WD40 is a kind of scaffold protein, which is widely involved in various processes such as plant growth and development, anthocyanin biosynthesis, and biotic/abiotic stresses. Using bioinformatics technology, we identified 258 HbWD40 family members from the rubber tree（Hevea brasiliensis） genome,and systematically analyzed their chromosomal distribution, protein conserved domains, tissue-specific expression, and expression patterns under low temperature stress. The results showed that HbWD40members were distributed on all 18 chromosomes; the HbWD40 family could be divided into 28 subfamilies according to different conserved domains. The results of expression pattern showed that 175 members were expressed in all 5 tissues, and 8 differentially expressed members were identified under low temperature stress. We further cloned HbWD40-168 gene by RT-PCR technique. The coding sequence（CDS） of this gene was 1 335 bp in length, encoding a putative protein of 444 amino acids. Conserved domain analysis showed that HbWD40-168 contained four WD40 repeat elements and two ZnF＿C3H1 zinc finger protein domains. Promoter analysis revealed the presence of multiple hormone-or stress-related cis-acting elements in the promoter region of HbWD40-168. Subcellular localization showed that HbWD40-168 localized in the nucleus. This study provides a basis for in-depth identification of the biological functions of the HbWD40 family in rubber trees.
Protoplasts are widely used in genetic transformation, physiological and biochemical fields, for it is easy to absorb exogenous substances. Also, an efficient protoplast isolation system and transient transformation system are the basis for molecular biology and related research. In recent years, plant protoplast isolation and transformation technology has achieved rapid development and application. Currently,the commonly used methods for plant protoplasts transient transformation include Agrobacterium infiltration, polyethylene glycol（PEG） mediation, electroporation, plant virus vectors and biolistic methods. This paper summarizes protoplast isolation steps, the factors affecting protoplasts isolation and purification,plant transient transformation methods and transient expression systems application fields, in order to provide reference for the establishment of more plant protoplast isolation systems and transient transformation systems.
In this paper, based on transcriptome library of Oncidium after soft rot infection, 72 WRKY protein sequences were identified, with a length distribution of 110–719 amino acids, an average length of 319.8 amino acids. Among the 72 WRKY protein sequences, 14 sequences were classified into group Ⅰ, 42sequences were classified into group Ⅱ, and 16 sequences were classified into group Ⅲ. Group Ⅱ could be further divided into 7 subgroups. Those 72 protein sequences contain at least one WRKY domain and C2H2 type zinc finger motif, of which the sequences from group I contain two WRKY domains. 12 WRKY genes were screened for fluorescence quantitative PCR analysis, results showed that the expression pattern of those 12 WRKY genes basically verified the high-throughput expression pattern, and the responses pattern could be divided into three types. Among them, OncWRKY44（Ⅰ）, OncWRKY69（Ⅱg） and OncWRKY70（Ⅱg） 3 WRKY genes from the first and third types could be induced by salicylic acid and methyl jasmonate,and showed the highest expression level in the infection site of symbiotic plants, which may be important node genes involved in the induced system resistance brought by Piriformospora indica.
The effect of Arabidopsis seed oil content on seed longevity was studied using the mutants with decreased seed oil content and overexpressing line with increased seed oil content. The results indicated that the wri1-1, wri1-4, fus3-T, AS11（dgat1-1） and ABX45（dgat1-2） mutants, which accumulates less triacylglycerol（TAG） in seeds, exhibit defect in seed longevity. On the contrary, the 35S-WRI1 overexpressing seeds with more accumulated TAG show an increase in seed longevity. Moreover, wri1-1 and wri1-4 are more sensitive to oxidative stress, compared to wild type. The treatment by ascorbic acid rescued the decrease of germination rate of these aged seeds. In summary, the storage TAG of Arabidopsis seeds may affect seed vigor through the reactive oxygen species（ROS） scavenging system.
