Molecular Cloning And Function Analysis Of Genes Involved In Insulin/PI3k/Akt Signaling Pathway In The Rice Brown Planthopper, Nilaparvata Lugens (Hemiptera: Delphacidae)

Brown planthopper(BPH, Nilaparvata lugens St?l) is one of the most destructive insect pests of rice. Breeding resistant rice is an important way to control BPH, but with the continuously cultivation of resistant rice varieties in large areas, some BPH populations have developed strong adaptations to the resistant varieties, so that some of the previouse resistant rice varieties can not control the BPH. Study the mechanism of the virulence variation in BPH is a key problems to explain the variation of BPH biotypes, and it is very important to develop the insect-resistant transgenic rice varieties to control the BPH. The Insulin/PI3K/Akt signaling pathway has been demonstrated to play a key role to regulate the organism’s growth, development, size and tolerance, etc. When this signaling pathway was inhibited, it can delay the organism growth and development, and the body size will become small, but the tolerance would increase at the same time. These characteristics were similar to the performances of the BPH in the adaption to the resistant rice varieties. Therefore, it is necessary to study the function of this pathway in BPH. In this study, three related genes Nl AKTIP, Nl PIK3R1 and Nl RPS6KA2 were cloned by rapid-amplification of c DNA ends(RACE) according to its gene sequence information available in BPH transcriptome. Then the real-time quantitative PCR(q PCR) was conducted to explore the expression pattern in different BPH developmental stages as well as in different populations in adapting to a resistant rice variety RHT. RNA interference(RNAi) technology was conducted to study the function of Nl AKTIP and Nl PIK3R1 in BPH. The results are reported as below:1. Molecular cloning, expression and function analysis of protein kinase B interation protein gene Nl AKTIP in the rice brown planthopperThe 964 bp full-length c DNA encoding protein kinase B interation protein in BPH(Nl AKTIP) was cloned by RACE according to its gene sequence information available in BPH transcriptome. Then the q PCR and RNAi experiment was conducted to explore the expression pattern and the function of Nl AKTIP. The q PCR showed that the Nl AKTIP gene was slightly expressed in nymphs and males,but strongly expressed in gravid females, it indicated that Nl AKTIP was very important to the gravid BPH. And the expression of Nl AKTIP was down-regulated in the progress of BPH adaption to RHT. RNAi experiment showed that with increasing concentrations of ds AKTIP, the body weight of treated BPH decreased to 1.36 mg(low concentration treatment), 1.34 mg(medium concentration treatment) and 0.77 mg(high concentration treatment), which were significantly lower(P<0.05) than the body weights of the untreated(2.21 mg) and the GFP(1.74 mg). RNAi also delayed the eclosion of BPH and affected ovarian development. Treatment with a high concentration of ds AKTIP(0.5 μg/μl) significantly delayed the eclosion for over seven days and restricted ovarian development to Grade II, whereas the ovaries of the untreated and the ds GFP control BPH developed to Grade IV. These results indicated that Nl AKTIP is crucial to the growth, development and the body size of BPH, and matched the performance of BPH reared on resistant rice varieties.2. Molecular cloning, expression and function analysis of phosphatidyl inositol 3 kinase p85α subunit gene Nl PIK3R1 in the rice brown planthopperThe 2694 bp full-length c DNA encoding phosphatidyl inositol 3 kinase p85α subunit gene in BPH(Nl PIK3R1) was cloned by RACE according to its gene sequence information available in BPH transcriptome. The q PCR showed that the Nl PIK3R1 gene was slightly expressed in nymphs and males,but strongly expressed in gravid females. Also the expression of Nl PIK3R1 was down-regulated in the progress of BPH adaption to RHT. This result indicated that Nl PIK3R1 was also associated to the virulence changes in BPH. RNAi experiment was conducted using feeding method, and the results showed that the Nl PIK3R1 gene expression was inhibited in the two ds Nl PIK3R1 treatment groups, especially in the high concentration treatment group. Feeding of ds Nl PIK3R1 led to the death of brown planthopper. The survival rate of the high concentration treatment group was significantly declined to 37.5% at day 7, and showed significant differences(P<0.01) compare with the blank group(87%) and the ds GFP group(76.67%). RNAi also resulted in slower eclosion and lighter body weight. The results suggest that the Nl PIK3R1 gene is very important to the survival, growth and development in BPH. RNAi results matched the performance of BPH reared on resistant rice varieties. This study indicates that Nl PIK3R1 gene play an important role in the virulence changes in BPH.3. Molecular cloning and expression analysis of a ribosomal protein s6 kinase gene Nl RPS6KA2 in the rice brown planthopperThe 2491 bp full-length c DNA encoding a ribosomal protein s6 kinase gene in BPH(Nl RPS6KA2) was cloned by RACE according to its gene sequence information available in BPH transcriptome. The q PCR showed that the Nl RPS6KA2 gene was slightly expressed in nymphs and males, but was strongly expressed in gravid females, and it indicated that this gene was very important to the gravid BPH. The q PCR also showed that the expression level of Nl RPS6KA2 was down-regulated when BPH from TN1 to RHT, and the expression level of Nl RPS6KA2 was up-regulated when BPH adjusted to RHT, It indicated that this gene also took part in the regulation progress of the virulence changes in BPH.This study makes contribute to the further study the function of Insulin/PI3K/Akt signaling pathway in BPH, and it also puts forward new ideas to explain the virulence changes.