發(fā)布時(shí)間：2018-01-15 17:20

  本文關(guān)鍵詞：重要森林鱗翅目害蟲種群遺傳調(diào)控相關(guān)基因研究　出處：《中國林業(yè)科學(xué)研究院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 馬尾松毛蟲 美國白蛾 轉(zhuǎn)基因 CRISPR/Cas9系統(tǒng) 性別決定 胚胎發(fā)育 Wnt-1 Abdominal-a

【摘要】：馬尾松毛蟲是我國最重要的森林食葉害蟲,美國白蛾是世界性重要檢疫害蟲,這兩種重要森林害蟲給林業(yè)生產(chǎn)和生態(tài)環(huán)境造成巨大損失,嚴(yán)重威脅我國森林生態(tài)系統(tǒng)的安全。為了達(dá)到長期持續(xù)控制害蟲的目的,急需開發(fā)新型的森林害蟲可持續(xù)控制方法,其中種群遺傳調(diào)控方法克服了傳統(tǒng)方法不能持續(xù)控制害蟲的缺點(diǎn),具有巨大的發(fā)展和應(yīng)用潛力。為了實(shí)現(xiàn)這一目標(biāo),我們分別從轉(zhuǎn)錄組測序篩選性別相關(guān)基因、克隆和研究性別決定關(guān)鍵基因、建立遺傳轉(zhuǎn)化和基因組編輯平臺(tái)等方面開展了研究,取得了如下結(jié)果:1.馬尾松毛蟲的轉(zhuǎn)錄組分析為了全面了解馬尾松毛蟲的遺傳學(xué)特性,選取了其4個(gè)發(fā)育時(shí)期和5齡幼蟲中期的5種組織,以及雌雄成蟲觸角這11組材料進(jìn)行了轉(zhuǎn)錄組分析。數(shù)據(jù)分析表明拼接得到510M reads,進(jìn)一步拼接得到50M的Transcripts,Transcripts平均長度為950.1051bp。結(jié)合基因同源性和從頭計(jì)算方法共注釋了54102個(gè)Unigenes,與NCBI-NR數(shù)據(jù)庫比對(duì)發(fā)現(xiàn)17985(33.2%)個(gè)可以有效地匹配。相對(duì)于卵期、蛹期和成蟲期,馬尾松毛蟲在幼蟲期基因數(shù)量變化最多。5齡幼蟲期的5種組織中,精巢和卵巢與頭、脂肪體和中腸差異大于非生殖器官之間的差異;其中,精巢和卵巢相比,卵巢高表達(dá)基因894個(gè),精巢高表達(dá)基因1344個(gè);雌雄成蟲觸角也是性二型的主要器官,雄性觸角高表達(dá)基因589個(gè),雌性觸角高表達(dá)基因227個(gè)。通過同源比對(duì)在馬尾松毛蟲中找到了21個(gè)性別決定相關(guān)的同源基因,包括性別決定初始信號(hào)X:A同源基因基因Da、Emc和Gro,計(jì)量補(bǔ)償途徑同源基因Msl3、Mle和Mof,體細(xì)胞性別決定途徑同源基因Sxl、Tra2、Dsx、Ix、Doa、Snf、Vir和Fl(2)d,求偶行為和種系分化途徑同源基因Fru、Dsf、Out和Ovo,搜索到與家蠶報(bào)道的文獻(xiàn)中提到的同源基因Imp、Hrp28、Psi和Piwi。其中精巢Top20基因中有11個(gè)未做功能注釋,可能也參與性別決定通路,尚待進(jìn)一步實(shí)驗(yàn)驗(yàn)證。2.馬尾松毛蟲Dsx,Tra2和Ix基因的克隆及功能研究為了探索馬尾松毛蟲性別決定機(jī)制,篩選并克隆了馬尾松毛蟲Dsx,Tra2和Ix三個(gè)基因,并對(duì)這三個(gè)基因進(jìn)行了RNAi。通過RACE擴(kuò)得三個(gè)基因的全長,獲得了六種DpDsx的剪接體形式,五種雌性剪接體全長分別為884bp、990bp、1148bp、1269bp和1962bp,分別表達(dá)兩種蛋白質(zhì),蛋白質(zhì)長度為243個(gè)氨基酸和252個(gè)氨基酸;一種雄性剪接體全長為1531bp,蛋白質(zhì)長度為275個(gè)氨基酸;dptra-2基因有四種剪接形式,無性別表達(dá)差異,其orf區(qū)全長分別為765bp、768bp、843bp和858bp,表達(dá)蛋白長度分別為254個(gè)、255個(gè)、280個(gè)和285個(gè)氨基酸;dpix基因有兩種剪接形式,分別命名為dpix-a和dpix-b,核苷酸長度分別為729bp和791bp,其中dpix-b比dpix-a多了一個(gè)外顯子,dpix-b的orf為576bp,編碼192個(gè)氨基酸;dpix-a在第三個(gè)預(yù)測的外顯子處含終止密碼子,dpix-a的的orf僅為222bp,編碼74個(gè)氨基酸。rnai結(jié)果表明任意干擾dsx,tra2和ix,都會(huì)對(duì)彼此產(chǎn)生影響,并未觀察到表型變化,需要更有效的基因編輯手段進(jìn)行基因功能研究。3.遺傳轉(zhuǎn)化分析為了試驗(yàn)轉(zhuǎn)基因操作在馬尾松毛蟲和美國白蛾上的可行性,我們利用pbac[a3-3*p3/egfp]和pbac[ie1/dsred]轉(zhuǎn)基因載體對(duì)胚胎期個(gè)體進(jìn)行顯微注射。熒光檢測和excisionassay結(jié)果表明piggybac轉(zhuǎn)座酶成功切割了轉(zhuǎn)基因載體,說明基于piggybac的轉(zhuǎn)座子可以用于構(gòu)建馬尾松毛蟲和美國白蛾的轉(zhuǎn)基因平臺(tái)�；趐bac[ie1/dsred]可以在馬尾松毛蟲和美國白蛾體內(nèi)瞬時(shí)表達(dá),在此基礎(chǔ)上我們進(jìn)行胚胎期注射來獲得可遺傳后代個(gè)體。對(duì)馬尾松毛蟲和美國白蛾注射pbac[ie1/dsred]、pbac[a3/hepler]和轉(zhuǎn)座酶mrna混合體系,僅獲得了馬尾松毛蟲g1代陽性個(gè)體,轉(zhuǎn)化效率僅達(dá)0.014%,并且熒光隨著昆蟲生長發(fā)育而逐漸消失,pcr檢測和inversepcr結(jié)果表明piggybac轉(zhuǎn)座子不能在馬尾松毛蟲體內(nèi)穩(wěn)定表達(dá),需要進(jìn)一步摸索實(shí)驗(yàn)條件,完善轉(zhuǎn)化體系。4.crispr/cas9基因組編輯為了驗(yàn)證crispr/cas9系統(tǒng)對(duì)馬尾松毛蟲和美國白蛾的基因組編輯效率,在胚胎期的同時(shí)注射cas9mrna和sgrnas。crispr/cas9技術(shù)對(duì)馬尾松毛蟲abd-a和wnt-1可以進(jìn)行高效切割,這兩個(gè)基因敲除均會(huì)導(dǎo)致g0代胚胎期高死亡率,分別為70.4%和77.5%,突變率分別為47.5%和55%,表型率分別是17.5%和32.9%。