Flowers are the reproductive organs of angiosperms, and flowering control is of great significance to the production of crops and ornamental plants. Flowers open in a variety of ways, the time required for flowers to open is also varies. The opening and closing of flowers are accompanied by a variety of physiological processes, such as cell expansion, elongation and changes in cell osmotic pressure,which are regulated by light, temperature, water, phytohormones and other factors. This paper reviews the flowering regulation mechanism from the aspects of physiological process, pollination effect and environmental factors, and provides a theoretical basis for further research on flowering regulation mechanism.
花椒(Zanthoxylum armatum)开黄花后不能正常结实,严重影响了相关产业发展.为探究花椒开黄花的生理特点及其防治关键药剂,本研究对不同时期花椒形态进行了观察,对多种植物激素含量进行了测定测定.以重庆多地九叶青花椒(Z.armatum var.novemfolius)正常株和黄花株为试材,采用石蜡切片法对花椒形态进行观察并对花椒黄花株和正常株花叶数量质量进行统计,采用高效液相色谱法和酶联免疫法对茎尖、花序的植物激素指标进行测定分析.结果表明:(1)花椒开黄花的生理实质为雌蕊的缺失和雄蕊的出现.花芽分化期黄花株花芽发育时间比正常株早,且分化程度更高.初花期和盛花期黄花株表现为雄蕊异常发育、雌蕊缺失特征,正常株则为雌蕊发育;且在盛花期时,黄花株花芽表现出发育时间早、花芽数量过多的特征.花椒黄花株小花质量、小花数量和花叶质量比分别是正常株的1.61倍、2.86倍和7.65倍,黄花株新生叶质量降低23.3％,数量下降55.6％.(2)脱落酸(ABA)和水杨酸(SA)在黄花株和正常株的秋季茎尖及次年春季花序中的含量差异显著.黄花株秋季茎尖中ABA含量下降了45.6％,SA含量上升了1.18倍.(3)喷施三碘苯甲酸(TIBA)药物能够改善花椒开黄花现象.喷施100 mg?L?1 TIBA后,春季黄花株中的SA含量较正常株已无显著差异;喷施500 mg?L?1 TIBA后,黄花株中ABA和SA含量较正常株均无显著差异.综上结果显示ABA和SA在花椒开黄花中起到关键性作用.
Cinnamate 4-hydroxylase（C4H）, a key enzyme in the phenylpropanoid metabolism of plants,plays an important role in the flavonoid biosynthetic pathway. Cold stress could significantly increase the total content of flavonoids which exhibit various pharmacological activities in Tetrastigma hemsleyanum.However, the exact mechanism is still unclear. In this study, the results of real-time quantitative PCR（qRTPCR） showed that the relative expression of C4H in T. hemsleyanum（ThC4H） increased under cold stress.Therefore, the full-length cDNA sequence of ThC4H was obtained by rapid-amplification of cDNA ends（RACE） and cloned into Saccharomyces cerevisiae. The activity of recombinant protein ThC4H was analyzed in vitro. The structural alignment and the molecular docking with substrate were also studied. The results showed that the cDNA of ThC4H contained an open reading frame of 1 518 bp, encoding 505 amino acids. Moreover, the ThC4H contained a conserved P450 domain and shared 91.88% sequence identity with the C4H of Parthenocissus henryana. Recombinant ThC4H displayed the ability to catalyze trans-cinnamic acid to p-coumaric acid in vitro. Molecular docking results showed that the formation of hydrogen bonds and the binding site location and orientation of C4H to trans-cinnamic acid were similar among T.hemsleyanum, liverwort and Sorghum bicolor, suggesting that they probably share a similar catalytic mechanism. Furthermore, the loop of number 2 and the diversity of two residues in the heme binding domain might contribute to the different properties of C4H. These results laid a foundation for the further functional exploration of C4H under low-temperature stress in T. hemsleyanum.
