| Pepper(Capsicum annuum L.)is a global vegetable.Nearly 3/4 of the world’s population regularly consumes peppers and pepper products.China’s pepper planting area ranks first in the world.The green peach aphid(Myzus persicae(Sulzer))not only directly harms plants,but is also a world-dangerous vector insect of 116 viruses,causing serious damage.The effectors in the salivary glands of aphids can inhibit plant defense responses,manipulate cells of host plants and promote their own infectivity to successfully complete colonization.But so far,there has been no research on the effectors of M.persicae that regulate the defense response of peppers.Therefore,this study combines physiological,biochemical and molecular biology.We have systematically carried out the screening of the effectors of M.persicae that can regulate the defense response of pepper,and created a technology system for gene silencing of M.persicae effectors.We explored the affinity of the M.persicae effector and the resistant and susceptible pepper varieties from the molecular level,and preliminarily analyzed the control mechanism of M.persicae effectors based on the salicylic acid and jasmonic acid signal pathways to regulate the defense response of pepper,which provided a theoretical basis for the in-depth understanding of the important role of the M.persicae effectors in the immune induction of pepper and the creation of a new germplasm resistant to aphid.The main findings are as follows:1.Cloned 4 effectors of green peach aphid,found that the green peach aphid effectors Mp10 and Mp C002 can regulate the defense response of pepper.Using the green peach aphid RNA as a template,we cloned 4 peach aphid effector genes Mp10(438bp),Mp C002(413bp),Mp42(453bp),Mp55(327bp)by PCR amplification with specific primers,and we confirmed that the sequence identity is 100%,99.72%,100%,99.78% by sequencing comparison and bioinformatics analysis,respectively.There were significant differences in the expression levels of Mp10 and Mp C002 in M.persicae after feeding resistant and susceptible pepper varieties(P<0.05),but there was no significant difference in the expression level between ZDC and DYJJ after 96 hours of feeding(P<0.05).The expression levels of Mp10 fed with ZDC 6 h,12 h,24 h,48 h and Mp C002 fed with ZDC 6 h,12 h,24 h were significantly higher than those with feeding-susceptible pepper DYJJ by 42.30%,30.98%,and 38.54%,49.82% and 67.07%,72.22%,86.49%,but the expression level of Mp C002 feeding ZDC and DYJJ for 48 h and 96 h was not significant different.2.Create a technology system for gene silencing of the M.persicae effectors Mp10 and Mp C002 to confirm that Mp10 and Mp C002 can regulate the defense response of pepper.The results of artificial feed showed that 20% sucrose water is the best artificial feed for M.persicae.The mortality rate of M.persicae after 8 days of feeding is 15.56%,which is significantly lower than that of compound feed,honey diluent and other concentrations of sucrose water.The results of the analysis of silencing efficiency and mortality showed that 15 ng/μL,4 d;30 ng/μL,6 d were the optimal treatment concentration and treatment time for gene silencing of M.persicae ds Mp10 and ds Mp C002,respectively.The silencing efficiency of Mp10 and Mp C002 were 82.12% and 64.70%,and the mortality of M.persicae was 33.33% and 26.67%,respectively.3.It was found that the M.persicae effector Mp10 can increase the content of salicylic acid and jasmonic acid and the expression of key genes by activating the salicylic acid and jasmonic acid signal pathways of the aphid-susceptible pepper variety DYJJ to regulate the defense response of pepper to the green peach aphid,but only by activating the ZDC salicylic acid signal pathway of the aphid resistant pepper variety ZDC to increase the content of salicylic acid and the expression of key genes so that it can regulate the pepper’s defense response to the green peach aphid.Compared with the normal M.persicae CK,the salicylic acid content in the ZDC leaf tissues significantly decreased by 12.76% at 12 hours after the Mp10 silenced M.persicae infestation,and the salicylic acid content in the DYJJ leaf tissues significantly decreased by 12.23% and 11.71% at 48 h and 96 h after the Mp10 silenced M.persicae infestation,while the salicylic acid content in the leaf tissues of ZDC and DYJJ at other time points after the Mp10 silenced M.persicae infestation were not significantly different from that of CK;Compared with CK,the key genes PR1;EDS5;PAD4 and PAL1 of the ZDC salicylic acid signaling pathway decreased significantly by 37.96% and 33.41% at 6 h and 12 h;29.25% at 12 h;14.50% at 12 h;25.54% and 27.18% at 12 h and 24 h after the Mp10 silenced M.persicae infestation.The key genes PR1;EDS5;PAD4 and PAL1 of the DYJJ salicylic acid signaling pathway decreased significantly by 35.04%,25.88%,34.61% and23.91% at 6 h,12 h,24 h and 48 h;40.26% and 27.39% at 12 h and 24 h;15.51%,26.57%and 27.14% at 6 h,12 h,24 h;17.04% at 24 h after the Mp10 silenced M.persicae infestation,and PR1,EDS5,PAD4 and PAL1 in leaf tissues of ZDC and DYJJ were not significantly different from CK at other time points,(P<0.05).Compared with M.persicae CK,the content of jasmonic acid in the leaf tissues of DYJJ significantly decreased by 17.66% and 14.11% at 24 h and 48 h after the Mp10 silenced M.persicae infestation.The content of jasmonic acid in leaf tissues of ZDC and DYJJ fed at other time points was significantly reduced.Compared with CK,the key genes LOX2 and AOC of the ZDC jasmonic acid signaling pathway decreased significantly by22.19% and 25.35% at 12 h after the Mp10 silenced M.persicae infestation;DYJJ’s AOC decreased by 29.54% at 6 h,but LOX2 of DYJJ was significantly increased by 55.53% and77.70% at 24 h and 48 h after the Mp10 silenced M.persicae infestation;LOX2,AOC,COI1,and OPR3 in ZDC leaf tissue and DYJJ leaf tissue at other time points after the Mp10 silenced M.persicae infestation were no significant different from CK(P<0.05). |
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