敲除abd-a和wnt-1得到相同的表型-腹節(jié)不正常發(fā)育。除此之外,敲除wnt-1還可以造成馬尾松毛蟲頭部和跗肢的缺失。crispr/cas9技術(shù)也可對(duì)美國白蛾wnt-1基因進(jìn)行高效編輯,胚胎期死亡率高達(dá)99.8%,突變率達(dá)62.5%,獲得體節(jié)融合、附肢缺失等表型,說明這個(gè)系統(tǒng)有效誘導(dǎo)馬尾松毛蟲和美國白蛾在特定位點(diǎn)的突變,對(duì)于非模式生物基因功能研究有很好的應(yīng)用前景。rt-pcr和免疫組化結(jié)果顯示美國白蛾胚胎發(fā)育類型符合短胚帶型和中間胚帶型。這些結(jié)果表明wnt-1在馬尾松毛蟲和美國白蛾胚胎發(fā)育早期體節(jié)分割和體軸形成中發(fā)揮關(guān)鍵作用,而Abd-a在胚胎發(fā)育后期的A2-A7腹節(jié)的體節(jié)決定中發(fā)揮作用。種群遺傳調(diào)控是有望實(shí)現(xiàn)害蟲可持續(xù)防治的新方法。本研究通過轉(zhuǎn)基因操作,探索遺傳轉(zhuǎn)化在馬尾松毛蟲和美國白蛾的應(yīng)用前景,發(fā)現(xiàn)piggybac轉(zhuǎn)座系統(tǒng)可以在馬尾松毛蟲和美國白蛾中應(yīng)用,為馬尾松毛蟲和美國白蛾的遺傳操作提供支持。CRISPR/Cas9系統(tǒng)對(duì)馬尾松毛蟲和美國白蛾可以進(jìn)行高效基因組編輯,這對(duì)于林業(yè)害蟲基因功能研究提供了有效手段,同時(shí)也對(duì)其他非模式生物的基因編輯研究提供新的思路。遺傳轉(zhuǎn)化和基于基因組編輯技術(shù)的基因功能研究是害蟲種群遺傳調(diào)控的基礎(chǔ),為害蟲可持續(xù)控制提供理論支持。
[Abstract]:The pine caterpillar is the most important forest insect pest in China, the American white moth is a worldwide important quarantine pest, the two important forest pests caused huge losses to forestry production and ecological environment, a serious threat to the safety of forest ecosystem in China. In order to achieve a sustainable long-term objective for pest control, it is imperative to develop the sustainable control of forest a new method of controlling pests, which overcomes the population genetic methods of pest control continued the traditional methods can not shortcomings, has great potential of development and application. In order to achieve this goal, we are screening of sex related genes from transcriptome sequencing, gene cloning and key decision on gender, the establishment of genetic transformation and genome editing platform to carry out the research results are as follows: 1. analysis of the transcriptome of Dendrolimus punctatus in order to fully understand the genetic characteristics of Dendrolimus punctatus, selected 5 the 4 developmental stages and 5 instar larvae in the middle, and the 11 groups of male and female adult antennae materials for transcriptome analysis. Data analysis showed that the splicing 510M reads, 50M Transcripts further spliced, the average length of Transcripts 950.1051bp. gene with homology and ab initio methods were 54102 Unigenes notes with the NCBI-NR database, 17985 (33.2%) can be effectively matched. Compared to the egg stage, pupal stage and adult stage, changes in the number of larvae of Dendrolimus punctatus gene up to 5.5 instar larvae in the testis and ovary and head, midgut and fat body differences greater than the differences between non reproductive organs; among them, compared with the testis and ovary, ovarian expression of 894 genes, the high expression of 1344 genes; the main organs of male and female adult antennae is type two, male antennae high expression of 589 genes, the female touch 227. High angle gene expression