4-hydroxy-3-methyl-2-butenyl diphosphate synthase（HDS） is a key enzyme in the monoterpene synthesis pathway. In order to reveal its effect in the synthesis of terpenes of Lilium ’Siberia’, the LiHDS gene of Lilium ’Siberia’ was cloned and analyzed by bioinformatics subcellular localization and virus induced gene silencing（VIGS） function verification, and the temporal and spatial expression of the gene was analyzed by real-time quantitative PCR（RT-qPCR）. The results showed that the open reading frame of LiHDS gene was 2 223 bp, the coding protein included 740 amino acids, the theoretical molecular weight was 81 967.95, and the theoretical isoelectric point was 5.76, which was highly similar to HDS protein such as Musa acuminata. LiHDS protein was mainly located in chloroplast. The highest expression of LiHDS was found in inner and outer tepals, and the lowest expression was in roots and leaves. In tepals, the expression of Li HDS gene was highest at the blooming stage and lowest at the senescence stage. After silencing the LiHDS gene in the petals by VIGS technology, the release of ocimene and linalool decreased significantly by 79% and 73% respectively, indicating that Li HDS gene played an important role in the monoterpene synthesis of Lilium ’Siberia’.
Enolase is a key enzyme involved in metabolism, growth, development of plants and responds to abiotic stresses. In this study, ZmENO1, a maize enolase gene was cloned, which protein sequence was highly similarity with sorghum XP＿021305192.1（SbENO1）. The expression of ZmENO1 was induced by drought stress, and the expression level was positively correlated with the drought tolerance of the material.Overexpression of ZmENO1 enhanced the tolerance of Arabidopsis to drought stress by stabilizing cell membrane osmotic potential and enhancing antioxidant enzyme activity through an ABA-dependent pathway. Interestingly, it was found that ZmENO1 enhanced the high temperature tolerance of overexpressing Arabidopsis lines possibly through an ABA-independent pathway. Yeast two-hybrid experiments showed that ZmENO1 was involved in carbon dioxide fixation, signal transduction and glyceride and sugar metabolism. This study clarified the function of ZmENO1 in response to drought and high temperature stress in Arabidopsis, and preliminarily predicted its potential interaction network, laying a foundation for further analysis of the mechanism of ZmENO1.
Populus euphratica leaf rust,caused by Melampsora larici-populina,is one of the serious fungal diseases that damages poplar seedlings and young forests,which also affects P.euphratica afforestation.However,very few reports have studied how P.euphratica physiologically responds to M.larici-populina infection.We used P.euphratica as the experimental material.We sprayed poplar with M.larici-populina exogenously,while we sprayed the control with distilled water.Subsequently,we collected the infected and control leaves at 7,14,and 21 d post-infection,and we aimed to explore the defense mechanism of P.euphratica against the leaf rust fungus by investigating the changes in the leaf reactive oxygen species（ROS）,antioxidant enzymes,hormones,and secondary metabolites like phenol and flavanol.The results show:（1） The ROS content decreased in the infected leaves.Nitrate reductase （NR） activity in infected leaves increased to 133.91 m U·g -1 at 14 d,with the NR activity in the infected leaves being higher than that of the control.As compared with control,the activities of superoxide dismutase （SOD）,peroxidase （POD）,catalase （CAT）,and ascorbate peroxidase （APX） all increased,with POD and APX activities increased 87%and 1.17 times at 21 d,respectively.Furthermore,the positive correlation between activities of NR and POD had increased post-infection.（2） Infected leaves had higher abscisic acid （ABA） and salicylic acid（SA） contents than the control post-infection,which reached 463.63 ng·g -1 and 39.51μg·g -1 at 14 d in the infected leaves.（3） The activities of cinnamate 4-hydroxylase （C4H） and leucoanthocyanidin reductase（LAR） reached 1.31 k U·g -1 ·min -1 and 54.70 U·g -1 ·min -1 in the infected leaves at 14 d.The trend changes of phenol and flavanol were similar,and they decreased to 2.18 mg·g -1 and 90μg·g -1 at 14 d,respectively,and subsequently increased.We found a significant positive correlation between phenol and flavanol contents.In conclusion,P.euphratica scavenged ROS by enhancing antioxidant enzyme activity to improve the disease resistance in infected leaves.The plant hormones ABA and SA played defensive roles in infected leaves and were related to the enhancement of antioxidant enzyme activities.Furthermore,the NR-mediated nitric oxide signal pathway may participate in plant defense against pathogens along with hormones.The secondary metabolites produced by the phenylpropane pathway,like total phenol and flavanol,also were essential in poplar defense.Therefore,we provided a reference for future studies focused on the defense mechanism of P.euphratica against the M.larici-populina fungus.
