摘要
由大丽轮枝菌（Verticillium dahliae Kleb.）引起的棉花黄萎病是一种土传性真菌维管束病害，是影响我国和世界棉花生产的最重要病害。其防治难度大，至今尚无理想的防治药剂，种植抗病品种是最为经济有效的方法。但陆地棉抗黄萎病机制复杂不清，抗病育种工作难度大。为此，开展陆地棉抗黄萎病机理研究，对抗病相关基因进行功能分析，将为通过基因工程手段培育抗病品种提供重要的工作基础和理论依据。
本研究以本组培育的陆地棉高抗黄萎病新品系中植棉KV1为材料，分别提取接种大丽轮枝菌强致病力落叶型菌株V991后0-96h棉株幼根总RNA，构建正、反向抑制差减杂交文库，经反向Northern杂交，筛选到147个信号强度差异明显的克隆，其中141条为高质量的EST序列，包括99个unigenes和42条重复序列。99个unigenes中包含92个上调表达基因和7个下调表达基因，涉及抗病及胁迫、蛋白质合成、信号传导、转录因子、代谢及次生代谢、细胞壁骨架、未知功能及新基因九大类功能，建立了黄萎病菌侵染初期陆地棉的抗病相关基因表达谱。其中Bet v1基因家族和UbI基因家族的基因高频表达，推测它们在陆地棉抗黄萎病的防卫反应和早期抗病信号传导途径中起重要作用。荧光定量PCR测定结果显示，5个Bet v1基因、2个UbI基因和1个衰老相关基因在接菌的抗病品种中植棉KV1与感病品种鄂荆1号中，表达量具有显著性差异。
棉花皱缩病毒诱导的VIGS技术为棉花抗病相关基因的功能验证提供了一个高效的技术平台。利用病毒诱导的基因沉默技术，在中植棉KV1中成功地沉默了与陆地棉抗病相关的2个泛素连接酶基因GhUbI1、GhSCF和1个功能未知基因GhEG。对沉默植株接种大丽轮枝菌V991，抗病性鉴定结果显示，野生型的中植棉KV1和转化空载体棉株的病情指数分别为17.47和19.64，沉默GhUbI1基因、GhEG基因和GhSCF基因的棉株病情指数分别为55.0、51.79和44.05。GhUbI1基因、GhEG基因和GhSCF基因沉默后，中植棉KV1对黄萎病的抗性丧失，证明了GhUbI1基因、GhEG基因和GhSCF基因在陆地棉抗黄萎病的过程中起重要作用。
成功构建了中植棉KV1接菌前后的均一化全长cDNA文库，库容量为7.5×10~5pfu/mL，重组率为96%。随机挑选了1000个克隆进行测序，得到平均长度为917.75bp的921条高质量EST序列，包括831个unigenes和90条重复序列。利用COG、KOG、KEGG和GO进行功能注释和代谢途径聚类分析，结果显示，169个基因被注释到24种COG分类中，352个基因被注释到24种KOG分类中，343个酶类映射到194个pathway。共获得6783个GO功能注释，平均每个基因有8.16个GO注释。其中3273个注释为生物过程，主要集中于细胞过程和代谢过程；2131个注释为细胞组分，主要集中于细胞和细胞部分；1379个注释为分子功能，主要集中于催化活性和蛋白结合。其中钙调蛋白基因、钙依赖性蛋白激酶基因、钙离子结合蛋白基因、延伸因子Tu基因、WRKY33基因、RIN4基因、丝氨酸-苏氨酸蛋白激酶PBS1基因、转录因子MYC2基因、茉莉酮酸酯ZIM结构域蛋白基因参与陆地棉与大丽轮枝菌互作途径。全长文库的成功构建为下一步克隆基因奠定了基础。
Verticillium wilt caused by the soil-borne fungus Verticillium dahliae, is a devastating disease ofcotton, leading to serious loss of lint yield worldwide. Because no efficient chemical control is availableagainst this pathogen, the usage of wilt-tolerant/resistant cultivars becomes the primary method tomanage this disease. However, the cotton genome is complicated, and the comprehensive understandingof the upland cotton defense response to V. dahliae still remains limited. The breeding of Verticilliumwilt resistant upland cottons has been unsuccessful. To study the molecular mechanisms and identify themolecular components that involve in the defense responses during upland cotton-V. dahliae interaction,will provide the possibility to improve plant disease resistance through plant genetic engineering.
