重要森林鳞翅目害虫种群遗传调控相关基因研究

发布时间：2018-01-15 17:20

本文关键词：重要森林鳞翅目害虫种群遗传调控相关基因研究　出处：《中国林业科学研究院》2016年博士论文　论文类型：学位论文

【摘要】：马尾松毛虫是我国最重要的森林食叶害虫,美国白蛾是世界性重要检疫害虫,这两种重要森林害虫给林业生产和生态环境造成巨大损失,严重威胁我国森林生态系统的安全。为了达到长期持续控制害虫的目的,急需开发新型的森林害虫可持续控制方法,其中种群遗传调控方法克服了传统方法不能持续控制害虫的缺点,具有巨大的发展和应用潜力。为了实现这一目标,我们分别从转录组测序筛选性别相关基因、克隆和研究性别决定关键基因、建立遗传转化和基因组编辑平台等方面开展了研究,取得了如下结果:1.马尾松毛虫的转录组分析为了全面了解马尾松毛虫的遗传学特性,选取了其4个发育时期和5龄幼虫中期的5种组织,以及雌雄成虫触角这11组材料进行了转录组分析。数据分析表明拼接得到510M reads,进一步拼接得到50M的Transcripts,Transcripts平均长度为950.1051bp。结合基因同源性和从头计算方法共注释了54102个Unigenes,与NCBI-NR数据库比对发现17985(33.2%)个可以有效地匹配。相对于卵期、蛹期和成虫期,马尾松毛虫在幼虫期基因数量变化最多。5龄幼虫期的5种组织中,精巢和卵巢与头、脂肪体和中肠差异大于非生殖器官之间的差异;其中,精巢和卵巢相比,卵巢高表达基因894个,精巢高表达基因1344个;雌雄成虫触角也是性二型的主要器官,雄性触角高表达基因589个,雌性触角高表达基因227个。通过同源比对在马尾松毛虫中找到了21个性别决定相关的同源基因,包括性别决定初始信号X:A同源基因基因Da、Emc和Gro,计量补偿途径同源基因Msl3、Mle和Mof,体细胞性别决定途径同源基因Sxl、Tra2、Dsx、Ix、Doa、Snf、Vir和Fl(2)d,求偶行为和种系分化途径同源基因Fru、Dsf、Out和Ovo,搜索到与家蚕报道的文献中提到的同源基因Imp、Hrp28、Psi和Piwi。其中精巢Top20基因中有11个未做功能注释,可能也参与性别决定通路,尚待进一步实验验证。2.马尾松毛虫Dsx,Tra2和Ix基因的克隆及功能研究为了探索马尾松毛虫性别决定机制,筛选并克隆了马尾松毛虫Dsx,Tra2和Ix三个基因,并对这三个基因进行了RNAi。通过RACE扩得三个基因的全长,获得了六种DpDsx的剪接体形式,五种雌性剪接体全长分别为884bp、990bp、1148bp、1269bp和1962bp,分别表达两种蛋白质,蛋白质长度为243个氨基酸和252个氨基酸;一种雄性剪接体全长为1531bp,蛋白质长度为275个氨基酸;dptra-2基因有四种剪接形式,无性别表达差异,其orf区全长分别为765bp、768bp、843bp和858bp,表达蛋白长度分别为254个、255个、280个和285个氨基酸;dpix基因有两种剪接形式,分别命名为dpix-a和dpix-b,核苷酸长度分别为729bp和791bp,其中dpix-b比dpix-a多了一个外显子,dpix-b的orf为576bp,编码192个氨基酸;dpix-a在第三个预测的外显子处含终止密码子,dpix-a的的orf仅为222bp,编码74个氨基酸。rnai结果表明任意干扰dsx,tra2和ix,都会对彼此产生影响,并未观察到表型变化,需要更有效的基因编辑手段进行基因功能研究。3.遗传转化分析为了试验转基因操作在马尾松毛虫和美国白蛾上的可行性,我们利用pbac[a3-3*p3/egfp]和pbac[ie1/dsred]转基因载体对胚胎期个体进行显微注射。荧光检测和excisionassay结果表明piggybac转座酶成功切割了转基因载体,说明基于piggybac的转座子可以用于构建马尾松毛虫和美国白蛾的转基因平台。基于pbac[ie1/dsred]可以在马尾松毛虫和美国白蛾体内瞬时表达,在此基础上我们进行胚胎期注射来获得可遗传后代个体。对马尾松毛虫和美国白蛾注射pbac[ie1/dsred]、pbac[a3/hepler]和转座酶mrna混合体系,仅获得了马尾松毛虫g1代阳性个体,转化效率仅达0.014%,并且荧光随着昆虫生长发育而逐渐消失,pcr检测和inversepcr结果表明piggybac转座子不能在马尾松毛虫体内稳定表达,需要进一步摸索实验条件,完善转化体系。4.crispr/cas9基因组编辑为了验证crispr/cas9系统对马尾松毛虫和美国白蛾的基因组编辑效率,在胚胎期的同时注射cas9mrna和sgrnas。crispr/cas9技术对马尾松毛虫abd-a和wnt-1可以进行高效切割,这两个基因敲除均会导致g0代胚胎期高死亡率,分别为70.4%和77.5%,突变率分别为47.5%和55%,表型率分别是17.5%和32.9%。敲除abd-a和wnt-1得到相同的表型-腹节不正常发育。除此之外,敲除wnt-1还可以造成马尾松毛虫头部和跗肢的缺失。crispr/cas9技术也可对美国白蛾wnt-1基因进行高效编辑,胚胎期死亡率高达99.8%,突变率达62.5%,获得体节融合、附肢缺失等表型,说明这个系统有效诱导马尾松毛虫和美国白蛾在特定位点的突变,对于非模式生物基因功能研究有很好的应用前景。rt-pcr和免疫组化结果显示美国白蛾胚胎发育类型符合短胚带型和中间胚带型。这些结果表明wnt-1在马尾松毛虫和美国白蛾胚胎发育早期体节分割和体轴形成中发挥关键作用,而Abd-a在胚胎发育后期的A2-A7腹节的体节决定中发挥作用。种群遗传调控是有望实现害虫可持续防治的新方法。本研究通过转基因操作,探索遗传转化在马尾松毛虫和美国白蛾的应用前景,发现piggybac转座系统可以在马尾松毛虫和美国白蛾中应用,为马尾松毛虫和美国白蛾的遗传操作提供支持。CRISPR/Cas9系统对马尾松毛虫和美国白蛾可以进行高效基因组编辑,这对于林业害虫基因功能研究提供了有效手段,同时也对其他非模式生物的基因编辑研究提供新的思路。遗传转化和基于基因组编辑技术的基因功能研究是害虫种群遗传调控的基础,为害虫可持续控制提供理论支持。
[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.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S763.42

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