In order to investigate the dwarfing effect of UV-B on trees, the recovery effect of IAA and the causes, the two years old ‘Luyou 3’ nectarine（Prunus persica var. nectarina） cultivated in greenhouse was used as material, and UV-B irradiation, UV-B+IAA and UV-B+TIBA（auxin transport inhibitor） were set up,the indexes related to growth and IAA metabolism were measured. The results showed that UV-B treatment significantly dwarfed the dry height, shortened the internode length of branches, reduced the number of leaves, decreased the content of tryptophan（TRP） and IAA in trees, and improved the resistance and IAAO activity. After the addition of IAA, the growth parameters, contents of IAA and TRP were significantly increased, reaching or even higher than the CK level, maintaining the previous high resistance, and the activities of two key synthetic enzymes were raised to the CK level, and the oxidative decomposition activity of IAAO was reduced to the CK level. TIBA treatment significantly restored stem diameter and leaf number, and continued to significantly decrease IAA content. None of the treatments significantly affected on the number of internodes. It can be seen that UV-B can reduce IAA content by decreasing TRP content and increasing IAAO activity, so as to dwarf the tree and shorten the branches. Exogenous IAA could not only offset the inhibitory effect of UV-B, but also play a role in promoting growth and maintaining high resistance. The results can provide reference and theoretical basis for the cultivation of strong seedlings in facilities and the regulation of tree growth in high UV-B areas.
Flower color is one of the main factors in assessing the ornamental and commercial value of horticultural plants. The prerequisite for breeding and developing varieties of flower color in ornamental plants is to understand the mechanism of flower color formation and its regulatory mechanism. Daylily（Hemerocallis spp.） are colorful and graceful, and are one of the three main perennial flowers. This paper outlined the research progress in daylily flower morphology, coloring types, pigment synthesis and its regulation, genetic modification, etc. to provide reference for the subsequent breeding of new varieties of daylily with different flower colors.
Sacha inchi（Plukenetia volubilis） is a promising woody oil plant, the seed oil of which is rich in α-linolenic acid. However, the lack of genetic transformation method greatly limited the investigation of gene function and molecular breeding in sacha inchi. In this study, based on the naked-eye visual reporter system RUBY, the effects of different Agrobacterium rhizogenes strains, infection methods, and vacuum treatment on the induction of hairy roots and genetic transformation efficiency were invesitgated. The results show that coating hypocotyle bases of seedlings with primary roots removed with A. rhizogenes K599 showed the highest effciency in inducing the hairy roots. Abundant composite plants with red hairy roots overexpressing RUBY were obtained by using this method. The calli were further induced in the callus-inducing media by directly using the transgenic red hairy roots as explants. Thus, in this study, we report the first A. rhizogenes–mediated hairy root transformation system in sacha inchi. This protocol would lay a foundation for further establishment of the efficient genetic transformation method in sacha inchi,and also facilitate the functional characterization of novel genes in the sacha inchi root system.
BBX transcription factors play vital roles in plant growth, development, and stress responses. In this study, CoBBX22 was cloned from Camellia oleifera by reverse transcription PCR（RT-PCR）. Sequence analysis showed that the open reading frame（ORF） of CoBBX22 gene is 891 bp, encoding 296 amino acids. It was predicted that the CoBBX22 protein is an acidic protein with 32 kDa molecular mass and 4.88isoelectric point. And the CoBBX22 protein contains two B-box domains. Phylogenetic analysis showed that CoBBX22 and CsBBX22 from C. sinensis are in the same branch, and the amino acid sequence identity is as high as 96.96%. Subcellular localization analysis revealed that CoBBX22 protein is localized in nucleus. Further studies showed that overexpression of Co BBX22 gene in Arabidopsis thaliana elevated plant sensitivity to drought stress. Therefore, the Co BBX22 from C. oleifera might act as a negative regulator in A.thaliana response to drought stress.
