Cloning And Characterization Of Prodh Gene CDNA From Leptinotarsa Decemlineata (Say) And Bacterial Expression Of DsRNA

Prodh exists in many insect species, which function is to generate alanine, carbon dioxide and water.by oxidation reaction. Many studies indicate that it involves in the generation of important neurotransmitter, glutamate and GABA. However, its another function, providing direct energy for the migration of bettles, has not been given due attention. We focus on the prodh gene of Colorado potato beetle. A putative CPB prodh gene was obtained using RT-PCR and RACE techniques. This gene produced three isoforms. The three isoforms share the same 5'-UTR and the coding region, but differ in the length of 3'-UTR sequences. Temporal expression of the three isoforms was determined in CPB adults before and after overwintering diapause. The mRNA level of isoform-1, which was similar to other two isoforms in CPB adults before overwinter, became the highest after overwinter. Moreove, we obtain lots of dsRNA by microbial fermentation technology. Our work is helpful for better understanding and prevention the migration of bettles.1. Amplication and RACE clone of Leptinotarsa decemlineata (Say) ProdhA pair of degenerating primers was designed according to the conserved region of proline dehydrogenase gene from other insect species and a cDNA fragment was isolated from Leptinotarsa decemlineata(Say) using RT-PCR.One shared 5'end and three different 3'end was acquired respectively by 5'RACE and 3'RACE.Its ORF(open reading frame) all encoded a 616 polypeptide of amino acids.The sequences were accepted and released by GenBank(accession number GU355892, GU355893, GU355894) and named respectively as Ld proline dehydrogenase transcript isoforms-1,2 and 3.The putative protein of this gene had an isoelectric point of 8.87 and a calculated molecular weight of 7.07×104. Alignment and phylogenetic tree showed it had high homologies with proline dehydrogenase gene from other insect species.2. Temporal expression analysis of Leptinotarsa decemlineata (Say) Prodh All assembled isoforms were validated by end-to-end PCR. Comparing cDNA to corresponding genomic DNA sequences, we found that the three isoforms resulted from alternative polyadenylation. Alternative use of polyadenylation sites produces mRNA isoforms with differnt 3'UTR sequences that contain different cis-elements. Consequently, this post-transcriptional processing plays some important roles in post-transcriptional regulation of mRNA nuclear export, cytoplasmic localization, translational efficiency and stability. Temporal expression of the three isoforms was determined in CPB adults before and after overwintering diapause. The mRNA level of isoform-1, which was similar to other two isoforms in CPB adults before overwinter, became the highest after overwinter. Our results raise the possibility that Ld PRODH expression is fine-tuned through 3'UTR to control proline catabolism for season-dependent activity.3. Cloning and phylogenetic analysis of sid-1 geneTo investigate the distribution of sid-1 homologue gene in insects, all the insect expressed sequence tags (EST) were downloaded from NCBI and a local BLAST database was build. In total, we chosen three important agricultural pests for further studying. In Nilaparvata lugens and Laodelphax striatellus, we got the full length of sid-1-like gene cDNA by RACE PCR. But in Leptinotarsa decemlineata, we only got the 5'RACE fragment. The two assembled sid-1-like gene cDNA were validated by end-to-end PCR. Alignment and phylogenetic tree of Leptinotarsa decemlineata sid-1-like gene showed it had high homologies with sid-1-like gene gene from other insect species, especially from Tribolium castaneum. These results indicated that Leptinotarsa decemlineata also has sid-1-like gene, which is not only a necessary foundation for entomological research by RNAi, but also the prerequisite to prevent Tribolium castaneum further spreading in China by dsRNA feeding technology, whose application is more and more widely.4. A preliminary study on techniques for bacterial expression of dsRNA from Prodh gene in Leptinotarsa decemlineata (Say)It had been reported that the methods, feeding dsRNA to insects, shows great prospects in gene function studying and insect pest control. However, dsRNA produced by conventional methods is expensive. So it is necessary to develop safe and effective techniques to produce large quantities of dsRNA. A possible way is to apply engineered bacterium to produce a massive amount of dsRNA in vivo. Here, a dsRNA-produced vector was constructed and transferred to Escherichia coli HT115. The engineered bacterium was cultured, and a great amount of dsRNA derived from Prodh gene in L. decemlineata was obtained. Moreover, the fermentation condition was optimized in order to get higher expression level of dsRNA. This is the first step to develop novel insecticides by RNA interference techniques.