Cultivation Of Insect Resistant Transgenic Maize Based On PTA Technology And RNAi Mechanism Of Mythimna Separata

Mythimna separata is an agricultural pest belonging to the Lepidoptera Noctuidae,which seriously threatens the food crops such as maize.RNAi technology is an important functional genetic tool,and host-induced gene silencing（HIGS）has opened up a new way to cultivate insect-resistant transgenic plants.RNAi efficiency is low in most lepidopteran insects,so improvement of RNAi efficiency is an urgent need for the control of lepidopteran pests based on RNAi technology.Ms Chi1 and Ms Chi2 are two genes encoding chitinase in Mythimna separata.In this study,transgenic rice expressing hp Ms Chi1 and hp Ms Chi2 was obtained to evaluate the HIGS effect on the recipient insects.The types and characteristics of the HIGS-induced si RNA were further analyzed by small RNA high-throughput sequencing.Furthermore,we developed a PTA expression system based on artificial micro RNA,and obtained transgenic maize which interfered with multiple genes simultaneously.The RNAi efficiency of Mythimna separata treated by hp RNA and PTA RNAi triggers was evaluated and compared.Finally,the biochemical characteristics of the RNAi mechanism core proteins such as Ms GAO2,Ms Dcr2 and Ms SID1 were analyzed,which provided a theoretical basis for the RNAi mechanism of Mythimna separata.The main findings are as follows:1.Analysis of HIGS effect and si RNA characteristics in Mythimna separataTransgenic rice hp Ms Chi1-Ox and hp Ms Chi2-Ox,which stably express the hp RNA expression cassettes of Ms Chi1 and Ms Chi2 genes,were obtained.The RNAi effect in Mythimna separata fed on transgenic rice was evaluated by target gene expression,mortality statistics,body weight and body length measurements.The results indicated that hp Ms Chi2-Ox rice had a more significant RNAi effect on Mythimna separata than hp Ms Chi1-Ox rice.In order to further analyze the abundance and characteristics of Ms Chi2-specific si RNA produced in Mythimna separata after feeding on transgenic rice,we performed small RNA high-throughput sequencing on the gut tissues of Mythimna separata fed on transgenic rice.Ms Chi2 specific si RNA was effectively accumulated in the hp Ms Chi2-Ox rice and Mythimna separata gut tissues feeding on hp Ms Chi2-Ox rice.The Ms Chi2 specific 20-24 nt si RNA has the following characteristics:（1）It is mainly distributed in the trigger region;（2）22 nt si RNA is the dominant si RNA;（3）It has a preference for the sense strand.2.Expression patterns of Ms Chi2 and biological activity of Ms Chi2Expression profile analysis showed that Ms Chi2 was mainly expressed in the body wall and intestinal tissues,which might be related to the degradation of chitin in the epidermis during molting.Ms Chi2protein was isolated from prokaryotic expression system and the purified protein was tested for chitinase activity.The chitin hydrolysis experiment showed that the purified Ms Chi2 protein had the chitin hydrolysis activity,further indicating that the Ms Chi2 protein is a bona fide chitinase.In order to verify the effect of chitinase on the growth of Mythimna separata larvae,the chitinase inhibitor Psammaplin A was mixed with artificial diet to feed Mythimna separata larvae,and ds Chi2 was mixed with artificial diet to serve as parallel control.Similar to the feeding effect of ds Chi2,Psammaplin A showed obvious insecticidal activity against the Mythimna separata.3.PTA expression system improves the silencing efficiency of target genes in Mythimna separataAmi RNA is a novel RNAi technology designed according to the expression characteristics and action principles of mi RNA.In this study,we introduced the PTA（Polycistronic-t RNA-ami RNA）expression system that interferes with multiple target genes.We constructed a PTA expression cassette for the Mythimna separata Ms Chi2,Ms Actin and Ms REase genes,and obtained a transgenic maize PTA-Ox that stably expresses PTA.Compared with hp Ms Chi2-Ox rice,feeding of PTA-Ox maize showed a higher and longer lasting RNAi effect,and simultaneously silenced three target genes.The Mythimna separata fed on PTA-Ox maize showed more obvious developmental defects in different growth stages and different organs.We speculate that the abnormal development may be related to the systematic silencing of the Ms Chi2 gene.The above results prove the effectiveness of our multi-target gene silencing system.4.Cloning and functional verification of RNAi core genes of Mythimna separataTo further explore the RNAi mechanism of Mythimna separata,we analyzed the biochemical activities of the RNAi mechanism proteins such as Ms AGO2,Ms Dcr2 and Ms SID1.In the electrophoretic mobility shift assay,Ms AGO2 bound and blocked the mobility of ³²P-labeled ss RNA in a concentration-dependent manner.Ms SID1 also specifically binds to ³²P-labeled ds RNA in a dose-dependent manner,and this ss RNA/ds RNA binding activity is eliminated by the unlabeled competitor.The Ms Dcr2protein cleaved ds RNA into si RNAs with a size of 20-24 nt,while the control GST protein did not show this activity.The biochemical activity of the RNAi mechanism core protein strongly indicates the existence of systemic RNAi in the Mythimna separata.In summary,we analyzed the characteristics of si RNA and the components of the RNAi mechanism to provide a strong theoretical basis for the RNAi mechanism of Mythimna separata,and the PTA system has important research significance for improving the RNAi control of lepidopteran pests.