褐飞虱红色复眼的突变机理研究

发布时间：2018-01-07 10:26

本文关键词：褐飞虱红色复眼的突变机理研究　出处：《扬州大学》2016年博士论文　论文类型：学位论文

【摘要】：褐飞虱(Brown planthopper, BPH), Nilaparvata lugens Stal,属半翅目飞虱科,是亚洲许多地区水稻上的重要害虫之一。褐飞虱正常眼色为褐色,在室内长期饲养的农药敏感品系中偶见红眼突变体。作为可见的遗传标记,褐飞虱红眼突变体具有多种潜在应用价值。明确突变体的生物学特征及突变机理将有助于此突变体应用价值的探索。前期研究结果表明,红眼表型受常染色体上一对隐性等位基因的调控,其眼黄素含量仅为野生型个体的36.3%,色素颗粒数量及电子致密度也显著下降。为了靶标基因的准确定位,本研究首先构建了褐飞虱褐眼野生型和红眼突变型近等基因系,然后以近等基因系为材料从基因及蛋白水平对褐飞虱红眼突变的机理进行了研究。主要研究结果如下：1.褐飞虱红眼突变体的种群参数比较本部分首先通过1代杂交和8代自交试验筛选了褐飞虱红眼突变型和褐眼野生型的近等基因系,命名为NIL-rr和NIL-BB。生物学试验结果表明,NIL-BB的生存力及繁殖力显著下降,而NIL-rr和普通的室内褐眼种群(BB)相似。其中,NIL-BB的单雌产卵量及卵孵化率两参数的显著降低是其种群适合度下降的主要原因。BB、NIL-rr和NIL-BB的种群趋势指数分别为52.18、43.80和4.19。另外,NIL-rr品系初羽化雌成虫的体重和体长也显著重／长于NIL-BB品系。相对于NIL-BB, NIL-rr较强的繁殖力及较大的体型可能是因为眼色突变基因或其紧密连锁基因对繁殖具有较强的补偿作用。本研究结果表明,NIL-rr并不具有生态风险,或可用作田间研究材料。意外获得的具有较低繁殖力和生命力的NIL-BB品系试虫可用作生殖相关信号通路的研究模型。2.褐飞虱眼色相关基因的克隆为了进一步定位突变基因,本部分在前期获得的4个基因片段的基础上,进一步利用生物信息学、热启动PCR和RACE技术克隆了10个眼色相关基因,其中有7个基因通过RACE技术获得了全长序列,包括5’-和3’-UTR,有3个基因还未获得全长序列。同源比对表明,这些基因都与其他昆虫的直系同源物具有较高的一致性,也存在典型的特征序列,因此认为它们编码蛋白就是相对应果蝇眼黄素合成通路的色氨酸加氧酶(vermilion)、犬尿氨酸甲酰胺酶、犬尿氨酸羟化酶(cinnabar)和酚额嗪酮合成酶(karmoisin),色素转运通路的White (white)和Scarlet (scarlet)半转运子,色素颗粒形成通路的接头蛋白复合体AP3的4个亚基(garnet/car mine/ruby/orange)。本部分的研究结果为筛选变异靶标基因奠定了基础。3.眼色基因变异位点的筛选为了寻找导致褐飞虱复眼眼色变异的分子机理,即突变位点,本研究对野生型和突变型近等基因系的10个眼色相关基因的编码区分别进行了克隆和对比分析。结果发现所有红眼突变个体的犬尿氨酸羟化酶(KMO)编码基因Nlkmo都存在三个同义突变位点、一个异义突变位点(L49I)和一个5碱基的插入突变位点。阅读框内5碱基的插入,使氨基酸翻译提前终止,缺少了115个氨基酸残基,破坏了两个跨膜螺旋。4. Nlkmo基因的眼色决定作用验证前人已证明,KMO在双翅目的果蝇和蚊子、鳞翅目的家蚕复眼着色过程中起关键作用,但其对半翅目昆虫复眼着色过程的影响还不明确。本章对褐飞虱野生型品系3龄若虫注射KMO双链RNA (dsKMO)后,Nlkmo表达水平显著下降,复眼眼色部分变为红色,且眼黄素也下降为对照个体的55.3%。这表明,同其他昆虫一样,褐飞虱KMO也是褐飞虱眼黄素合成通路关键酶之一。此结果还说明,Nlkmo作为标记基因可用于褐飞虱及相近昆虫的卵期RNAi技术的研发。5. Nlkmo基因的时空表达特征及其在突变体中的表达变化为了深入了解褐飞虱Nlkmo基因的分子特性,本部分测定了其时空表达特征,并对比分析了其在红眼突变体中的表达变化。Nlkmo基因在所有生育期及组织内都有表达,包括卵、若虫、成虫、卵巢、中肠、马氏管、体壁和脂肪体。Nlkmo转录本水平在NIL-BB和NIL-rr两品系间并无显著差异,推断Nlkmo存在的一个异义突变位点和一个5nt的插入位点并不影响转录本水平,可能是在蛋白质翻译及修饰阶段对眼色产生影响。6. Nlkmo编码蛋白的催化活性分析本章首先提取NIL-BB和NIL-rr两品系褐飞虱头部的总蛋白,分别命名为KMO-BB(?)KMO-rr。利用液相色谱技术测定了两种总蛋白转化犬尿氨酸(KYN)为3-羟基犬尿氨酸(3-HK)的催化活性。结果表明,KMO-BB的催化活性约为KMO-rr的9.03倍。为了精准分析,本章利用昆虫杆状病毒表达系统对野生型和突变型KMO酶进行了外源表达,分别命名为rKMO-BB和rKMO-rr,其中rKMO-rr未引入点突变位点(L49I),只引入了5碱基的插入突变位点。利用相同方法测定重组蛋白的催化活性,结果表明,rKMO-BB的活性远远高于KMO-BB,而rKMO-rr的活性小于KMO-rr,以致未能检测到。以上结果说明,Nlkmo既是褐飞虱红眼突变表型产生的靶标基因,而5碱基的插入是红眼突变体KMO催化活性下降或丧失的主要突变方式。在褐飞虱头部,除了KMO,也许还存在其他同工酶或备选通路可将KYN转化为3-HK,这还需进一步研究证明。综上所述,Nlkmo基因5碱基的插入突变方式是褐飞虱红眼表型产生的分子基础。褐飞虱红眼突变机理的揭示将有助于褐飞虱及相关昆虫卵期RNAi技术及转基因体系的构建,从而推动飞虱后基因组学的发展。
[Abstract]:The brown planthopper (Brown planthopper BPH), Nilaparvata lugens Stal, belong to Hemiptera Delphacidae, is one of the most important pests in many parts of Asia. The rice brown planthopper at normal Brown pesticide susceptible strains of long-term breeding in the interior of the eye. Occasionally seen in the mutants as genetic markers can be seen, the eye has a variety of mutants of brown planthopper the potential application value. The biological characteristics of mutant specific and mutation mechanism will help explore the application value of the mutant. Our previous study showed that the eye phenotype by regulation of autosomal recessive alleles, the xanthommatin was only wild genotype 36.3%, pigment particle number and electron density significantly decreased. In order to accurately locate the target gene, we firstly