Function Analysis Of The Candidate Gene Ca21394 For Resistance To Phytophthora Capsici By VIGS In Pepper

Pepper(Capsicum annuum L.)is a popular vegetable,which widely grown around the world.Pepper Phytophthora blight caused by Phytophthora capsici,which can occur during all stages of pepper and resulting in severe death,and obviously affect the yield production.Practice has proved that,breeding new cultivars with resistant genes is fundamental to prevent plants from diseases.The preliminary results in our laboratory show,Ca21394 for resistance in pepper blight of candidate genes,but without functional verification;Virus-induced gene silencing(VIGS)is a method of rapid silencing of target gene.It is more and more use for functional analysis of candidate genes in plants.In this study,the resistance to Phytophthora capsici leon.in capsicum materials 'G43' as test material,using VIGS analysis if Ca21394 involved in the resistance response of pepper to Phytophthora capsiei.The results were as follows:Constructing the VIGS recombinant vector pTRV2:Ca21394.Using a milder effect on host tobacco rattle virus(TRV)as a viral vector,with pepper leaf DNA as a template,design primer in Ca21394 gene sequences of the 5 'end of a significant sub region cloned to 438 bp fragment size.In pTRV2 carrier of the XbaI and BamHI double digestion sites insert the fragment when detection is correct by double enzyme cut detection and sequence to finish the construction of recombinant vector pTRV2:Ca21394.And transform the recombinant vector constitution to GV3101(Agrobacterium tumefaciens)by heat shock transformation method.Optimized Agrobacterium inoculation of pepper.VIGS recombinant vector need frozen bacteria liquid activation,LB liquid medium,inducing culture medium(IM)culture before it inoculates plants.This test uses the recombinant vector of eight hydrogen tomato red element desaturase(PDS)gene pTRV2:PDS as the materials,study on OD600 value and culture time of Agrobacterium tumefaciens GV3101.When liquid LB culture medium concentration of bacterium suspension OD600 value was 1.15,faster IM culture bacterium liquid concentration(OD600=0.50).This test got the relationship of liquid LB medium and IM culture time and Agrobacterium concentration.It can cut the times of measure Agrobacterium concentration.Identified the correlation of candidate genes Ca21394 and resistance to Phytophthora capsici.In Phytophthora blight of pepper materials 'G43' inoculation pTRV2:Ca21394 recombinant vector Agrobacterium bacterium liquid.When the plants inoculated pTRV2:PDS,to be albinism symptoms,inoculation Phytophthora capsici to them.Real time quantitative RT-PCR showed that Ca21394 expression decline in the plants of inoculation pTRV2:Ca213945 which 4#silenced plants efficiency highest was 72%,plant disease symptoms significantly.In vitro leaves inoculation of Phytophthora display that not inoculated with Agrobacterium leaf(WT)without obvious disease blade incidence of shape,inoculated with empty vector(pTRV2:00)comparatively light,silent Ca21394 gene(inoculation pTRV2:Ca21394)the onset of leaf is more serious,after inoculated four days lesion covered with leaves.By these phenomenons further illustrates the Ca21394 gene is pepperresistance gene about Phytophthora capsici.This research VIGS recombinant vector pTRV2:Ca21394 by analyze the sequence of pepper Ca21394 gene,preliminary shows that the candidate gene Ca21394 involved in the resistance to Phytophthora capsici reaction,provide scientific basis for further studies on pepper Phytophthora blight gene function.