Screening Of Squash Leaf Curl China Virus Zucchini Resistant Germplasm And Development Of Virus Vectors

Squash leaf curl China virus(SLCCNV)cause mosaic and leaf curling symptoms after infecting cucurbit plants,which has had a huge impact on the zucchini industry in China in recent years.SLCCNV belongs to the Begomoviruses in the Geminiviridae,and is transmitted by whiteflies.Breeding resistant varieties is the most economical and effective method to control plant virus disease.Screening of disease-resistant varieties is an important part of breeding disease-resistant varieties.Plant viruses have the advantage of using host cell replication rather than integrating into the host genome.The high copy level of geminiviruses based vector is widely used in genetic engineering.In this study,the whole genome of SLCCNV WF-zucchini was analyzed,and the infectious clone of SLCCNV was successfully constructed.The infectious clone of SLCCNV was used to screen the germplasm of Cucurbita pepo resistant to SLCCNV.At the same time,the SLCCNV virus vector was developed,and the SLCCNV transient expression vector,gene silencing vector and gene editing vector were constructed.The specific results are as follows :(1)The complete genome sequence of SLCCNV WF-zucchini isolate was obtained,and the phylogenetic relationship between SLCCNV WF-zucchini isolate and other isolates on NCBI was clarified.The DNA-A and DNA-B of SLCCNV WF-zucchini isolates were obtained by PCR amplification,and the whole genome sequence was obtained by sequencing.The phylogenetic analysis of DNA-A and DNA-B of SLCCNV WF-zucchini isolate showed that the DNA-A of SLCCNV WF-zucchini isolate was close to the genetic distance of Shandong Weifang Shouguang SLCCNV-SDSGC cucumber isolate,the DNA-B was close to the genetic distance of Shandong Zibo SLCCNV-SDZBZ zucchini isolate,and the DNA-A and DNA-B were distantly related to SLCCNV in India,Cambodia,Malaysia,Indonesia and Vietnam.(2)Construction of SLCCNV infectious clone and analysis of their infectivity on various plants.The infectious clones of SLCCNV were constructed : p CB1.4SL-A and p CB1.5SL-B.After 8 days of inoculation with Cucurbita pepo,the leaves of the system showed symptoms such as mosaic and leaf curl,and the infection efficiency was 100 %.In order to analyze the infectivity of SLCCNV WF-zucchini isolate on various plants,nine plant varieties,including Cucurbita pepo,Cucumis melo,Citrullus lanatus,Cucumis sativus,N.benthamiana,Solanum lycopersicum,Brassica rapa,Raphanus sativus and Capsicum annuum were inoculated.The infectivity was determined by symptom observation and PCR detection.Obvious mosaic and leaf curl symptoms were observed on Cucurbita pepo and Cucumis melo,and the Cucumis melo plants were dwarfed.PCR detection showed that the target bands could be detected in Cucurbita pepo,Cucumis melo,Citrullus lanatus,Cucumis sativus,N.benthamiana,but not in Solanum lycopersicum,Brassica rapa,Raphanus sativus and Capsicum annuum.The results showed that the SLCCNV WF-zucchini isolate was infectious on most Cucurbitaceae plants and some Solanaceae plants.(3)Screening the germplasm of zucchini resistant to SLCCNV.The infectious clone of SLCCNV was inoculated on zucchini by Agrobacterium infiltration inoculation,and a total of 46 zucchini germplasms were inoculated.Screening of zucchini disease-resistant germplasm according to its incidence.The results showed that 14 squash germplasms were highly resistant.(4)SLCCNV transient expression vector was constructed to improve the expression of target protein in cucurbitaceae plants.LIR,Ca MV 35 S promoter-target gene-NOS terminator,SIR,AC3-AC2-AC4-AC1-LIR were inserted to p Cambia1300 vector in turn to construct SLCCNV transient expression vector.GFP and Fluc were expressed in N.benthamiana,and the protein expression of GFP and Fluc was higher than that of the control group.It was further verified by fluorescence and Western blot detection on N.benthamiana,N.tabacum,Cucurbita pepo and Cucumis melo.The results showed that the SLCCNV transient expression vector could increase the expression of the target protein in Cucurbitaceae plants.At the same time,P19 can further increase the expression of the target protein.(5)SLCCNV-based gene silencing vector was constructed to silence PDS in melon and zucchini.The AV1 protein in the SLCCNV infectious clone p CB1.4SL-A of SLCCNV was replaced with the PDS conserved sequence of melon and zucchini : gene Cu PDS to obtain the gene silencing vector p CB1.4SL-A-Cu PDS.Melon and zucchini were co-inoculated with p CB1.4SL-A-Cu PDS and p CB1.5SL-B.After 21 days of co-infiltration inoculation,the melon and zucchini showed obvious whitening phenomenon.The silencing efficiency was detected by RT-q PCR.The silencing efficiency was 83.2 % on melon,and69.6 % on zucchini.(6)The SLCCNV-based gene editing vector was constructed to edit the PDS gene of N.benthamiana.The gene editing vectors p C1300-SLC-35S-Sp Cas9 and p C1300-SLC-35S-Nb-g RNA(HinfⅠ)were obtained by using SLCCNV transient expression vector to express the guide RNA(g RNA)of N.benthamiana PDS and Sp Cas9,respectively.After 4-6 days of co-infiltration inoculation,the total DNA of N.benthamiana inoculated leaf plants was extracted,and the PDS gene editing region of N.benthamiana was amplified by PCR(the editing region of N.benthamiana endogenous gene PDS gene has a Hinf Ⅰenzyme site),digested with restriction enzyme HinfⅠ,and the uncut part was connected to p MD18-T vector for sequencing.The sequencing results showed that gene editing occurred in2 of the 12 sequenced samples.The same SLCCNV transient expression vector was used to express the g RNA of N.benthamiana PDS and Sp Cas9,and the gene editing vector p C1300-SLC-Nb-g RNA(HinfⅠ)-Sp Cas9 was obtained.N.benthamiana was inoculated and detected by the above method.The sequencing results showed that 7 of the 10 sequenced samples had gene editing.In summary,the SLCCNV gene editing vector can achieve gene editing on N.benthamiana,and the gene editing efficiency on the same vector is high.