constructed a brown Eyed brown planthopper and wild type red mutant Nils, and near isogenic lines as materials from The gene and protein levels were studied on the mechanism of red brown planthopper mutations. The main results are as follows: 1. the population parameters of brown planthopper red mutant comparison of this part of the first through the 1 generation and the 8 generation hybrid self screening test of BPH red brown eyed mutant and wild type of near isogenic lines, named NIL-rr and NIL-BB. the biological test results show that the survival and fecundity of NIL-BB decreased significantly, while NIL-rr and common indoor brown eyed population (BB) similar. Among them, NIL-BB significantly decreased the hatching eggs per female and egg rate two parameters are the main cause of population fitness decreased.BB, NIL-rr and NIL-BB index of population trend. For 52.18,43.80 and 4.19. in NIL-rr strains of newly emerged female adult body weight and body length were significantly longer than that of NIL-BB / heavy lines. Compared with NIL-BB, NIL-rr strong fecundity and large size may be Because the eye color mutation gene or closely linked genes has strong effect on reproductive compensation. The results of this study show that NIL-rr has no ecological risk, or can be used as field research materials. The NIL-BB strain unexpectedly has lower fecundity and vitality of the test insects can be used as a research model of.2. clones of brown planthopper genes related to reproductive related glances the signal pathway in order to further locate the mutant gene, 4 gene fragments obtained in the prophase of this part, further use of bioinformatics, hot start PCR and RACE cloning of 10 color related genes, including 7 genes, the full-length sequence by RACE technology, including 5 '- and 3' -UTR, 3 genes have not obtained the full sequence. Homologous comparison showed that these genes have high consistency with the ortholog of other insects, there is also a characteristic sequence of typical column, because This is that they correspond to protein encoding tryptophan oxygenase synthesis pathway in Drosophila eye Flavin (vermilion), formamidase, kynurenine hydroxylase (cinnabar) and the amount of phenol in chalcone synthase (karmoisin), pigment transport pathway of White (white) and Scarlet (scarlet) - transporter, 4 subunits of pigment particles formed joint protein complexes of the AP3 pathway (garnet/car mine/ruby/orange). This part of the study results for screening the target gene mutation screening for.3. laid a wink gene variant in order to find the molecular mechanism that caused the eye glances of brown planthopper variation, mutation sites, the study of wild type the mutant and isogenic lines of 10 genes encoding region glances were analyzed and compared. The results showed that all clones eye mutation of kynurenine hydroxylase (KMO) gene encoding Nlkmo are Three synonymous mutations, a nonsynonymous mutation (L49I) and a 5 nucleotide insertion mutation. Insert the reading frame of 5 BP, the amino acid premature termination, lack of 115 amino acid residues, destroying two transmembrane helix.4. Nlkmo gene has been shown to verify the previous decision function at in KMO, Diptera flies and