| The brown planthopper (BPH), Nilaparvata lugens (N. lugens), is the most devastating rice pest in extensive areas throughout Asia. In recent years, N. lugens outbreaks have occurred more frequently in the Yangtze River Delta areas and in the South of China. As the genomes of more insect species are sequenced, RNAi is likely to become an ever-more powerful tool to ascribe functions to the many newly identified genes by means of direct feeding, micro injections or germ-line transformations. At present, RNA interference (RNAi) is an effective tool to control important insect pests via gene silencing to open new areas of basic investigation of insect physiology. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin1(ACT), muscle actin (MACT), ribosomal protein S11(RPS11), ribosomal protein S15e (RPS15), alpha2-tubulin (TUB), elongation factor1delta (EF),18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation). In addition, we cloned the full-length cDNA of N. lugens AK gene (nlak), analyzed the expression profile of nlak in different developmental stages and different tissue parts of N. lugens, constructed the recombinant plasmid to express N1AK recombinant protein, and investigated nlak gene function by RNAi. The results were as follows:1. Selection of reference genes in N. lugens(1) Developmental stage:According to the results of RefFinder, the stability ranking from the most stable to the least stable in the developmental stages was RPS15, RPS11, TUB, EF,18S, AK, ACT, and MACT. According to geNorm, four reference genes (RPS15, TUB,18S, and EF) should be required for a suitable normalization in the different developmental stages;(2) Body part:According to the results of RefFinder, the stability ranking from the most stable to the least stable gene in different body parts was RPS11, TUB, RPS15,18S, ACT, MACT, EF, and AK. According to geNorm, three stable reference genes (RPS11,18S, and RPS15) should be required for a suitable normalization in the different body parts;(3) Geographic population:According to the results of RefFinder, the stability ranking from the most stable to the least stable gene in the two different populations was TUB, RPS11, EF, RPS15, AK, ACT,18S, and MACT. According to geNorm, three reference genes (RPS11, EF, and RPS15) should be required for a suitable normalization in the two different geographic populations;(4) Temperatural press:From the results of RefFinder, the stability ranking from the most stable to the least stable gene in the temperature-stressed samples was RPS15, TUB, EF, RPS11, AK, MACT,18S, and ACT. According to geNorm, three reference genes (RPS15, TUB, and EF) should be required for a suitable normalization in the different temperature treatment samples;(5) Pesticide treatment:According to RefFinder, the stability ranking from the most stable to the least stable in the pesticide-stressed samples was RPS11, EF, TUB, RPS15,18S, AK, MACT, and ACT. According to geNorm, three reference genes (RPS11, EF, and TUB) should be required for a suitable normalization in the pesticide-stressed samples;(6) Diet treatment:According to RefFinder, the stability ranking from the most stable to the least stable in the different diets treatments was RPS15, TUB, RPS11, EF, AK,18S, ACT, and MACT. According to geNorm, four reference genes (RPS15, TUB, EF, and RPS11) should be required for a suitable normalization in the different diets treatments;(7) Stravation:According to RefFinder, the stability ranking from the most stable to the least stable in the starvation treatments was RPS11, TUB, RPS15, AK,18S, EF, ACT, and MACT. According to geNorm, three reference genes (RPS11, AK, and EF) should be required for a suitable normalization in the starvation treatments. After identifing the ranking of N. lugens reference genes across all of the investigated treatments, the stability ranking from the most stable to the least stable across the different developmental stages, body parts, populations, and stressors was RPS11, RPS15, EF, TUB, AK,18S, ACT, and MACT. We believe that these results make an important contribution to gene analysis studies in N. lugens and form the basis of further research on stable reference genes in N. lugens and other organisms.2. The molecular characterization of arginine kinase Gene in N. lugens(1) Cloning and sequence analysis of nlakUsing PCR, TA cloning, sequencing to obtain CDS sequence of nlak, and using5’-RACE and3’-RACE to obtain5’-and3’-UTR (Untranslated Regions) of nlak. The nlak cDNA is1377bp long and contains a1071bp unique ORF (open reading frame) which encodes356amino acides. The arginine kinase activity center-CPTNLGT located from270to276. According to the result of Prosite, the calculated molecular mass of N1AK is46.6kDa, and the isoelectric point is5.69. (2) Expression of N1AK recombinant proteinThe N1AK recombinant protein was successful expressed by Tranferring the recombinant virus Ac NPV-N1AK into healthy insect cell line sf21. We detected the the N1AK recombinant protein through Western Blotting by using6-His antibodies.(3) Expression profile of nlak of N. lugensThe relative expression of nlak was investigated in all developmental stages of BPH via qRT-PCR and AK enzyme activity. The expression of nlak in egg was the lowest in all developmental stages, the expression in1st and2nd instar were higher than any other stages, and the expression in2nd instar was the highest. In addition, the relative expression of nlak was also investigated in all tissue parts of BPH. Whether in the male adults or in the female adults, the expression of nlak in head was the highest, followed by the expression in thorax. The expression in abdomen was significantly lower than the expression in head and thorax. And, the expression of nlak in male adults was higher than the expression in female adults.(4) RNAi of nlakIn this study, we transferred dsRNA of nlak into the BPH by the method of feeding artificial diet. According the results of mortality, qRT-PCR, and AK enzyme activity, we found that the effect of nlak RNAi was more obvious in the early feeding. After ingestion of different concentrations of nlak dsRNA and different fragments of nlak dsRNA, the mortality of BPH increased from the day after feeding dsRNA. At the same time, the relative expression of nlak gene and the activity of NLAK decreased. After two days, the mortality of BPH tended to be more flat. And, the relative expression of nlak gene also tended to be flat after three days. With the extension of RNAi, the relative expression of nlak gene fell down first and then rose up.Taken together, the results indicated that all the programs identified both RPS11and RPS15as the same ideal reference genes for most of the experimental conditions assessed here; the relative expression of nlak was investigated in all developmental stages and all body parts of N. lugens, and knockdown of nlak expression in vivo RNAi generated the lethal effect in N. lugens. These techniques will provide powerful tools for evaluation of reference genes in insect and functional genomics of N. lugens. |
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