Silencing Chitin Synthase A Gene Of Cnaphalocrocis Medinalis Using RNA Interference

Rice is one of the most important food crops and the staple food for more than half of people in the all world. However, insect pest is one of the main reasons causing reduction of rice yield. The rice leaf folder, Cnaphalocrocis medinalis(Lepidoptera: Pyralidae), commonly known as leaf roller, bracts worm etc, is one of the most seriously pest of rice. Even though chitin synthase is the last enzyme in the chitin synthesis pathway, and it is a key enzyme that plays an important role for the growth and development of insect. It could be an ideal target for the pest control.RNA interference(RNAi) refers to a phenomenon in which RNA molecules inhibit gene expression which typically by causing the degradation of the homologous mRNA molecules, and the gene silencing process induced by double-stranded RNA(dsRNA), which is the highly evolutionarily conserved mechanism of gene regulation.The main purpose of this article is to obtain transgenic rice which expresses dsRNA of chitin synthase A gene(CmCHSA) by transgenic RNAi and injection in vitro RNAi technology from C. medinalis, so as to achieve the purpose to control C. medinali.Which laying the foundation for biological control of C. medinalis. The main results are as follows.1. Cloning and expression profiling of CmCHSA gene in C. medinalisCmCHSA gene was cloned using reverse transcription-polymerase chain reaction(RT-PCR) combined rapid amplification of cDNA ends-polymerase chain reaction(RACE-PCR) from C. medinalis. The full-length cDNA of this gene is 5223 bp contains a 4695 bp of open reading frame(ORF) which encoding 1564 amino acids.Bioinformatics analysis showed that chitin synthase A includes 16 transmembrane domains, but did not have any signal peptide of amino acids, with isoelectric point of6.63. Homology modeling prediction shows that the tertiary strcture of CmCHSA gene contains 5 ?-helices and 4 ?-sheets. Homology analysis of CmCHSA amino acid sequences displayed that it had 96% similarity with CHSA of Omphisa fuscidentalis,92% with CHSA of Spodoptera exigua, 92% with CHSA of Mamestra brassicae and91% with CHSA of Helicoverpa armigera. The expression profile analyses showedthat CmCHSA gene was expressed throughout all the developmental stages of C.Medinalis, with the highest and the lowest expression level in pupa and egg,respectively. In all the tissues tested the expression levels in descending order is:epidermis > head > muscle > midgut > ovary > malpighian tubule > fat body >testis.2. Interference the expression of Cm CHSA gene by injection of dsRNAThe target sequence specific to CmCHSA gene was designed for RNAi. Designed two RNAi target sites —CmCHSA-? and CmCHSA-?, two double-stranded RNAs(dsRNAs) were synthesized in vitro and were separately introduced into the 3rd instar larvae through microinjection. RNAi effects were detected by phenotype observation and real-time quantitative PCR(RT-qPCR). After injection of dsRNA, the larvae developed loss of appetite, growth retardation, and malformation; some died.RT-qPCR showed that at the 96 th h after injection of CmCHSA-? and CmCHSA-? dsRNAs, the mRNA expression of CmCHSA decreased by 59% and 64%, respectively,in comparison with the control. At the 7th d after injection of CmCHSA-? and CmCHSA-? dsRNAs, the larval mortality rates were 80% and 83.33%, respectively,increased by 66.67% and 70%, compared to the control. Moreover, the corrected mortality rate of each group is 76.92% and 80.77%, respectively.3. Construction and identification of recombinant RNAi expression vectorUsed the same sequence specific to CmCHSA-? and CmCHSA-?, synthesized with BamH I and Spe I restriction enzyme cutting sites at both sides, respectively.After double enzyme digestion, these fragment were inserted into vector p1301 to construct recombinant expression vector p1301-CmCHSA-?* and p1301-CmCHSA-?*. The results showed that the recombinant vector had been successfully constructed by PCR, double enzyme digestion and sequencing identification. Then p1301-CmCHSA-?* and p1301-CmCHSA-?* were to transformed into Agrobacterium tumefaciens LBA4404 by liquid nitrogen freezing and thawing method to construct genetic engineering bacteria.4. Acquirement of the transgenic rice and detection of RNAi effectsThe calli of rice Zhonghua 11 were infected by A. tumefaciens LBA4404 which contained p1301-CmCHSA-?* and p1301-CmCHSA-?* plasmid, respectively. Thenthe calli were screened by higromycin, differentiation of the resistant calli and plant regeneration, 20 transgenic rice plants were obtained. And 6 rice plants were found to be positive ones containing CmCHSA-?* and 7 rice plants were found to be positive ones containing CmCHSA-?* through RT-PCR detection. Used the C. medinalis feeding on normal rice as controls, observed each C. medinalis feeding on transgenic rice found that its life activity decreased significantly, growth and development lagged,presented the molt blocked and the worm varied small, deformity even death, etc.Using real time quantitative PCR detected of ramets Cm CHSA mRNA expression levels in C. medinalis feeding on different transgenic rice and counted its deaths. The results showed that the average expression level of CmCHSA in C. medinalis feeding on CmCHSA-?* transgenic rice decreased by 56.8% and feeding on CmCHSA-?'*transgenic rice decreased by 61%, compared to the control groups. And the mortality was also increased 36.00% and 38.00%, respectively. The results indicated that the transgenic rice plants containing the interference sequence of CmCHSA gene which could strongly silence CmCHSA in C. medinalis that fed on these transgenic rice.