Verticillium wilt resistant upland cotton (Gossypium hirsutum) Zhongzhimian KV1was used inthis study. RNA from the root tissues of Zhongzhimian KV1inoculated with V. dahliae strain V991from6to96h or water mock were used to construct forward and reverse subtractive cDNA libraries.Based on the results from dot blot analysis,147clones were clearly induced by V. dahliae and selectedfrom the SSH libraries for sequencing.141qualified expressed sequence tag (EST) sequences withinsertions longer than100bp were used in the Blast search. The results showed that141qualifiedsequences represented99unigenes and other42sequences were repeats. Among99genes,92wereup-regulated and7were down-regulated. All these genes were divided into9categories, which involvedisease/stress responses, protein synthesis and biosynthesis, signal perception/transduction, transcriptionfactor, metabolic process, secondary metabolism, cell wall/cytoskeleton, unclear functions, and novelESTs. Two important clues regarding wilt-resistant G. hirsutum were obtained from this study. One wasBet v1family; the other was UbI gene family that may play an important role in the defense reactionagainst Verticillium wilt. The result from real-time quantitative reverse transcription polymerase chainreaction showed that the transcriptional level of two UbI genes, five Bet v1gene andsenescence-associated protein gene expressions changed significantly in Zhongzhimian KV1andEjingNo1within96h after V. dahliae inoculation.
Cotton leaf crumple virus-induced gene silencing (VIGS) is a powerful tool for studing genefunctions which participate in the cotton-pathogen interaction.VIGS was used to silence endogenousgenes in resistant upland cotton cultivar Zhongzhimian KV1via targeting the fragment of two E3ubiqutin ligase genes GhUbI1and GhSCF, and a function unkown gene GhEG. V991was used toinoculate silence cotton plants, in order to identify disease resistance. The results showed that, thedisease indice of wild type Zhongzhimian KV1and VIGS with vector control plants were17.47and19.64respectively. The disease indice of silencing GhUbI1, GhEG and GhSCF plants were55.0,51.79and44.05respectively. These results confirmed that GhUbI1, GhEG and GhSCF gene are related toVerticillium resistance in cotton.
A normalized full-length cDNA library of Zhongzhimian KV1was constructed with tissues ofZhongzhimian KV1inoculated with V991from24h and water mock. The titer of library was7.5×105pfu/mL, and the recombination ratio was96%. Random selected1000clones were sequenced, and921 ESTs represented831unigenes and other90sequences were repeats. The average size of cDNAinserts was917.75bp. The reults of COG, KOG, KEGG and GO functional annotations showed that169genes were annotated to24COG functional categories,253genes were annotated to24KOGfunctional categories, and343enzymes were mapped to194pathways.6783GO functional annotationswere obtained, an average of8.16GO annotation for each gene. Among these GO annotations,3273GOannotations belong to biological process, which mainly foucus on cellular process and metabolicprocess.2131GO annotations belong to cellular component, which mainly foucus on cell and cell part.1379GO annotations belong to molecular function, which mainly foucus on catalytic activity andbinding. Calmodulin, calcium-dependent protein kinase, calcium-binding protein CML, elongationfactor Tu, WRKY transcription factor33(WRKY33), RPM1-interacting protein4(RIN4),serine/threonine-protein kinase PBS1, transcription factor MYC2and jasmonate ZIM domain-containingprotein were involed in the the defense responses during upland cotton-V. dahliae interaction. Thislibrary could be well used for gene cloning.
引文
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