Silencing Chitin Synthase B Gene Of Cnaphalocrocis Medinalis Using RNA Interference

Almost half of the people in the world use rice as their staple food and rice as one of the world’s most important food and cash crops.The prevention of pests and diseases has always been a crucial issue.Cnaphalocrocis medinalis mainly feeds on the leaves of grasses such as rice,and it is also a very serious type of pest.It belongs to the family Lepidoptera(Pyralidae),commonly known as leaf worm,scraper,and leaf hopper.Chitin synthase is a key enzyme in the pathway of chitin synthesis in insects and plays an important role in the growth and development cycle of insects.Therefore,this enzyme is also often used by scientists as an ideal target for pest control.RNA interference(RNAi)refers to the highly conserved and highly specific degradation of homologous mRNAs induced by double-stranded RNA(dsRNA)in the course of evolution.The main purpose of this dissertation is to clone and analyze the full-length cDNA sequence of the chitin synthase B gene(CmCHSB)in C.medinalis.Using transgenic RNAi combined with in vitro RNAi technology,transgenic rice plants with significant silencing effect on the chitin synthase B gene of C.medinalis were obtained.The developmental and lethal effects of the C.medinalis feeding on this transgenic plant can be achieved through the use of safe and effective biological control methods to control the endangerment of rice leaf folder,providing a certain theory and practice for biological control of the pest.The main findings of this study are as follows: 1.Cloning and expression profiling of chitin synthase B gene from rice leaf folderFirstly,RT-PCR was used to amplify the fragment of the open reading frame of rice leaf folder,and then combined with RACE-PCR to clone the full-length cDNA of chitinase synthase B gene(CmCHSB)from Cnaphalocrocis medinalis.Then the two fragments were spliced to obtain the full-length 4878 bp chitinase synthase B gene and 4578 bp open reading frame,encoding a total of 1525 amino acids.Bioinformatics analysis of the enzyme using related online software showed that the enzyme has 17 transmembrane helix regions,the theoretical isoelectric point is 5.89,no signal peptide.Homologous modeling predictions show that the three-dimensional molecular space structure of CmCHSB contains 6 alpha helices,6 beta sheets,and no tag sequences.The results of amino acid sequence homology analysis showed that the amino acid sequence of CmCHSB had the highest agreement with the amino acid sequence of chitin synthase B of Ostrinia furnacalis,reaching 78%;with Manduca sexta and Helicoverpa armigera.The amino acid sequence identity of the chitin synthase B was 73%.The amino acid sequence identity of chitin synthase B with more than a dozen species of lepidopteran insects has reached more than 69%.The results of gene spacetime expression analysis showed that CmCHSB was expressed during the entire growth stage of C.medinalis,the egg stage was extremely low,almost no expression could be detected,and the expression was highest in the adult stage.The expression pattern of CmCHSB showed a certain regularity.The adult tissues detected by CmCHSB also detected expression,with the lowest expression in the head and the highest expression in the midgut.The level of its expression pattern shows a certain degree of regularity.2.Injecting dsRNA interferes with the expression of chitin synthase B gene in Cnaphalocrocis medinalisAccording to the full-length cDNA sequence of the chitin synthase B gene of C.medinalis,a corresponding target point was designed and named as CmCHSB.The dsRNA of this gene was synthesized in vitro using the kit.The third-instar healthy and active larvae of normal breeding were introduced into mice by in vitro microinjection.The target sites and blanks of Aequorea victoria green fluorescent protein(GFP)were designed in the same way as controls.After being introduced into the larvae by in vitro microinjection,the RNA interference effects were investigated by phenotyping of C.medinalis and RT-qPCR techniques to detect mRNA expression levels of C.medinalis.After 48 h and 72 h of dsRNAs injected with CmCHSB and GFP respectively,the number of white strips fed by rice leaf folders in the blank group and the GFP group was significantly higher than that in the experimental group(injection of dsRNA),and the white bars between the two control groups.There is no significant difference in the number.With the passage of time,the two controls were more and more severely fed by rice leaf folders,but there was no significant change in feeding in the experimental group.This shows that after injection of dsRNA,the activity of rice leaf folder was significantly reduced.Seven days after the injection treatment,the RNA extracted from the survived C.medinalis in the straight tube was extracted and real-time fluorescence quantitative detection was performed.RT-qPCR results showed that after injection of dsRNA for 7 d,the expression level of CmCHSB decreased significantly,compared with the GFP and the blank control,decreased by 18.11% and 37.83%,respectively.3.Construction and identification of recombinant RNA interference plant expression vectorsAccording to the full-length cDNA sequence of the chitinase synthase B gene of C.medinalis,a 400 bp segment was designed as a target site.After adding two BamH I and Spe I sites to the ends of the target site,Artificial synthesis was carried out,and after double digestion,the plant expression vector p1301 was ligated and the p1301-CmCHSB* recombinant plant expression vector was constructed after successful ligation.The constructed recombinant expression vector was identified by PCR,double enzyme digestion and sequencing.The identification results showed that the recombinant expression vector p1301-CmCHSB* was successfully constructed,and the constructed plant expression vector was transferred into Agrobacterium tumefaciens EHA105 by liquid nitrogen freezing-thawing method.Successfully constructed genetic engineering bacteria.4.Obtaining and indoor testing of transgenic riceUsing a previously constructed A.tumefaciens EHA105 containing p1301-CmCHSB* plasmid to infect the calli of Suihui No.527 japonica rice,10 strains of transgenic plants were successfully obtained through a series of steps including callus resistance selection,differentiation,and plant regeneration plants.For some reasons during planting,only 8 plants survived.By RT-PCR detection of the target site,the results showed that the 8 transgenic plants that survived were all positive plants containing CmCHSB*.