lancifolium . At present, the effect of the continuous cropping of L. lancifolium on soil fungal community structure is not clear. In this study, Illumina MiSeq was used to study the fungi of the rhizosphere soil associated with L. lancifolium subjected to three treatments: no continuous cropping, continuous cropping for 3 years, and continuous cropping for 5 years. The results showed that continuous cropping of L. lancifolium could increase the fungal richness and diversity in the rhizosphere to different degrees. Ascomycota was the dominant phylum, and its abundance increased after continuous cropping. In addition, the abundance of beneficial fungi, such as Chaetomium , decreased, and the abundance of harmful fungi, such as Fusarium and Colletotrichum, greatly increased with the duration of continuous cropping. Overall, continuous cropping changed the composition of soil fungal communities, reduced the abundance of beneficial fungi, and increased the abundance of harmful fungi. Thus, continuous cropping increased the potential for soil-borne diseases and endangered the bulb growth of L. lancifolium .

近年来,卷丹百合的连作问题愈发突出,严重限制了百合产业的可持续发展.连作所产生的障碍主要表现在:土壤病虫害严重,植株的生长受限,生理功能逐渐衰退、品质变差.基于此,针对卷丹百合重茬病的主要成因、危害及其作用机理进行了研究,重点探讨了改进卷丹百合的种植模式、施肥方法和土壤病虫害的防治等这类治理措施产生的积极作用.将近年来在连作障碍防治方面具有广泛应用价值的几种方法作一综述.

λ干扰素作为一种多效性细胞因子属于Ⅲ型干扰素,研究表明其能够对Ⅰ型和Ⅱ型干扰素逃逸的病毒起作用.为了利用家蚕杆状病毒表达系统高效表达出具有高抗病毒活性的羊λ3干扰素(OvIFN-λ3),首先将OvIFN-λ3基因优化后合成,将其克隆至转移载体pVL1393中,得到重组质粒pVL1393-OvIFNλ3,再将重组质粒与失活拯救型家蚕杆状病毒穿梭载体reBmBac DNA共转染家蚕卵巢传代细胞Bm-N,得到含OvIFN-λ3基因的重组杆状病毒.随后利用重组杆状病毒感染五龄起蚕,待其发病后收集蚕血淋巴.采用微量细胞病变抑制法在羊肾细胞上进行重组表达绿色荧光蛋白的水疱型口炎病毒(recombinant vesicular stomatitis virus expressing green fluorescent protein,VSV-GFP)的攻毒实验,测定OvIFN-λ3抗病毒活性可达(6.5±0.27)×105 U/mL;利用空斑筛选法筛选重组病毒,感染家蚕后,测定其效价最高可达(3.1±0.42)×106 U/mL,表达量明显提高.家蚕杆状病毒表达系统为羊λ3干扰素产品的大量生产应用提供了新方法.

开放式教学是在教师引导下,积极发挥学生主体作用,发展自我的一种教学方式,符合完全学分制的精髓,从开放植物学课堂内容和质量评价体系方面,探讨了开放式教学在植物学课堂教学中的实际应用,能够有效提升学生的学习能力和学习效果.