by homology to Dendrolimus punctatus found 21 homologous genes related to sex determination, including sex determination of the initial signal X:A homologous genes Da, Emc and Gro, the measurement means of compensation for the homeobox genes Msl3, Mle and Mof, somatic sex determination pathway homologous genes Sxl, Tra2, Dsx Ix, Doa, Snf, Vir, and Fl (2) d, courtship behavior and species differentiation pathway homologous genes Fru, Dsf, Out and Ovo, to search for homologous gene Imp mentioned and silkworm reported in the literature Hrp28, Psi and Piwi. in testis Top20 gene in 11 without functional annotation, may also be involved in the sex determination pathway, pending further experimental verification of.2. pine caterpillar Dsx, cloning and functional analysis of Tra2 and Ix gene of Dendrolimus punctatus in order to explore the mechanism of sex determination, screening and cloning of the pine caterpillar Dsx and Ix three, Tra2 gene, and the three genes were R NAi. through the RACE expansion and full-length three genes, obtained six kinds of DpDsx splicing forms, five female splicing length were 884bp, 990bp, 1148bp, 1269bp and 1962bp, respectively. The expression of two proteins, protein length of 243 amino acids and 252 amino acids; a male spliceosome was 1531bp, the protein is 275 amino acids long; dptra-2 gene has four alternative splicing, no gender difference expression of the full-length ORF region were 765bp, 768bp, 843bp and 858bp, the expression of protein length were 254, 255, 280 and 285 amino acids; dpix gene has two alternative splicing, named dpix-a and dpix-b, the nucleotide length were 729bp and 791bp, which dpix-b dpix-a more than one exon, dpix-b ORF is 576bp, encoding 192 amino acids; dpix-a in third predicted exon containing the termination codon, dpix-a ORF is only 222bp, The results show that.Rnai encoding 74 amino acids of DSX tra2 and IX of any interference, and will affect each other, did not observe phenotypic changes, the need for more effective gene editing methods for gene function research of.3. genetic transformation analysis in order to test operation in transgenic pine caterpillar and the feasibility of American white moth, we use pbac[a3-3*p3/egfp] and pbac[ie1/dsred] transgenic the carrier of individual embryo microinjection. Fluorescence detection and excisionassay results show that the piggyBac transposase cutting the transgenic vector, piggyBac transposon can be used to construct transgenic platform of Dendrolimus punctatus and American white moth. Based on pbac[ie1/dsred] in the pine caterpillar and American white moth in transient expression based on the basis of our embryo injection to obtain genetic offspring. The pine caterpillar and American white moth Injection of pbac[ie1/dsred], pbac[a3/hepler] and mRNA transposase mixed system, for Dendrolimus G1 generation