摘要
褐飞虱Nilaparvata lugens (brown planthopper, BPH)是水稻上的重要害虫之一,在我国南方部分地区和长江中下游地区近年来褐飞虱经常暴发成灾,对我国水稻生产和粮食安全构成严重威胁。目前,对于褐飞虱的控制以化学防治为主,然而化学药剂导致的环境污染问题日益严重,因此当前害虫防治的研究热点集中在寻求环境友好的防止新途径。与此同时,RNAi技术已经广泛地应用于各种生物体基因功能的研究,这一技术成功地阐明了多种昆虫的基因功能。利用该技术分析基因功能的同时,也为农业害虫的生物防治开辟了RNAi沉默靶标基因的新途径。在RNAi的研究过程中,利用实时荧光定量PCR分析靶标基因mRNA水平是检测基因沉默效果的重要手段,本研究详细分析评估了不同实验条件下(不同发育时期、不同组织部位、不同地理种群、不同温度胁迫、不同药剂处理、不同食物饲喂和饥饿处理)褐飞虱8种常用持家基因actin1(ACT)、muscle actin (MACT)、ribosomal protein S11(RPS11)、ribosomal protein S15e (RPS15)、alpha2-tubulin (TUB)、elongation factor1delta (EF)、18S ribosomal RNA (18S)和arginine kinase (AK)的表达稳定性。并且,克隆褐飞虱nlak基因的全长cDNA,分析nlak基因mRNA的组织分布和发育表达模式,构建昆虫杆状病毒重组质粒表达N1AK重组蛋白,通过RNAi探讨nlak基因作为分子靶标的可能性,为深入研究精氨酸激酶基因的表达与调控规律,阐明昆虫体内能量的代谢、贮存和利用等调控机制提供重要的理论依据,并且为利用RNAi这一新技术有效防治褐飞虱提供理论基础。研究结果如下：
一、褐飞虱内参基因的筛选
不同发育时期各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS15, RPS11, TUB, EF,18S, AK, ACT, MACT。若要采用多内参校正,需要选取RPS15. TUB、18S和EF这四个基因；不同组织部位各内参基因稳定性的综合排序从稳定到不稳定依次为：PS11, TUB, RPS15,18S, ACT, MACT, EF, AK。若要采用多内参校正,需要选取RPS11、18S和RPS15这三个基因；不同地理种群各内参基因稳定性的综合排序从稳定到不稳定依次为：TUB, RPS11, EF, RPS15, AK, ACT,18S, MACT。采用多内参校正时,需要选取RPS11、EF和RPS15这三个基因；不同温度胁迫下各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS15, TUB, EF, RPS11, AK, MACT,18S, ACT。采用多内参校正时,需要选取RPS15、TUB和EF这三个基因；不同药剂处理下各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS11, EF, TUB, RPS15,18S, AK, MACT, ACT。采用多内参校正时,需要选取RPS11、EF和TUB这三个基因；不同食物饲喂时各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS15, TUB, RPS11, EF, AK,18S, ACT, MACT。采用多内参校正时,需要选取RPS15、TUB、EF和RPS11这四个基因；饥饿处理下各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS11, TUB, RPS15, AK,18S, EF, ACT, MACT。采用多内参校正时,需要选取RPS11、AK和EF这三个基因；对所有处理的样品数据进行综合分析,结果发现各内参基因稳定性的综合排序从稳定到不稳定依次为：RPS11, RPS15, EF, TUB, AK,18S, ACT, MACT。此结果对于褐飞虱中基因表达分析具有重要意义,并且为进一步分析褐飞虱和其他生物内参基因稳定性奠定了基础。
二、褐飞虱精氨酸激酶基因的分子特性研究
1.褐飞虱精氨酸激酶基因(nlak)的克隆与分析
采用PCR. TA克隆、测序和拼接,获得了nlak基因的完整编码区序列,并通过5'-RACE和3'-RACE获得5’端和3’端非翻译区(Untranslated Regions, UTR)序列。克隆到的nlak基因cDNA全长为1377bp,含有一个完整的1071bp的开放阅读框序列(Open reading frame, ORF),编码356个氨基酸残基。其中,CPTNLGT位于nlak氨基酸序列第270-276位,是精氨酸激酶的活性中心。根据Prosite软件分析得出其蛋白质分子量为39.81kDa,等电点为5.69。
2.重组Ac NPV-N1AK在昆虫杆状病毒系统中的表达
将同源重组的Ac NPV-N1AK病毒转染健康的昆虫sf21细胞系,获得第一代重组Ac NPV-N1AK病毒。该重组病毒能够继续感染新的健康的sf21细胞,在感染后的第7天可以收集到重组Ac NPV-N1AK蛋白。利用抗6-His兔多克隆抗体进行Western Blotting,检测到重组NPV-N1AK蛋白在sf21细胞中成功表达。
3. nlak基因在褐飞虱体内的表达分析
qRT-PCR和酶活性检测结果表明nlak基因在褐飞虱各个发育时期均有表达,褐飞虱卵期的nlak基因表达量远低于其他各个时期,1龄若虫和2龄若虫中表达量显著高于其它龄期若虫,并且在2龄若虫中的表达量为整个发育时期最高。此外,在褐飞虱成虫的各组织部位中nlak基因均有表达,无论在雌成虫还是雄成虫体内,头部的nlak基因表达量最高,其次是胸部,而腹部的表达量明显低于头部和胸部。并且,对于整个虫体而言,nlak基因在雄成虫体内的表达量远大于雌成虫。
4. nlak基因沉默后对褐飞虱的影响
采用饲喂法将体外合成的nlak基因dsRNA通过人工饲料导入褐飞虱体内,通过对死亡率的统计、qRT-PCR和酶活性检测发现,在饲喂初期dsRNA对nlak基因干扰效果比较明显。不同浓度和不同片段的dsRNA饲喂结果均表明,摄取dsRNA的褐飞虱第2天出现死亡率上升、mRNA水平下降和酶活力降低的现象。第3天开始死亡率变化趋于平缓,第4天开始mRNA水平变化也趋于平缓,nlak基因的表达量随着RNAi处理时间的延长呈现先降低后升高的趋势。
综上所述,褐飞虱RPS11和RPS15基因在大部分试验条件下表现出较高的稳定性,可以作为qRT-PCR的内参基因使用；nlak基因在褐飞虱各发育时期和各组织部位中均有较强的表达水平,该基因RNAi能对褐飞虱具有明显的致死效应,可作为防治褐飞虱的新途径,为昆虫内参基因筛选和褐飞虱基因功能研究提供了理论指导。
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 nlak
Using 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 protein
The 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. lugens
The 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 nlak
In 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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