Farmland ecosystem is the most active and one of the terrestrial ecosystem carbon pools. It has a big carbon sequestration potential, in which bacteria play a major role in microbial CO2 fixation processes. In this study, the composition and diversity of CO2-assimilating bacteria were investigated using cloning and sequencing, terminal restriction fragment length polymorphism(T-RFLP) and quantitative PCR. Soil samples were collected from the lime concretion black soil in Mengcheng, Anhui province with four treatments: CK(control, no fertilizer), CK-F(mineral NPK fertilizers alone), W-NF(wheat straw residue incorporation, no mineral NPK fertilizers), and W-F(mineral NPK fertilizers combined with wheat straw residue). The carbon fixation microorganisms mainly include Nitrosomonas, Mesorhizobium, and Bradyrhizobium, etc. In the treatment of CK and W-NF, the cbb L-containing bacterial communities were dominated by obligate autotrophic bacteria such as Nitrosomonas, Nitrobacter and Nitrosococcus. While in the treatment of CK-F and W-F, the obligate autotrophic bacteria decreased sharply and the facultative autotrophic bacteria increased sharply. In CK and W-NF treated soil, the dominant cbb L-containing bacterium was Nitrosomonas, and the relative abundances were 73.2% in CK and 72.4% in W-NF. In CK-F treated soil, the dominant cbb L-containing bacteria were Nitrosomonas and Mesorhizobium, and their relative abundances were 33.3% and 17.6%, respectively. In W-F treated soil, the dominant cbb L-containing bacteria were Nitrosomonas and Bradyrhizobium, and their relative abundances were 26.4% and 24.5%, respectively. Fertilization and straw incorporation both increased the diversity of cbb L-containing bacterial communities. Fertilization had a stronger effect on soil bacterial diversity and structure than straw incorporation. Fertilization or straw incorporation both increased bacterial cbb L abundance(1.32×107-3.29×107 copies·g-1 soil) with the highest cbb L copy number(3.29×107 copies·g-1 soil) in the W-F treated soil. All above results indicated that fertilization and straw incorporation significantly influenced the CO2 fixation bacterial community structure as well as increased bacterial abundance and diversity.

Soil bacteria are important drivers of nearly all biogeochemical cycles in terrestrial ecosystems and participate in most nutrient transformation processes in the soil. Thus knowledge about the shift of microbial community structure and diversity following different agricultural management practices could improve our understanding of soil processes and help us to develop agricultural management strategies. Because most soil bacteria are nonculturable, and traditional molecular biology methods, such as DGGE, are partial, the researches on soil bacterial are limited. High-throughput sequencing technology provides an effective tool for microbial molecular biology research. In this study, the technology of high-throughput sequencing on Illumina MiSeq platform was adopted to investigate the effects of fertilization and straw residue incorporation on bacterial communities in lime concretion black soils. The 16S rRNA genes of topsoil bacteria in lime concretion black soil were sequenced by high-throughput sequencing on Illumina MiSeq platform and related biological analysis conducted to investigate the changes in soil bacterial composition, diversity and structure under 4 different fertilization practices at wheat tillering stage. Soil samples were collected in lime concretion black soils in Mengcheng, Anhui Province, China with 3 treatments (CK: no fertilization, no straw return; F: chemical fertilization without straw return; W: straw return without chemical fertilization). The results showed that 173 323 reads of 14 873 OTUs (operational taxonomic unit) were generated at 3% cutoff level under all treatments with an average length of 439 bp. Bacterial OTUs were classified into 19 different known phyla and 41 classes. Proteobacteria, Acidobacteria, Actinobacteria and Bacteroideteswere the dominant phyla (with relative abundance > 10%) in lime concretion black soils. Then Alphaproteobacteria, Betaproteobacteria, Sphingobacteriia, AcidobacteriaandGammaproteobacteriawere thedominant classes (with relative abundance > 10%). The total number of dominant genera (with relative abundance > 1%) in all 3 treatments was 47, of which 21 genera were found in all treatments, and the largest number (39) of dominant genera occurred under F treatment. The dominant phylum, class and genus with the highest relative abundances were Proteobacteria(38.7%-43.1%), Alphaproteobacteria(14.5%-18.1%) andSphingomonas (4.6%-7.7%) in all 3 treatments. The richness indexes (Chao1 and ACE indexes) were significantly lower for F treatment than for CK and W treatments. ACE index decreased by 22.8% under F treatment compared with CK. The richness indexes (Chao1 and ACE indexes) of CK and W treatments were not significantly different from each other. The Shannon index of W treatment was significantly higher than that of CK and F treatments, it increased by 4.1% compared with CK. Then the Shannon indexes of CK and F treatments were also not significantly different from each other. The Simpson index of F treatment was significantly higher than that of CK and W treatments. The Simpson index of F treatment increased by 38.1% compared with CK treatment. The Simpson index of W treatment was lowest among 3 treatments, decreasing by 23.8% compared with CK treatment. Hierarchical cluster analysis showed that CK and W treatments were in the same cluster group, while F was in a different cluster group. All the above findings suggested that chemical fertilization had a stronger effect on the composition, relative abundance of the dominant bacterial group and bacterial structure in soils than incorporation of straw residue. While chemical fertilization decreased soil bacterial richness, it increased bacterial predominance. On the contrary, straw residue incorporation increased soil bacterial diversity, but decreased bacterial predominance.

