【作者】 张国彦；

【导师】 翟保平；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 东方粘虫Pseudaletia separata(Walker)是我国和其它亚洲国家粮食作物的主要害虫之一,广泛分布于我国各地。东方粘虫具有迁飞性、暴食性等特点,容易暴发成灾。我国科学家曾对其宏观迁飞规律、迁飞行为机制、飞行与生殖的关系等进行了深入研究,但对其种群分子遗传学却很少涉及。本研究着力于东方粘虫微卫星标记的分离和鉴定,为以后种群遗传学研究准备工具；同时,本研究对该物种基因组微卫星特征和变异进行了较深入地分析,并优化了鳞翅目成虫DNA提取的技术方法。将传统的蛋白酶K+SDS+苯酚抽提方法和CTAB方法结合起来,筛选出从东方粘虫成虫自然种群的乙醇保存标本中提取高质量、高产量基因组DNA的方法,用于制备分离微卫星标记所需的DNA样品。对比研究结果表明,用CTAB+传统方法提取DNA时,取腹部中段10-20 mg组织样品进行实验,不同种群DNA产品合格率68.42%-95.28%,提取合格DNA量5.59-10.04 mg/g,显著高于传统蛋白酶K+SDS裂解及苯酚抽提方法的7.69%-40%和2.83-5.78 mg/g。另外,用热NaOH法快速制备用于大规模基因分型的DNA样品的研究结果表明,对于胸部和腹部组织样品,用热NaOH溶液处理20 min,就能够提取出微卫星PCR扩增反应所需的DNA样品；对于头部组织样品,用热NaOH溶液处理40-60 min是比较合适的。对比研究结果显示,来自热NaOH方法的DNA样品在构建微卫星富集文库过程中失败,但两种样品作为模板进行微卫星PCR扩增的产物在遗传分析仪上检测结果相同。利用生物素5’标记的(CA)15、(GA)15、(ATG)12、(GAT)12、(TAGA)8和(GTGA)8作为探针,采用磁珠富集法构建东方粘虫微卫星富集文库。经过基因组DNA酶切、300-700bp片段回收、连接接头、纯化、连接产物变性、已结合探针的磁珠捕捉目标片段、目标片段洗脱、PCR扩增、连接pGEM-T载体并转化DH5α高效感受态细胞、蓝白筛选等过程,最后构建成功包含8531个克隆的粘虫微卫星富集文库。从文库随机抽取866个克隆进行测序,并分析序列中是否含有微卫星,统计结果表明,所建文库中平均微卫星阳性克隆率35.8%,高于以往对棉铃虫、马尾松毛虫等鳞翅目昆虫进行同类研究的结果。用REPEATMASKER软件和人工分析相结合,对本研究已筛选出的一批东方粘虫微卫星的类型、各类丰度、变异情况等进行深入分析,并与InSatDb数据库中5种全基因组测序昆虫(黑腹果蝇、冈比亚按蚊、赤拟谷盗、意大利蜜蜂、家蚕)基因组中微卫星的特征和变异情况进行比较。本研究已发现369个东方粘虫微卫星序列、46个低复杂度区域、6个转座元件和1个tRNA.所得微卫星分属于21个不同重复基序,以TTG/CAA基序的微卫星最多,占全部微卫星总数的37.13%,其次是CA/TG类和CAT/ATG类,分别占30.89%和15.72%。这三类微卫星合起来占全部微卫星的83.74%。所得微卫星序列还具有短型偏多、不完全型(存在突变)偏多、AT富集的偏多、复合型较少等特点。这些特点与5种全基因组测序昆虫微卫星的统计结果基本一致。对不完全型微卫星的重复序列变异情况进行统计,替换碱基比例一般0.6%-11.7%,个别达到15.6%-26.1%；缺失碱基比例0.5%-10.6%,插入碱基比例0.3%-11.3%,平均每处插入／缺失碱基1-1.8,个别达到3-6个碱基。进一步的分析发现,东方粘虫微卫星重复序列的变异还存在如下特点：碱基替换在突变中占据优势,在全部微卫星基因座中发生比例达到92.38%,而缺失和插入微卫星比例分别只有27.62%和25.24%；替换+插入、替换+缺失的发生比例接近,均为20%稍多,而缺失+插入、替换+缺失+插入的比例较低,均为12%稍多；两类数量最多的微卫星TTG/CAA(137个)、TG/CA (114个)的重复序列变异中,转换和颠换发生的频率并不相同,转换要多于颠换；重复序列长度101-150bp和151-200bp的微卫星发生变异的概率较大,变异碱基数较多,而小于50bp和大于200bp的微卫星变异比例和碱基变异比例均明显减小；TTG/CAA类的变异微卫星比例高于TG/CA类,而变异碱基比例则明显低于后者；变异微卫星重复序列近5’端(将一个重复序列分为五段,即5’端、近5’端、中段、近3’端、3’端)变异碱基数最多,其次是中段,而两极则相对较少发生变异,而且变异最多区域出现在近5’端的微卫星数量也最多。另外,在各类微卫星基因座侧翼序列比较中,仅发现11类侧翼序列相同的多拷贝微卫星基因座,涉及微卫星基因座24个,占所发现全部微卫星基因座的6.50%。但并没有发现侧翼序列高度相似的微卫星家族。在含有微卫星的克隆序列中,经过序列选择、引物设计和合成、Touchdown PCR初选(24个基因组DNA样品)、优化PCR优选(24个基因组DNA样品)、遗传分析仪再选(48个基因组DNA样品)等三轮筛选,共筛出8个多态性微卫星标记(GenBank登录号FJ896055-FJ896062).对8个微卫星基因座在48个样品中的等位基因分布情况进行统计,并用GenePop软件进行检测,经Bonferroni校正,均符合哈迪-温伯格平衡(Hardy-Weinberg proportions),微卫星基因座之间也没有检测出显著的配子相不平衡(gametic phase disequilibrium)。用Cervus软件统计计算,各基因座有11-31个等位基因,期望杂合度0.747-0.931,显示出各基因座都具较好多态性,可用做东方粘虫种群研究的遗传标记。本研究的创新之处主要表现在以下几个方面：一、改进了鳞翅目成虫基因组DNA提取方法,使之适合微卫星富集文库构建、微卫星标记筛选和分型的需要；二、国内首次成功构建东方粘虫微卫星富集文库,抽样检测微卫星阳性克隆率较高；三、首次对东方粘虫基因组微卫星序列进行特征和变异分析,丰富了鳞翅目昆虫基因组微卫星序列的信息；四、首次筛选出8个东方粘虫微卫星标记,填补了东方粘虫微卫星遗传标记的空白。更多还原

