Silencing Trehalase Gene Of Cnaphalocrocis Medinalis Using Transgenic RNA Interference

Rice Oryza sativa is one of the most important food crops and is staple food for nearly half of people in the world. The insect pest is one of the main reasons causing reduction of rice yield. The rice leaffolder(RLF), Cnaphalocrocis medinalis(Lepidoptera: Pyralidae), is an economically important pest of rice in Asia. Trehalase is a key enzyme in insects’ trehalose metabolism and plays important roles in development and energy regulation of insects. It could be an ideal target for pest control. RNA interference(RNAi) refers to a biological phenomenon in which RNA molecules inhibit gene expression, typically by causing the degradation of homologous m RNA molecules. RNAi is a gene silencing process induced by double-stranded RNA(ds RNA) molecules and is a highly evolutionarily conserved mechanism of gene regulation. The main purpose of this study is to obtain transgenic rice that expresses ds RNA of RLF soluble trehalase gene(Cm Tre1) by transgenic RNAi technology; after RLF feeds on these rice leaves, Cm Tre1 will be silenced.This research will be laying the foundation for biological control of C. medinalis. The main results are as follows.1. Cloning and expression profiling of Cm Ttre1 in C. medinalisCm Tre1 from C. medinalis was cloned using rapid amplification of c DNA ends-polymerase chain reaction(RACE-PCR). The full-length c DNA of Cm Tre1 is2364 bp containing a 1704 bp of open reading frame(ORF) that encodes 567 amino acids. Bioinformatics analysis indicated that RLF trehalase included a signal peptide of 16 amino acids but did not have transmembrane domain, with isoelectric point of5.32. Homology modeling analysis demonstrated that tertiary strcture of the trehalase contained 18 α-helices and 2 β-sheets. Homology analysis of Cm Tre1 amino acid sequences displayed that it had 74% similarity with soluble trehalase(Tre1) of Omphisa fuscidentalis, 67% with Tre1 of Heliconius melpomene, and 66% with Tre1 of Danaus plexippus. Analyses of gene expression profiling showed that Cm Tre1 was expressed throughout all developmental phases of C. medinalis, with the lowest expression level in pupa and the highest level in adult; that Cm Tre1 was expressed in all the adult tissues tested, with such expression levels in descending order:midgut>cuticle>malpighian tubule>head>ovary>fat body>muscle>testis.2. Construction and identification of recombinant RNAi expression vectorThe target sequence specific to Cm Tre1 was designed for RNAi and synthesized with Bam H I and Spe I restriction enzyme cutting sites at both sides, and was named Cm Tre1*. After double enzyme digestion, this fragment was inserted into vector p1301 to construct recombinant expression vector p1301-Cm Tre1*. The results of PCR, enzyme digestion, and sequencing showed that the recombinant vector had been successfully constructed. Then p1301-Cm Tre1* was transformed into Agrobacterium tumefaciens LBA4404 to construct genetic engineering bacteria which would be used for transgenic anti-insect rice breeding.3. Acquirement of the transgenic rice and detection of RNAi effectsThe calli of rice Zhonghua 11 were infected by A. tumefaciens LBA4404 that contained p1301-Cm Tre1*. Then the calli were screened by higromycin. After differentiation of the resistant calli and and plant regeneration, 74 transgenic rice plants were obtained. Twenty-six rice plants were found to be positive ones containing Cm Tre1* through GUS staining as well as PCR and RT-PCR detection. Cm Tre1 m RNA expression levels in RLFs that fed on these transgenic rice were detected with real time quantitative PCR(RT-q PCR) and trehalase activities in vivo were tested by the Trehalase Kit, using RLFs that fed on non-transgenic rice as controls. The results showed that the average expression level of Cm Tre1 in RLFs feeding on transgenic rice decreased by 44.79% and the average trehalase activity in vivo fell by 14.94%,compared to the control groups. The findings indicated that the transgenic rice plants containing the interference sequence Cm Tre1* could strongly silence Cm Tre1 in RLF that fed on these transgenic rice.