通过对小麦/玉米秸秆还田土壤样品进行富集培养,利用刚果红纤维素培养基筛选得到产纤维素酶的真菌J-25,并对该菌株进行形态学观察和18S-ITS-5.8S区序列系统发育分析,初步鉴定为格孢腔菌属(Phaeosphaeria),定名为Phaeosphaeria sp.XJ-25.在研究液体发酵培养基中碳源、氮源、培养时间、起始pH、培养温度、接种量以及通气量对菌株XJ-25的羧甲基纤维素酶(CMC)的酶活及滤纸酶(FPA)的酶活影响的基础上,利用响应面法对发酵条件进行优化.结果显示,CMC酶活达到最大的条件为玉米秸秆粉1.34 g·L-1(麸皮0.16 g·L-1)、尿素10.06 g·L-1、培养基初始pH 3.25、接种量4.13％,CMC酶活达到23.871 U·mL-1. FPA酶活最大的发酵最佳条件为玉米秸秆粉1.34 g·L-1(麸皮0.16 g·L-1)、尿素10.12 g·L-1、培养基初始pH 3.41、接种量4.22％,FPA酶活为8.653 U·mL-1.粗酶液的最适反应温度为50℃,酶液的热稳定较差,当温度超过40℃,该酶活力显著下降.最适反应pH为5,在pH 4～6的范围内酶活性较稳定.

In order to explore the regulation and control of the gene expression of ATP sulfurylase(APS),the key enzyme related to selenium metabolism,we cloned the APS DNA fragments from tea plant by PCR,and cloned the 5′ flanking sequences of APS genes by application of the genome walking technology.Using the online promoter prediction tool of PLACE,Plant CARE to analysis the gene structure,the results showed that the gene sequence contains the specific structures of a promoter,such as TATA-box,CAAT-box,etc.The results established a certain theoretical foundation for the expression of the key enzyme gene related to selenium metabolism.

In order to investigate the molecular mechanism of Vc accumulation,661 bp cDNA encoding L-galactono-1,4-lacton dehydrognease(GalLDH) fragment was cloned from fruit of Actinidia eriantha by the method of RT-PCR,and then translated into amino acid sequences.In GenBank,compared with Actinidia delicisa,the amino acid sequence of GalLDH gene sequence showed 99% identity with those of Actinidia delicisa and they were both classified in the same family.The amino acid sequence of GalLDH gene sequence showed 91% identity with Vitis vinifera and more than 80% identity with Fragaria xananassa,Rosa roxburghii,Ipomoea batatas,citrur unshiu,Arabidopsis thaliana and Cucumis melo.These results suggested that the cDNA fragment cloned in this stady was from GalLDH gene of Actinidia eriantha.

Glutamate-1-semiadhyde aminotransferase (GSAT) is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase (UPMT), a novel red fluorescent protein. In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli, we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU. The expressed UPMT was fused histidine tag at N terminus. We also amplified E. coli hemL gene encoding GSAT by PCR reaction, eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid. The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU. The expressed GSAT has the extra Strep-TagII at N terminus. Compared to overexpression upmt gene alone, coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT, as estimated by western blot analysis, or the constitution of red fluorescent materials, as shown by UV/visible light scanning analysis, but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm. The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine, the GSAT inhibitor, suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen III, and thus enhanced the red fluorescence of the E. coli cells conferred by the recombinant UPMT.