In order to establish a high-efficiency regeneration technology system of Sanguisorba officinalis,the effect of different plant growth regulator combinations on the induction of bud clusters,the proliferation of buds clusters,the growth of shoots,and the rooting of shoots were investigated by taking the stem segments with shoots as explants.The results show that when explants were cultured on MS+1.5 mg·L -1 6-BA+0.5 mg·L -1 NAA+30 g·L -1 sucrose+7 g·L -1 carrageenan medium for 30 days,the axillary buds expanded into brown hard callus with clump buds differentiated on the surface,an average of 7.6 buds were induced per axillary bud and the inductivity of explants was 100%.The callus with buds was transferred to MS+3.0 mg·L -1 6-BA+0.5 mg·L -1 NAA+30 g·L -1 sucrose+7 g·L -1 carrageenan medium to proliferate for30 days,the average of 10.1 sprouts were produced per generation,and the average proliferative multiple of bud cluster increased to 3.26 times.After the bud cluster were divided into small pieces,they could be cultured on the proliferation medium for four generations,and the multiplication ratio of each generation was 2.4 to 2.6,with an average of 10 sprouts per generation.Sprouts could be transfer to 1/2MS+0.05–0.1 mg·L -1 NAA+0.1 mg·L -1 IBA+15 g·L -1 sucrose+0.5 g·L -1 activated carbon+7.0 g·L -1 carrageenan medium to rooting.After 30 days,the rooting rate reached 100%,and more than seven adventitious roots with an average root length of more than 12 cm were induced at the base of the sprouts,and the seedling height could reach more than six cm.This study established the asexual rapid propagation technology system of S.officinalis,which provides a basis for the large-scale production of S.officinalis plantlets.
The pathological research on radish clubroot is very preliminary. In this study, we used Plasmodiophora brassicae to infect the roots of radish seedlings to observe the infection pattern, and the metabonomic detection technique based on LC-MS/MS was used to detect metabolites in the roots of infectious diseases and healthy radish. The results showed that P. brassicae completed lateral root infection within 72 h after inoculation, symptoms mainly appeared in the cortex splitting stage of radish. It is speculated that P. brassicae initially arrivals the spore proliferation stage during the division of the cortex cells. The root biomass of susceptible radish decreased significantly in the later stage of growth, the vascular tissue in the diseased root disappeared, and the cells were filled by spores. The metabonomic detection of radish at cortex splitting stage showed there was a significant separation between control and susceptible samples; a total of 6 354 different expressed metabolites were detected, mainly including arachidonic acid, terpenoids, starch and other substances.
Plant lipoxygenases（LOXs） catalyze lipid degradation and produce physiologically active metabolites. Lipoxygenases are involved in the LOX pathway which is a series of enzyme-catalyzed reactions.Plant lipoxygenases can be divided into two subfamilies, 9-LOX and 13-LOX, based on the catalytic position specificity of the substrates. The rice LOX-1（encoded by OsLOX1） is an unconventional lipoxygenase with dual position specificity. In this study, through the bioinformatics analysis of the rice lipoxygenase family, the amino acid sequence similarity and conserved domain were analyzed, and the dual position-specific conserved sites were compared. And the rice OsLOX1 gene was cloned, heterologously expressed, and purified, and recombinant LOX lipoxygenase exhibited enzyme activity. In this study, the bioinformatics analysis and heterologous expression of rice lipoxygenases provide a basis for revealing the mechanism of dual position specificity, and for applying in agriculture, food, and chemical industry.
It is difficult to propagate Begonia peltatifolia by leaf cuttings which rarely regenerate shoots. For better protection and utilization of this species, its leaves are used as explants for efficient in vitro propagation. The results indicated that the optimum callus and shoots induction medium for the leaves were MS+6-BA 1.00 mg·L -1 +NAA 0.15 mg·L -1 , with 97.67% and 71.00% induction rate. The proliferation coefficient of clustered shoots reached 20.10 in the MS+6-BA 1.25 mg·L -1 +NAA 0.05 mg·L -1 as optimum medium. The optimum rooting medium for clustered shoots was 1/2MS+NAA 0.50 mg·L -1 （single shoot） and 1/2MS+NAA 0.10 mg·L -1 （cluster shoots）. The rooting rate of them was 91.11% and 100.00%, respectively.The shoots in the shoot-cluster state were dark green, and the roots were robust. After being transplanted,about 90.63% of plantlets survived.
