Study On Silencing Efficacy Of Chitinase In Mythimna Separata By Maize-mediated RNA Interference

Mythimna separata(Walker)(Lepidoptera: Noctuidae)is a typical seasonal long-distance migratory pest which harms mainly grain crops such as maize and wheat.Host-induced gene silencing which is an important reaearch area in pest control has received extensive attention in recent years.In this study,the effect of Chitinase gene silencing in M.separata was investigated by maize-mediated RNAi using TRV as the expression vector.1.Temporal and spatial expression patterns of Chitinase in M.separata larvae.(1)The expression level of Chitinase genes in the head,epidermis,foregut,midgut,hindgut,Malpighian tubules,and fat body of 5th instar larvae were determined by RT-q PCR.The results showed that Chitinase 1(Chi 1)and Chitinase 2(Chi 2)genes were expressed in all tissues.Chi 1 and Chi 2 expressed mainly in the head and epidermis.The expression level in head were extremely significantly higher than that in other tissues(P<0.01).The expression level in epidermis was second to that in head,with weak expression in the other tissues.(2)The expression levels of Chitinase gene in 5th instar larvae were determined by RT-q PCR.Tests were performed every 12 h from 4th instar larvae after ecdysis.The results showed that the trend of Chitinase expression first decreased and then increased in 5th instar stage.The Chi 1 and Chi 2 expressed mainly at 12 h and 72 h.The Chi 1 m RNA level was extremely significantly higher at 72 h than that at other moments(P<0.01),followed by 12 h,and the lowest was 48 h.The Chi 2 m RNA level was extremely significantly higher at 12 h than that at other moments(P<0.01),followed by 72 h,and the lowest was 48 h.2.Construction of HIGS system for silencing Chitinase genes of M.separata in maize.The specific primers for the interfering fragments adding Bam HI(CGGGATCC)and Kpn I(CGGGGTACC)restriction sites were designed use Primer-Blast based on the Chi 1 and Chi2 gene sequences,the interference fragments of Chi 1 and Chi 2 were obtained by PCR based on armyworm c DNA template.The recombinant plasmids were digested respectively with two restriction enzymes,Kpn I and Bam HI.The interference fragments of Chi 1 and Chi 2with restriction sites were inserted into the Kpn I and Bam HI sites of p TRV2 to obtain p TRV2 recombinant plasmids(p TRV2-Chi 1 and p TRV2-Chi 2).p TRV2 recombinant plasmids were transformed into maize germ(Zhengdan 958)by Agrobacterium tumefaciens GV3101.The transformation effect of p TRV2 recombinant plasmid was detected at DNA and RNA level.The results showed that 100% maize plants were successfully transformed and expressed in 20 d treated maize seeldings.3.Study on silencing efficacy of Chitinase in M.separata by maize-mediated RNA interference.Blank control group(GV3101),negative control group(p TRV2-GFP)and experimental group(p TRV2-Chi 1 and p TRV2-Chi 2)were established.Three biological replicates were set in each group,10 3rd instar larvae were treated in each replicate.(1)Chitinase expressions were examined at 12 h after 4th,5th,and 6th instar larvae finishing molting respectively.The results showed that there were no significant difference in Chitinase m RNA level between negative and blank control group(P>0.05).The expression levels of Chi 1 and Chi 2 in experimental group(p TRV2-Chi 1)were extremely significantly lower than those in negative and blank control group(P<0.01).(2)There was no significant difference in the developmental duration between negative and blank control group(P>0.05).The developmental duration in experimental group was extremely significantly longer than that in negative and blank control group(P<0.01).There was no significant difference in the death rate between negative and blank control group(P>0.05).The death rate was no significant difference between p TRV2-Chi 1 and p TRV2-Chi2 experimental group(P>0.05).The mortality rates of experimental group(p TRV2-Chi 1)were extremely significantly higher than those of negative and blank control group(P<0.01),with the mortality rates of with 33.3%,56.7% and 73.3% for 4th,5th,and 6th instars larvae,respectively.The mortality rate of 4th instar larvae in experimental group(p TRV2-Chi 2)was extremely significantly higher than that in blank control group(P<0.01),and significantly higher than that in negative control group(P<0.05).The mortality rates of 5th(60.0%)and6th(70.0%)instars larvae in experimental group(p TRV2-Chi 2)were extremely significantly higher than that in negative and blank control group(P<0.01).(3)It was found that the larvae in experimental group were smaller than those in negative and the blank control group at the same age.Some larvae could not molt into next instar and died.In this paper,we constructed the temporal and spatial expression patterns of Chitinase in M.separata larvae,and established HIGS system in maize,and verified the efficacy of Chitinase silencing gene in M.separata using maize mediated RNAi technology.The results provide reference date for RNAi technique in insect mediate by maize for the future research,and provide an alternative methods for plant-insect interaction,and also provide some basic information for pest control.