positive individuals, the conversion efficiency was only 0.014%, with the growth and development of insects and the fluorescence disappeared, PCR detection and inversepcr results show that the piggyBac transposon cannot express in Dendrolimus punctatus body stable, need to further explore the experimental conditions, improve the transformation system of.4.crispr/cas9 in order to verify the crispr/cas9 genome editing system editing efficiency of Dendrolimus punctatus and American white moth genome injection in the embryonic period, while the cas9mrna and sgrnas.crispr/cas9 technology can be an efficient cutting of the pine caterpillar Abd-A and Wnt-1, the two knockout will lead to G0 generation of embryonic mortality were 70.4% and 77.5% respectively, the mutation rate 47.5% and 55% of phenotypic rates were 17.5% and 32.9%. knockdown of Abd-A and Wnt-1 to the same The phenotype abdominal segment does not develop normally. In addition, deletion of.Crispr/cas9 Wnt-1 knockout technology can also cause the head and limbs of the pine caterpillar tarsal can also be efficient editing of American white moth Wnt-1 gene, the embryonic mortality rate as high as 99.8%, the mutation rate of 62.5%, obtained somite fusion, appendage deletion phenotype, indicating mutation this system effectively induced by Dendrolimus punctatus and American white moth in a specific location, for the study of non model organism gene function has the application prospect of.Rt-pcr and immunohistochemical results show good hyphantriacunea embryonic development with short germ band type and intermediate type germ band. The results indicated that Wnt-1 in pine caterpillar and American white moth the early stages of embryonic development play a key role in somite segmentation and body axis formation, and the role of Abd-a in the late embryonic development of A2-A7 abdominal segments somite decision. Population genetic regulation is expected to achieve harm A new method of insect prevention sustainable. This study through genetic manipulation, explore the application prospect in genetic transformation of Dendrolimus punctatus and American white moth, piggyBac transposon system in pine caterpillar and American white Ezhong application for pine caterpillar and hyphantriacunea GA.CRISPR/Cas9 system can provide support for efficient genome editing of Dendrolimus punctatus and American white moth, which for forest pest gene function research provides an effective means, but also provides new ideas for gene editing studies on other non model organisms. Genetic transformation and gene function research of genome editing technology is based on the genetic basis of pest control, to provide theoretical support for sustainable pest control.

【學(xué)位授予單位】：中國林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S763.42

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