钾素(K)是保障梨果高产和优质的关键元素,土壤缺钾会导致果实品质严重下降,而钾离子通道基因在草本植物响应外界供钾水平、控制体内钾离子吸收和运输中起关键作用,但在木本植物中该基因的功能和分子机制尚不清楚.本研究前期通过RNA-Seq技术从'黄冠梨'中筛选到一个中柱外向整流型钾离子通道基因PbSKOR,生物信息学表明PbSKOR蛋白质含有1个典型的离子跨膜结构域PF00520,在烟草的瞬时表达发现该蛋白质定位在细胞膜上.系统发育分析表明该蛋白与苹果MdSKOR同源关系最近.研究发现在高钾和低钾胁迫下,PbSKOR-过表达拟南芥植株根系中PbSKOR基因的表达水平都显著高于叶片,表明PbSKOR基因具有组织特异性表达的特点.无论低钾胁迫还是高钾胁迫下,PbSKOR-过表达拟南芥植株根系和叶片中的生物量、钾素含量和钾素积累量都显著高于野生型拟南芥和skor突变体拟南芥,这说明PbSKOR过表达能够增强拟南芥植株应对外界高钾和低钾胁迫的能力,提高K+吸收,调节植株体内K+的平衡和渗透势.基于以上分析推测该基因将会在梨树钾素高效吸收新品种选育和应对外界非生物胁迫中发挥重要作用.
In order to investigate the effect of plant growth promoting bacterium Pseudomonas putida UW4 on the root growth and physiological characteristics of chrysanthemum cuttings, the cuttings seedlings of ‘Jinling Chixing’, ‘Jinling Honghe’ and ‘Jinling Meiguijing’ were irradiated by P. putida UW4. And the root growth index, chlorophyll content, antioxidant enzyme activities and other physiological indexes were investigated. The results showed that, the root morphology and growth indexes of different chrysanthemum were significantly improved after treating the growth-promoting bacteria. Compared with the control（CK）,the growth-promoting bacteria significantly improved the root volume of ‘Jinling Chixing’ by 193%, and significantly improved the root surface of ‘Jinling Honghe’ and ‘Jinling Meiguijing’ by 38% and 30% respectively, the root activity of ‘Jinling Meiguijing’ increased by 50% significantly. Growth-promoting bacteria could promote photosynthesis and growth metabolism of different chrysanthemum cuttings, the content of total chlorophyll, soluble protein and free amino acid in cuttings leaves were significantly improved by 11%–157% compared with the control. Growth-promoting bacteria reduce the content of peroxides in cuttings and increase the activities of antioxidant enzymes to alleviate mechanical damage during cutting. The mitigation effects of growth-promoting bacteria were significantly different among different varieties, the growth-promoting bacteria significantly reduced the contents of MDA and H2O2 by 17% and 24% respectively, and increased the activities of antioxidant enzymes by more than 50%. By principal component analysis, 2 principal components were extracted in 19 indexes. It was indicated that the factors with higher loading were root growth factor of principal component 1, and antioxidant factor photosynthetic pigment factor and growth metabolism factor of principal component 2. Combined with the clustering heatmap,the rooting ability and physiological level of different chrysanthemum after treating growth-promoting bacteria were ‘Jinling Meiguijing’ ＞ ‘Jinling Honghe’ ＞ ‘Jinling Chixing’, and the growth-promoting effects of growth-promoting bacteria on different chrysanthemum were significantly different. According to the effects of growth-promoting bacteria on various indexes of chrysanthemum cuttings, it was found that the growth promoting effect on ‘Jinling Meiguijin’ cuttings was the best, and the root growth（total root length,root surface area, root volume, root tip number, branch number） and antioxidant capacity（SOD, POD, APX and GR） of all varieties were significantly improved, which provided a theoretical basis for chrysanthemum reproduction.