mosquitoes, which plays a key role in Lepidoptera silkworm eye coloring process, but its effect on insect compound eye coloring process is not clear. This chapter of the brown planthopper in wild type strain 3 instar nymphs of KMO injection of double stranded RNA (dsKMO), the expression level of Nlkmo decreased significantly, ommateum look into the red part, and also show that this xanthommatin decreased as the control of individual 55.3%., like other insects, one of brown planthopper KMO is brown planthopper xanthommatin biosynthesis pathway key enzyme. The results also show that the Nlkmo gene can be used as marker Research and development of.5. Nlkmo gene of brown planthopper in space-time and similar insect eggs of the RNAi expression characteristics and the change of its expression in the mutant in order to in-depth understanding of molecular properties of brown planthopper Nlkmo gene, expression characteristics of the space studied in this part, and comparative analysis of their eye in mutant expression changes in expression of.Nlkmo gene and in all growth stages and tissues including egg, nymph, adult, ovary, midgut, Malpighian tubules, integument and fat body of the.Nlkmo transcript level in NIL-BB and NIL-rr had no significant difference between the two lines, a Nlkmo insertion sites are inferred the presence of the mutation site and a 5nt does not affect the transcript level. May be in the stage of protein translation and modification effect analysis of catalytic activity of.6. produced at Nlkmo encoding protein firstly extracted total protein of brown planthopper in head NIL-BB and NIL-rr two strains, respectively. Named KMO-BB (?) two total protein conversion of kynurenine was determined by KMO-rr. liquid chromatography (KYN) for 3- hydroxykynurenine (3-HK) catalytic activity. The results showed that the catalytic activity of KMO-BB is about 9.03 times of KMO-rr. In order to accurate analysis, this chapter uses the rod like insects the baculovirus expression system of exogenous wild-type and mutant KMO enzyme expression, named rKMO-BB and rKMO-rr, in which rKMO-rr has not introduced the point mutation site (L49I), only introduced 5 nucleotide insertion mutations. The catalytic activity of the purified protein was determined by using the same method, the results showed that the activity of rKMO-BB was higher than that of KMO-BB. While the activity of rKMO-rr is less than KMO-rr, which failed to detect. The above results suggest that Nlkmo is the target gene mutation of red brown planthopper phenotype, and inserted into the 5 base mutation is the main red eye mutant KMO catalytic activity decreased or lost. In Nilaparvatalugens head, in addition to KMO, there may be other isozymes or alternative pathway can convert KYN to 3-HK, which need further research are also proved. In summary, the Nlkmo gene 5 nucleotide insertion mutation is the molecular basis for producing brown planthopper eye phenotype. To reveal the mechanism of red brown planthopper mutation will help to build the eggs RNAi and related techniques of brown planthopper insect and transgenic system, thus promoting the development of genomics planthopper.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.112.3

【相似文献】