Gene Expression Proifles In Resistant Upland Cotton Responding To Verticillium Dahliae Miectiou And Function Analysis Of Resistant Related Gene

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.