【作者】 吴巧娟；

【导师】 喻德跃；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 大豆食叶性害虫是影响大豆产量和品质的因素之一。本文的研究目的之一是筛选鉴定大豆抗感虫材料。采用室内喂养斜纹夜蛾的方法，利用网室采集的58份大豆资源的叶片，室内喂养刚孵化的斜纹夜蛾幼虫。根据虫体的反应，以幼虫重和蛹重为指标，鉴定了大豆资源对斜纹夜蛾单一虫种的抗性表现。鉴定结果表明：两指标在各材料间差异极显著；且幼虫重和蛹重间相关显著。综合2001年和2002年的抗性鉴定结果根据标准品种分级法所划分的抗性等级筛选出4份表现高抗的材料：黄皮小青豆、日本、矮杆黄、P1227687；表现高感的材料3份：监利牛毛黄、沔阳白毛豆、大浦大粒黄。可作为抗性鉴定的标准材料，用于抗性分级；也可作为抗感亲本，用于大豆对斜纹夜蛾抗生性的遗传研究或育种应用；也可作为克隆抗虫基因和鉴定基因功能的材料。 本研究的另一个目的是克隆抗虫基因。根据大豆KUNITZ型胰蛋白酶抑制剂基因保守域设计特异性引物，用同源序列克隆法从大豆材料Lamar(抗)和89-29(感)中分离KUNITZ型胰蛋白酶抑制剂基因。序列分析表明，该基因与已报导的序列高度同源：其核苷酸序列及推导的编码氨基酸序列的同源率分别为99.3％和99.1％。对从抗感材料获得的蛋白酶抑制剂基因进行分析，可知抗感材料间存在着3个核苷酸的差异并引起2个氨基酸残基的变化。 根据大豆凝集素基因设计特异性引物，从大豆材料皖82-178(抗)和通山薄皮黄豆甲(感)中获得凝集素基因。序列分析表明，皖82-178中克隆到的基因片段大小为999bp，没有内含子，编码一条长为285个氨基酸、分子量约31KD的肽链。其中N-端31个氨基酸是信号肽。与已报道的序列高度同源：其核苷酸序列及推论的编码氨基酸序列的同源率分别为99.6％和99.3％。系统进化分析表明，大豆凝集素基因最先与亲缘关系较近的其他豆科植物先聚类，再和亲缘关系较远的禾本科、十字花科等植物聚类。说明了这些植物间的亲缘关系，也说明对基因表达产物功能的推测是可靠的。通山薄皮黄豆甲中获得的lec序列编码一条长为253个氨基酸的肽链。结构分析表明只含有Legume lectin beta domain，而皖82-178中得到的lec基因含有Legume lectin beta domain和Legume lectin alpha domain。这些变化对多肽的抑制活性可能会有影响，但结果还有待于进一步验证。 根据大豆胰蛋白酶抑制剂基因设计特异性引物，从科丰l号(杭)、1 1 38一2(感)组合的重组自交系的亲本中分别获得大豆蛋白酶抑制剂基因.经序列分析表明，克隆到的基因片段大小为1 376bP，含835bp的内含子。经分析亲本间有酶切位点(B stBI)的差异.从185个家系中随机筛选了104个家系进行以Ps分析.但根据所得的分子数据和现有养虫的杭性数据进行分析，发现还不能将此C人PS标记与杭虫性结果相联系，需要进一步研究.更多还原

【Abstract】 Soybean leaf-feeding insects affect the yield and quality of soybean. One objective of this study was to screen resistant and susceptible soybean accessions. The 58 accessions were tested for the antibiosis to cotton worm (Prodenia litura Fabricius) under inoculation condition according to the 10 days larval and pupal weight. The result indicated that the larval weight and pupal weight were all significantly different among varieties. Low larval and pupal weight indicates the high level of antibiosis. Larval weight was positively related to pupal weight. According to the result of 2001 and 2002, four genotypes, N20922, N21565, N21249 and N21551 were identified to be highly resistant to cotton worm; Three genotypes, N3108, N4029-3 and N3155-1 were identified to be highly susceptible to cotton worm. These genotypes can be used in inheritance study, breeding program and functional study.In this study, Soybean kunitz trypsin inhibitor was cloned through candidate gene homology-based method by specific primers designed on the basis of KTi3. DNA sequence analysis indicates that the cloned DNA fragment composed of 700bp has 99.3%homologies in nucleotide sequence and 99.1% in amino acid sequence respectively compared with previously published sequence. There are three bases differences, which lead to two amino acids changes between sequence of KTI from Larma and 89-29.With the same method, the soybean lectin was also cloned. The sequence from Wan82-178 show that the cloned fragment is 999bp. Like most plant lectin genes, there is no intron in the amplified sequence. The open reading frame of the fragment codes a 31KD polypeptide, which contains 285 amino acid residues. There is a signal sequence with 31 amino acid residues at its NH2 terminus. The cloned DNA fragment has 99.6% and 99.3%homologies in nucleotide sequence and amino acid sequence compared with Led. Molecular evolution studies of Lec reveals that Lec clusteres with legumes first, thenclusteres with Gram ineae and mustard family. This shows the genetic relationship among plants and suggests that the inference is reliable about the function of the product of gene. The sequence from Tong Shan bo pi huang dou jia show that the cloned fragment is 1000bp. The open reading frame contains 253 amino acid residues. Structure analysis show that it only contains Legume lectin beta domain. And the sequence from Wan 82-17S contains Legume lectin beta domain and Legume lectin beta domain. These changes maybe affect the activity of polypeptide. But this requires further study to be carried out.The soybean trypsin inhibitor was obtained from two parents of recombinant inbred population (RIL) from the cross of kefeng No 1 (resistance) and 1138-2(susceptible). DNA sequence analysis indicates that the cloned fragment is 1376bp with an 835bp intron. After the BstB I digestion, the digestion products showed polymorphism between the two parents. 104 lines were screened randomly from RIL to digest the PCR products with BstB I. The data analysis showed that the current CAPS marker data are not relevant to the resistance data. This requires further study to be carried out更多还原