Transcriptional Regulation Of ZYMV Virus Resistance In Cucurbita Pepo

Zucchini(Cucurbita pepo L.)is an important vegetable widely cultivated worldwide.The average annual cultivated area and yield of zucchini in our country are among the highest in the world,and it is still increasing year by year.Now,Zucchini industry has become an important pillar to increase farmers' income and adjust rural industrial structure.However,the production of zucchini is seriously damaged by viral diseases,among which the zucchini yellow mosaic virus(ZYMV)is one of the main diseases of squash and other melon vegetables.After infection of ZYMV,zucchini plants had the symptoms of mosaic,distortion,plant dwarfing,rugged shape of fruit,stiff and bitter flesh,which leads to the decline or even loss of the commercial traits of the fruit.ZYMV infection can lead to yield loss of more than 60%,even no production,resulting in huge economic losses.Breeding resistant varieties is the most economical,safe and effective way to control viral diseases.However,the research on antiviral breeding of zucchini is lagging behind,and there are few resistant resources available,resulting in the lack of virus-resistant zucchini varieties in the production.Therefore,screening the ZYMV virus disease resistance-related genes by transcriptome sequencing is helpful to explore and reasonably evaluate the disease-resistant genetic resources,to provide genetic reserve for the molecular breeding of zucchini virus resistance,and to ensure the safe production of zucchini.It also provides theoretical support for the molecular mechanism analysis of virus disease resistance.Using the artificial inoculation identification at seedling stage,we selected and obtained the zucchini inbred lines that are highly resistant and highly susceptible to ZYMV,and analyzed the inheritance of ZYMV resistance.Total RNA was extracted after inoculation of ZYMV virus,and the transcriptome library was sequenced by Solexa high-throughput sequencing technology.Bioinformatics analysis was performed on the obtained transcriptome data to screen the ZYMV-induced genes and to analyze the expression of virus-induced genes.The main results are as follows:1.Acquisition inbred material with high resistance and high sensitivity to ZYMVThe artificial inoculation identification and evaluation system of zucchini ZYMV virus disease at seedling stage was established.Six high-resistance materials and 10high-sensitivity materials were obtained by inoculation and trait identification of more than 200 germplasm materials of zucchini.2.The ZYMV resistance of zucchini is controlled by a pair of dominant genes.High resistant inbred line CPZR1 and high susceptible sensitivity inbred line CPZS1 were used as parents to prepare six-generation segregated populations.The ZYMV viruses were inoculated twice by cotyledon friction inoculation method to identify resistance and susceptibility.The results showed that the segregation ratio of resistant-and susceptible plants in each generation was in accordance with the Mendelian theoretical segregation ratio,and the ZYMV resistance of zucchini was controlled by a pair of dominant resistance genes.3.Obtaining high quality transcriptome sequencing data of more than 12GTwenty-four hours after inoculation,the virus-induced and blank buffer inducted(control)leaves of disease-resistant and susceptible materials were used to extract total RNA to form 4 sample cells.After high-throughput sequencing,high-quality sequencing data of more than 3G were obtained,respectively.The GC content of zucchini transcriptome was about 47%.The number of Unigenes and the long Unigenes(?1K)was 73,148 and 11,108,respectively..The average length of Unigenes was582.6 bp.The coding region,protein sequence,functional annotation and expression abundance of Unigenes were predicted,and 2287 reliable SNP sites and 4129 SSR sites were obtained between the two materials.4.Thirty-three candidate genes related to ZYMV disease resistance were obtainedThere were 402 differentially expressed genes induced by ZYMV virus between resistant and susceptible materials(the expression difference was more than 1 time)and104 differentially expressed genes between virus induction and control treatment..After cluster analysis of differentially expressed genes,functional annotation,and comparison of expression levels among treatments,33 candidate ZYMV disease-related genes were obtained,which were mainly pathogenesis-related genes,cell wall forming factors,ATPase subunit encoding genes and auxin related genes.The expression profiles of 15 genes were verified by qPCR,which proved the reliability of transcriptome sequencing results and further clarified the expression characteristics of these genes at different time after virus induction.5.Construction of RNAi and plant over-expression vectors of two genesThe full-length sequences of two ZYMV resistance related genes(CpGp1 and CpPr1)were cloned by RACE technology,and the RNAi and plant over-expression vectors of two genes were constructed.