【Abstract】 The oriental armyworm Pseudaletia separata(Walker) is one of the most important agricultural pests in China and other Asian countries, and occurs throughout China except for Xinjiang and Tibet provinces. It has the habits of migration and gluttony, frequently leading to a large-area outbreak. Great efforts have been made by Chinese scientists in the study of its migration regularity, migratory behavior mechanism and the relationship between flight and reproduction, however molecular genetics of oriental armyworm was seldom involved. In this work some polymorphic microsatellite loci of this Lepidopteran insect were characterized which can be used as markers for genetic study. we have firstly construct six microsatellite-enriched libraries, then screen microsatellite markers. Furthermore, the extraction methods of genomic DNA from Lepidoptera adults were optimized, and the features and variations of microsatellite sequences were analyzed.CTAB-based method was combined with routine phenol extraction (called "CTAB-combined method" below) to obtain high-quality and high-yield DNA from various populations of ethanol-preserved adult oriental armyworms collected in the field. 10-20 mg tissue is appropriate to extract DNA in a 2 ml microcentrifuge tube, with a qualified DNA products proportion of 68.42%-95.28% and a DNA yield of 5.59-10.04 mg/g, significantly greater than 7.69%-40% and 2.83-5.78 mg/g of DNA products prepared with a route method (tissue digestion with SDS and proteinase K, DNA purification with phenol:chloroform:isoamyl alcohol (25:24:1) and DNA precipitation with alcohol). Simultaneously, hot NaOH solutions were successfully used to isolate genomic DNA which is suitable for temples in PCRs when abdominal or thoracic tissue was heated for 20 min, or cephalic tissue was heated for 40 min. It was demonstrated that two kinds of DNA samples were both good enough for microsatellite genotyping, but DNA preparations extracted with NaOH-based approach failed in microsatellite isolation. Six microsatellite-enriched libraries were constructed with a magnetic-bead enrichment method, in which 5’biotin-labelled (CA)15, (GA)15, (ATG)12, (GAT)12, (TAGA)8 and (GTGA)8 were used as probes. This involved a series of successive steps, i.e.enzyme restriction of genomic DNA, the recovery of 300-700bp fragments, adapter ligation, purification and denaturation of ligation products, capture of target fragments with magnetic beads combined with probes, elution of target fragments, PCR amplification, vector ligation and transformation into DH5a competent cells, blue-white screening. Finally six microsatellite-enriched libraries were achieved containing 8531 clones. In 866 clones randomly sampled from libraries and sequenced, microsatellite-positive clones account for 35.8%, greater than results of similar research in cotton bollworm Heliothis armigera and masson pine moth Dendrolimus punctatus.369 microsatellites were found from 866 sequenced clones by Repeatmasker software and manual analysis. In addition,46 low complexity regions,6 mobile elements and 1 tRNA were recognized from them. These microsatellites were divided into 21 types of repeat motif, of which TTG/CAA motif is the most common with a proportion of 37.13%, secondly CA/TG (30.89%), thirdly CAT/ATG (15.72%), only 16.26% from other types. Among all microsatellites, short (repeat sequence length<50bp) type or imperfect (existence of mutations among repeat sequences) type or AT-rich type is in the majority, and the