Genetic Transformation And Functional Analysis Of Powdery Mildew And Downy Mildew Related Susceptible Genes In Cucumber

Powdery mildew and downy mildew are two serious diseases in the production of cucumber(Cucumis sativus L.).Planting resistant varieties is the most cost-effective way to control powdery mildew and downy mildew.Researcher have been focused mainly on the study of genetic inheritance.However,the breeding of disease-resistant varieties should not be limited to the research of disease-resistant genes.The mechanism of susceptible diseases and research on susceptible genes should also be paid attention.Therefore,research on the susceptible gene is an attractive option for plant disease resistance breeding.This laboratory has collaborated with the Plant Breeding Department of Wageningen University in The Netherlands,homologous sequence of powdery mildew and downy mildew candidate susceptible genes in cucumber have been selected based on the reported Mlo,Pmr and Dmr genes from Arabidopsis and cucumber whole-genome sequence information.RNAi expression vectors of powdery mildew susceptible genes CAD1(Csa4M332620),PMR4-9(Csa6M128000)and Downy mildew candidate susceptible genes DMR1(Csa7M025730)and DMR6-2(Csa5M146870),and CRISPR/Cas vectors were constructed for the susceptible genes of CAD1,PMR4-9 and DMR1,respectively.Agrobacterium-mediated genetic transformation methods were used to transduce the two vectors into cucumber plants and the functions of the four candidate genes were verified.The results of current study are as follows:1.By comparing the regeneration frequency of RNAi vector and CRISPR vector of the same target gene,the regenerative ability of each target gene on the two vectors is not significant difference.More regeneration buds were obtained by genetic regeneration of RNAi vector than CRISPR vector,and more regenerative plants can be obtained from RNAi vector.2.Agrobacterium transformation method was used to transform the RNAi vectors and CRISPR vectors into cucumber.A total of 189 T0 transgenic plants were obtained,of which 166 transgenic plants with RNAi vectors and 23 plants with CRISPR vectors.3.Agrobacterium transformation method was used to transform the RNAi vectors of powdery mildew candidate genes CAD1 and PMR4-9 into cucumber.After acclimation culture,49 transgenic plants with CAD1 and 32 plants with PMR4-9 transgenic plants were obtained.12 and 7 transgenic plants with powdery mildew resistance were obtained by PCR and showed resistant to powdery mildew in field test and by q RT-PCR.4.Agrobacterium transformation method was used to transform the RNAi vectors of the candidate downy susceptible genes DMR1 and DMR6-2 into cucumber.After acclimation culture,38 transgenic plants with DMR1 and 47 plants with DMR6-2 transgenic plants were obtained.By PCR testing and showed resistant to powdery mildew in the field of T0 and T1 transgenic plants,8 familiy lines with DMR1 and 12 familiy lines with DMR6-2 of transgenic plants had powdery mildew resistance.It shows that powdery mildew resistance has a certain correlation with downy mildew resistance.5.Field investigation on the resistance of To(24 T1 families were formed from)and T1 plants showed resistance to powdery mildew.These families maintained high genetic stability.6.The downy mildew inoculation test was conducted on 38 families of T1 generation form downy mildew candidate gene DMR1 and DMR6-2.The results showed that 9 Family lines had strong resistance to downy mildew,and most of these families maintained disease resistance.Among five family lines in 9 family lines,T0 plants showed reduced expression of the target genes tested by q RT-PCR analysis.Five families were new materials with a stable inheritance of downy mildew in cucumber.