本文關(guān)鍵詞：褐飛虱紅色復眼的突變機理研究　出處：《揚州大學》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： 褐飛虱 紅眼突變體 種群適合度 犬尿氨酸羥化酶 插入突變 轉(zhuǎn)錄本水平 RNA干擾 催化活性

【摘要】：褐飛虱(Brown planthopper, BPH), Nilaparvata lugens Stal,屬半翅目飛虱科,是亞洲許多地區(qū)水稻上的重要害蟲之一。褐飛虱正常眼色為褐色,在室內(nèi)長期飼養(yǎng)的農(nóng)藥敏感品系中偶見紅眼突變體。作為可見的遺傳標記,褐飛虱紅眼突變體具有多種潛在應用價值。明確突變體的生物學特征及突變機理將有助于此突變體應用價值的探索。前期研究結(jié)果表明,紅眼表型受常染色體上一對隱性等位基因的調(diào)控,其眼黃素含量僅為野生型個體的36.3%,色素顆粒數(shù)量及電子致密度也顯著下降。為了靶標基因的準確定位,本研究首先構(gòu)建了褐飛虱褐眼野生型和紅眼突變型近等基因系,然后以近等基因系為材料從基因及蛋白水平對褐飛虱紅眼突變的機理進行了研究。主要研究結(jié)果如下：1.褐飛虱紅眼突變體的種群參數(shù)比較本部分首先通過1代雜交和8代自交試驗篩選了褐飛虱紅眼突變型和褐眼野生型的近等基因系,命名為NIL-rr和NIL-BB。生物學試驗結(jié)果表明,NIL-BB的生存力及繁殖力顯著下降,而NIL-rr和普通的室內(nèi)褐眼種群(BB)相似。其中,NIL-BB的單雌產(chǎn)卵量及卵孵化率兩參數(shù)的顯著降低是其種群適合度下降的主要原因。BB、NIL-rr和NIL-BB的種群趨勢指數(shù)分別為52.18、43.80和4.19。另外,NIL-rr品系初羽化雌成蟲的體重和體長也顯著重／長于NIL-BB品系。相對于NIL-BB, NIL-rr較強的繁殖力及較大的體型可能是因為眼色突變基因或其緊密連鎖基因?qū)Ψ敝尘哂休^強的補償作用。本研究結(jié)果表明,NIL-rr并不具有生態(tài)風險,或可用作田間研究材料。意外獲得的具有較低繁殖力和生命力的NIL-BB品系試蟲可用作生殖相關(guān)信號通路的研究模型。2.褐飛虱眼色相關(guān)基因的克隆為了進一步定位突變基因,本部分在前期獲得的4個基因片段的基礎上,進一步利用生物信息學、熱啟動PCR和RACE技術(shù)克隆了10個眼色相關(guān)基因,其中有7個基因通過RACE技術(shù)獲得了全長序列,包括5’-和3’-UTR,有3個基因還未獲得全長序列。同源比對表明,這些基因都與其他昆蟲的直系同源物具有較高的一致性,也存在典型的特征序列,因此認為它們編碼蛋白就是相對應果蠅眼黃素合成通路的色氨酸加氧酶(vermilion)、犬尿氨酸甲酰胺酶、犬尿氨酸羥化酶(cinnabar)和酚額嗪酮合成酶(karmoisin),色素轉(zhuǎn)運通路的White (white)和Scarlet (scarlet)半轉(zhuǎn)運子,色素顆粒形成通路的接頭蛋白復合體AP3的4個亞基(garnet/car mine/ruby/orange)。本部分的研究結(jié)果為篩選變異靶標基因奠定了基礎。3.眼色基因變異位點的篩選為了尋找導致褐飛虱復眼眼色變異的分子機理,即突變位點,本研究對野生型和突變型近等基因系的10個眼色相關(guān)基因的編碼區(qū)分別進行了克隆和對比分析。結(jié)果發(fā)現(xiàn)所有紅眼突變個體的犬尿氨酸羥化酶(KMO)編碼基因Nlkmo都存在三個同義突變位點、一個異義突變位點(L49I)和一個5堿基的插入突變位點。閱讀框內(nèi)5堿基的插入,使氨基酸翻譯提前終止,缺少了115個氨基酸殘基,破壞了兩個跨膜螺旋。4. Nlkmo基因的眼色決定作用驗證前人已證明,KMO在雙翅目的果蠅和蚊子、鱗翅目的家蠶復眼著色過程中起關(guān)鍵作用,但其對半翅目昆蟲復眼著色過程的影響還不明確。本章對褐飛虱野生型品系3齡若蟲注射KMO雙鏈RNA (dsKMO)后,Nlkmo表達水平顯著下降,復眼眼色部分變?yōu)榧t色,且眼黃素也下降為對照個體的55.3%。這表明,同其他昆蟲一樣,褐飛虱KMO也是褐飛虱眼黃素合成通路關(guān)鍵酶之一。此結(jié)果還說明,Nlkmo作為標記基因可用于褐飛虱及相近昆蟲的卵期RNAi技術(shù)的研發(fā)。5. Nlkmo基因的時空表達特征及其在突變體中的表達變化為了深入了解褐飛虱Nlkmo基因的分子特性,本部分測定了其時空表達特征,并對比分析了其在紅眼突變體中的表達變化。Nlkmo基因在所有生育期及組織內(nèi)都有表達,包括卵、若蟲、成蟲、卵巢、中腸、馬氏管、體壁和脂肪體。Nlkmo轉(zhuǎn)錄本水平在NIL-BB和NIL-rr兩品系間并無顯著差異,推斷Nlkmo存在的一個異義突變位點和一個5nt的插入位點并不影響轉(zhuǎn)錄本水平,可能是在蛋白質(zhì)翻譯及修飾階段對眼色產(chǎn)生影響。6. Nlkmo編碼蛋白的催化活性分析本章首先提取NIL-BB和NIL-rr兩品系褐飛虱頭部的總蛋白,分別命名為KMO-BB(?)KMO-rr。利用液相色譜技術(shù)測定了兩種總蛋白轉(zhuǎn)化犬尿氨酸(KYN)為3-羥基犬尿氨酸(3-HK)的催化活性。結(jié)果表明,KMO-BB的催化活性約為KMO-rr的9.03倍。為了精準分析,本章利用昆蟲桿狀病毒表達系統(tǒng)對野生型和突變型KMO酶進行了外源表達,分別命名為rKMO-BB和rKMO-rr,其中rKMO-rr未引入點突變位點(L49I),只引入了5堿基的插入突變位點。利用相同方法測定重組蛋白的催化活性,結(jié)果表明,rKMO-BB的活性遠遠高于KMO-BB,而rKMO-rr的活性小于KMO-rr,以致未能檢測到。以上結(jié)果說明,Nlkmo既是褐飛虱紅眼突變表型產(chǎn)生的靶標基因,而5堿基的插入是紅眼突變體KMO催化活性下降或喪失的主要突變方式。在褐飛虱頭部,除了KMO,也許還存在其他同工酶或備選通路可將KYN轉(zhuǎn)化為3-HK,這還需進一步研究證明。綜上所述,Nlkmo基因5堿基的插入突變方式是褐飛虱紅眼表型產(chǎn)生的分子基礎。褐飛虱紅眼突變機理的揭示將有助于褐飛虱及相關(guān)昆蟲卵期RNAi技術(shù)及轉(zhuǎn)基因體系的構(gòu)建,從而推動飛虱后基因組學的發(fā)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.

【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.112.3

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