This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) from tea plant [Camellia sinensis (L.) O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP (cDNA amplified fragment length polymorphism), we have isolated some transcript-derived fragments (TDFs) occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp (EF011075) cDNA was obtained via rapid amplification of cDNA ends (RACE). It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

The cDNA-amplified fragment length polymorphism approach was used to identify genes expressed differentially during early and late flower bud development in tea plant(Camellia sinensis).The result showed that there was evident differential expression in flower buds between the early and late stages.A transcript derived fragment,TDF53,was obtained via selective amplification with E12/M20 primer pair.TDF53 was specifically expressed only in the flower buds at the late stages by RT-PCR.The complete cDNA was recovered by rapid amplification of cDNA ends,then cloned and sequenced.It was found that the full-length cDNA of 2 079 bp(GenBank accession number DQ887753) included a 1 701 bp open reading frame predicting a 567-aminoacid polypeptide of 63 kDa.Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with pollen specific proteins from tobacco and Brassica napus.Thus,we propose that this cDNA encodes the first pollen specific protein described in tea plant,designated CsPSP1.

SCAR marker is one of new molecular markers based on RAPD technology.It has such merit as low price,high stability,site-specific and so on compared with other markers,and it may provide a new way for molecular breeding of tea.In this experiment,the special segment with random primer in different tea cultivars genomic DNA was recovered,cloned and sequenced.According to its sequence,a pair of special primers was synthesized and the RAPD marker was successfully converted into SCAR marker.It improves stability of molecular identification and affirm applied prospect of SCAR technology in genetic resources of tea.

【Objective】This study was conducted to analyze differential gene expression pattern during the early and late stages of flower bud at molecular levels. 【Method】cDNA-amplified fragment length polymorphism (cDNA-AFLP) was employed to analyze the gene expression pattern during different developmental stages of flower bud. The complete cDNA sequence of 14-3-3 gene was isoslated from flower bud at the late stage using rapid amplification of cDNA ends (RACE). The expression of 14-3-3 protein was analyzed through reverse transcription polymerase chain reaction (RT-PCR) and Western-blot. 【Result】One hundred and twenty-two (10.9%) from approximately 1 100 cDNA fragments analyzed were differentially expressed. The full-length cDNA sequence of 14-3-3 gene is 1 072 bp (GenBank accession number DQ444463). It contained a 780-nucleotides-long open reading frame starting at position 68 and terminated by a stop codon (TAA) at position 848-850, and a 193-nucleotide-long 3’-untranslated region plus a 29-monomers-long poly (A)-tail. The encoded protein of 260 amino acids in length had a predicted molecular mass of 29.4 kD. Homology searches with the deduced 260 amino acid residues indicated that 14-3-3 of tea (Camellia sinensis) had a high similarity to other plant 14-3-3 proteins. RT-PCR and Western-blot analyses showed that the 14-3-3 gene and protein could be found in the flower buds at the late stage, but not at the early stage, root, stem, and leaf. 【Conclusion】 The protein of 14-3-3 expressed especially in tea flower bud at the late stage. It further indicated that cDNA-AFLP technique is a useful tool for study of geneisolation during tea flower bud development.

Plant expressing vectors of key enzyme genes related to selenium metabolism were constructed by employing the vector pBI121,one of the most effective expressing vectors for dicotyledon in transformation process.In the construction the GUS in pBI121 was substituted by ATP sulfurylase genes(APS1 and APS2)in tea plant,while the selenocysteine methyltransferase of Camellia sinensis(CsSMT)was directly attached to that of GUS,so that expressing vectors of pBI-APS1,pBI-APS2 and pBI-CsSMT were obtained respectively.These engineering bacteria were acquired by introducing recombinant vectors into Agrobacterium tumefaciens LBA 4404(pAL4404)through method of triparental-mating.This practice provides a fundamental investigation for producing enriched-selenium product by the way of the gene engineering.

