The DNA Cloning and RNA Interference of Some Functional Genes in Laodelphax Striatellus (Fallen)
|School||Nanjing Agricultural College|
|Course||Agricultural Entomology and Pest Control|
|Keywords||Laodelphax striatellus Functional genes Full-length cDNA cloning qRT-PCR Internal reference genes Stability in expression RNA interference Leathal effect|
The small brown planthopper, Laodelphax striatellus (Fallen), is an important pest of food crops including rice and wheat. It can not only sucking the sap of crop plants but also spread harmful viruses, causing rice stripe disease and gray-streaked dwarf etc. After decades of latency from the 1960s, the outbreak of L. striatellus occurred in the late 1990’s and continued to now, resulting heavy rice stripe disease in the Huaihe area since 2000. The control of this pest for long time mainly depends on pesticides, which lead to not only a serious environmental pollution, but also resistance to pesticides. It is urgently needed to expore new alternitives to control L. striatellus. RNA interference (RNAi) is a post-transcriptional gene silencing phenomenon induced by double-stranded RNA (dsRNA), widely existing in fungi, plants and animals. It is a process that intracellular dsRNA induce and degrade the specific mRNA matched with the dsRNA. RNAi could be used in pest cotrol by the transgenic crops or transgenic insecticidal microbes that express the dsRNA of functional genes from insect pests. This new RNAi mediated insect control strategy attracts attentions of scientists all over the world.To explore the RNAi mediated control technique of L. striatellus, we in this study, cloned full-length cDNAs of some important function genes from L. striatellus, and subsequently screened out some lethal genes by RNAi experiments with dsRNA feeding of the 2d-old nymphs. In addition, the expresson stabilities among different day-old nymphs of some 6 house keeping genes from L. striatellus were measured and compared. The main results were listed as follows:1. By analysing the transcriptome data and carrying out RACE strategy,6 full-length and 12 5’end cDNA of functional genes from L. striatellus were obtained. These 6 genes with full lengh cDNA obtained were three tubulin genes, an ADP ribosylation factor (ARF) gene, a 60s ribosomal protein L9 (RPL9) gene, a glucose transporter (ST) gene and a juvenile hormone binding protein (JHBP) gene, with the names (Genbank accession number) of LstrTUBl (JF728809), LstrTUB2 (JF728810), LstrTUB3 (JF728811), LstrARF (JF728807), LstrRPL9 (JF728806), LstrST (JN050856) and LstrJHBP (JF728808), respectively.2. RNAi experiments of 15 functional genes were carried out by dsRNA feeding of 2d-old L. striatellus nymphs. The results showed that 7 genes including CHI7, RPL9 and ST displayed high lethal effect, with the corrected nymph mortality> 40% at 120h after the dsRNA feeding. Among these 7 genes, CHI7 showed the highest corrected mortality of 64%. ATPase andα2-TUB showed lowest mortalites (< 20%), and other 6 genes presented the corrected mortalities ranged from 20% to 40%. The expression level was determined by qRT-PCR in RNAi experiment with dsCHI7. It showed that the expression of CHI7 decreased by 57% at 3d after the treatment, indicating the validity of the RNAi procedure used.3. As the juvenile hormone binding protein (JHBP) plays important roles in insect development and metomorphsis, LstrJHBP obtained in this study was further studied in terms of subclassification, expreesion pattern and RNAi effect. LstrJHBP was assumed to be a cytosolic JHBP, according to analysis of signal peptide, amino acid sequence and evolutional tree. LstrJHBP was expressed throuthout nymph stage to adult stage, but significantly highly expressed in nymph of 4th instar. The expression levels between male and female adults were not different statisticly. RNAi expreiment indicated that feeding with dsRNA for 5 days at concentration of 56.65 ng/μl could not result in significant nymph mortality, comparing with control group.4.β-Actin is often used as internal reference gene in qRT-PCR, however its expression is not very stable in L. striatellus as well as some other insects. To find a more stable reference gene,6 house keeping genes cloned in this study were selected to measure their expression levels in 5 nymph instars by qRT-PCR, and their stabilities in expression were evaluated by geNorm and NormFinder. As a result, ARF and RPL9 showed higher stabilities thanβ-Actin, and thus were recommended as the alternatives of internal reference. It also indicated that combined use of two internal reference ARF-RPL9 resulted in minimum error.