compound type is small in number. Statistical results of imperfect microsatellites show that the rate of base substitution, base deletion and base insertion are 0.6%-26.1%, 0.5%-10.6% and 0.3%-11.3%, respectively.Further analysis showed that mutations in repeat sequences of oriental armyworm genome have other characteristics as follow:base substitution holds overwhelming superiority in imperfect microsatellites with a proportion of 92.38%, microsatellites containing deletion or insertion only account for 27.62% and 25.24%, respectively; the proportions of microsatellites containing substitution+insertion and substitution+deletion are both a little more 20%, deletion+insertion and substitution+deletion+insertion types have similar ratios, a little more 12%; the occurance rate of transition is greater than transversion in two most abundant types in this study; microsatellites with 101-150 bp and 151-200bp repeat sequences possess greater mutation rate and more mutational bases than those with<50 bp and> 200 bp repeats; compared with TG/CA type, TTG/CAA type has more imperfect members, but a less percentage of mutational bases; in a mutation-containing microsatellite repeat sequence, mutations mainly occur in 5’proximal sequences, secondly in middle segment, least in two extremes. But no microsatellite family was found in comparison of all microsatellites, only 11 types of multicopy microsatellites were discovered involving 24 (0.6%) microsatellite loci. These features above are mostly consistent with that of 5 species which have been whole-genome sequenced.Among DNA fragments containing microsatellites,8 polymorphic microsatellite loci (GenBank Acc. no. FJ896055-FJ896062) were finally identified after three rounds of screening:primary screening of 24 DNA samples with touchdown PCRs, optimization PCRs of 24 DNA samples and the third-round selection on ABI Prism 3100 genetic analyzer. Genotypic data of 48 adult samples were analysed with GenePop 4.0. No significant deviation from Hardy-Weinberg proportions was detected after Bonferroni correction, and exact tests for gametic disequilibrium between loci were all nonsignificant. The number of alleles per locus ranged from 11 to 31, and the expected heterozygosity ranged from 0.747 to 0.931 (Cervus version 3.0.3). The availability of these 8 informative loci for P. separata will provide useful markers for studies of population genetics.The major innovations of this study were as follows:the extraction methods of genomic DNA were improved from Lepidoptera adults for microsatellite marker isolation, characterization and large-scale genotyping.For the first time 6 microsatellite-enriched libraries of oriental armyworm were constructed which were of a higher average rate of microsatellite-positive clones.The situations of motif, abundance and mutation of microsatellite loci of oriental armyworm were first analyzed enriching the information of Lepidoptera microsatellites.8 polymorphic microsatellite markers were identified, filling a gap in microsatellite genetic markers of oriental armyworm.更多还原