Plants have relatively high sensitiveness to air pollution. When plants assimilate pollutant to the extent that they are poisoned,their appearance and physiological activity will be influenced. Hydrogen peroxidase which distributes in plant widely is an important enzyme. Activity of hydrogen peroxidase would decrease obviously when plants were polluted. Four sampling points were selected in Hefei city,and leaves were gathered from Ligustrum lucidum Ait. respectively. Atmosphere pollutional current situation could be reflected basically by analyzing activity of Hydrogen peroxidase in leaves. It could help us monitor and appraise atmosphere quality in Hefei city.And we put forward some advice about utilizing plant to monitor atmosphere pollution.

A comprehensive transcriptome analysis by means of cDNA-amplified fragment length polymorphism(AFLP) and modified cDNA-AFLP technique were performed in order to gain further understanding of the development in molecular level of tea reproductive organs: tea floral bud.Fragments detected among the young and the mature tea floral bud were grouped into three differential expression patterns :(Ⅰ) The young-specific expressed fragments;(Ⅱ) Fragments detected only in the mature tea floral bud;(Ⅲ) Fragments occurring in both.Some transcript-derived fragments(TDFs) were isolated by cDNA-AFLP and its modified technique.The majority of the TDFs are still function unknown according to BLAST alignment search in public data bases.Some of them showed homology with known genes.They are anthranilate phosphoribosyl transferase(AnPRT),beta-primeverosidase,Cytchrome(P450),DnaJ-like protein,Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit(RbcS),alpha-tubulin and Carbonic anhydrase by cDNA-AFLP technique.We isolated pollen coat protein gene(Pcp) by modified cDNA-AFLP method.The full length of three genes,pollen coat protein(Pcp),DnaJ-like protein and RbcS,have been cloned and submitted to Gen Bank.

The gene of β-1,3-glucanase(β-1,3-glu) extracted from tea is found to be difficultly translated in prokaryotic cell,for some sequences binding with the RBS(ribosome binding site) compete with the ribosome.However,it gets much easier to be translated in E.coli by mutating these two sequences.To make sure that the primary structure of protein are not altered,experiment techniques such as PCR in the team of synonymous codons are conducted.Molecular mass of the translated protein proved to be 75.8 kD by SDS-PAGE as expected.In addition,its enzyme activity was detected by Salicylic acid method.

DDRT-PCR technique was used to explore the differential gene expression profiles analysis of tea plant induced by tea looper (Ectropic oblique) attack. Results showed that 222 differential expression fragments were obtained, which was 32.8% of total fragments, by amplified in anchor primers A3, A4, A6, A7, A8 and random primers R1-R8,R12, respectively. Twenty fragments of which were re-amplified by RT-PCR. Based on the BLASTx search in GenBank, it were found that eight of the fragments shared no homology, five of the fragments shared homology with the unknown proteins, and seven of the fragments shared higher score homology with the known proteins, which can be divided into six groups related to photosynthetic pigment protein in photosystem II(C15), abiotic resistance induced protein(D22), glucoside metabolism enzyme(A45), nucleic acid and protein transcription and regulation factors(A12, D63), auxin-regulated protein(E94) and VIP2 protein(D73). The fragments of C15, A45, D22, D73 and E94 were firstly found in the studies of molecular mechanism of plant-insect interaction, up to day.

For studies of differential gene expression, cDNA-AFLP was analyzed in tea plant at the stage of bud flower development. The result showed that there were evident differential expression in flower between the early stage and the late stage. Of the 1110 fragments inspected, 35 and 87 specially displayed in the early and late stages; 15 fragments were cloned and sequenced. Sequence analysis indicated that among 15 squenced fragments, nine were sequence-similar to proteins in GenBank, i.e. oxidoreductase, chromosome 13, 14-3-3 protein, Amyrel gene, calreticulin interacted protein, ATP sulfurylase, catalytic / iron ion binding, desiccation-related protein and ntp302, two were similar to hypothetical proteins, and no similar sequence were found in GenBank for the other 4 fragment.