【作者】 廖芳；

【导师】 李关荣； 鲁成；

【作者基本信息】 西南农业大学 ， 生物化学及分子生物学， 2003， 硕士

【摘要】 家蚕是重要的经济昆虫，也是分子遗传学研究中重要的模式生物。蚕学研究的深入促进了家蚕近缘种—野桑蚕的研究。野桑蚕作为丰富的野生蚕业资源，是可开发利用的宝贵基因库，用于增加家蚕的遗传多样性和进行品种复壮，同时也是研究家蚕起源分化的重要材料。迄今已对野桑蚕进行了广泛的生物学上的研究、染色体结构及变异研究、DNA多态性研究、家蚕育种上的应用研究以及作为桑园害虫防治上的研究。α-淀粉酶是家蚕生物学上非常重要的功能酶类，已有大量的研究报道，但迄今还没有野桑蚕α-淀粉酶及基因的相关报道。本文对野桑蚕α-淀粉酶基因进行全序列测定与分析，并作了基于Intron Ⅰ的家蚕和野桑蚕分子系统学初步研究。 1．重庆野桑蚕α-淀粉酶基因全序列的测定 根据家蚕α-淀粉酶全基因序列设计了的7对引物，对野桑蚕α-淀粉酶基因进行了分段扩增，并逐一克隆测序和序列拼接，得到8093bp的野桑蚕α-淀粉酶基因全序列。 野桑蚕α-淀粉酶基因全序列的AT％为51.61％，包含5个外显子和4个内含子，外显子的AT％平均值为49.35％，内含子的AT％平均值为63.23％。 在Intron Ⅱ中有一个完整的non-LTR retrotransposon，其内编码一个reverse transcriptase；Intron Ⅱ还有一段序列与另外一种反转座子的部分序列高度同源。 2．家蚕和野桑蚕α-淀粉酶基因序列比较分析 家蚕和野桑蚕α-淀粉酶基因具有相似的基因结构，都包含5个exon和4个intron，各内含子、外显子长度在家蚕和野桑蚕间较类似，外显子和内含子AT％的平均值差异不大。家蚕和野桑蚕α-淀粉酶基因核苷酸序列相似性为90％，推定氨基酸序列相似性为98％。 家蚕、野桑蚕α-淀粉酶基因Intron Ⅱ中的(野桑蚕)non-LTR retrotransposon之间序列长度相等，相似性为98％，其内编码reverse transcriptase的基因，序列长度相等，核苷酸序列和氨基酸序列相似性达98％。 野桑蚕相对于家蚕来说，在α-淀粉酶基因外显子区域发生了13次转换、4次颠换、12bp插入，在内含子区域发生了362次转换、343次颠换、94bp插入和56bp缺失，全基因(包括UTR)共发生 MMrttoopMI－MgZX＄ffatt 了380次转换、350颠换、113hp插人和57hp缺失。 家蚕、野桑蚕a－淀粉酶基因密码子碱基组成统计结果表明：H者密码子3个位点的碱基组成差 异非常小。 3．五种昆虫的Q－淀粉酶基因序列的比较分析 野桑蚕 a－淀粉酶基因核苦酸序列与家蚕、草地贪夜蛾（Spodotera户u归rdaX玉米螟（Ostrinia nubilalis on 25）和果蝇（Dnsophila subobsc。）相似性分别为 90o、12％、12O、50，推定氨基酸 序列相似性分别为98％、80％、78％、58％，表明：野桑蚕与家蚕的亲缘关系最近，果蝇与野桑蚕的 亲缘关系相对较远。 4．家蚕、野桑蚕a－淀粉酶基因密码子碱基组成统计比较 对密码子进行了碱基组成的统计比较，结果表明：家蚕与野桑蚕间密码子三个位点的碱基组成非 常相似。密码子位1都表现出明显的瞟吟碱基偏好性（家蚕59．88％，野桑蚕59．90％）；密码子位2亦 表现较弱的瞟吟偏好性（家蚕55．07％，野桑蚕54．64％）；密码子位3有强烈的啼陡偏好性，家蚕和 野桑蚕的CT％分别为67．77％和67．17O，其中家蚕的Co为35．79o，野桑蚕的C％为35．59％，特别 占优势。位点2的ATO／较高，位点1、3差异极小。 5．根据 Intron序列进行了初步的分于系统学研究 基于六种家蚕品种C108、大造、白夏B、BH863、赤塾和重庆野桑蚕的 Intron的序列，以草地 贪夜蛾（坯m加teraMiperda）和果蝇（Dnaophila subobscura）为外群，采用 UPGMA法构建的分子 系统树表明：家蚕与野桑蚕之间有非常近的亲缘关系，家蚕中BH863似乎与野桑蚕之间有最近的亲 缘关系，这一初步研究结果还有待进一步证实。更多还原

【Abstract】 Bombyx mori is both an economically important insect and a model organism for the study of molecular genetics. The extensive study on Bombyx mori spurred the investigation of its consanguinity variety-Bombyx mandarina. As an abundant wild sericultural resource Bombyx mandarina is a treasure with much potential in research and development. It could increase the genetic diversity and be used as an important material for the study on the origin of silkworm. Up to now, extensive biological, chromosomal structure and variation, DNA polymorphism, breeding application as well as pest control studies have been conducted. Alpha-amylase is a functionally important enzyme for silkworm that feed on mulberry leaves. Although there are many studies on alpha-amylase of Bombyx mori, no reports on the alpha-amylase gene of Bombyx mandarina have been seen so far. In this investigation, the whole alpha-amylase gene of Bombyx mandarina has been cloned, sequenced and analyzed with that of the Bomyx mori. Additionally, based on the sequence of Intron I of alpha-amylase, a preliminary molecular systematics study has been performed on Bombyx mori and Bombyx mandarina.1. Sequencing of the whole alpha-amylase gene of Bombyx mandarina (Chongqing)Based on the published nucleotide sequence of alpha-amylase gene of Bombyx mori, seven pairs of primers were designed for the sectional amplification and cloning of the alpha-amylase gene of Bombyx mandarina. The combined sequence, i.e. the whole alpha-amylase gene of Bombyx mandarina, is 8903bp in length. Its AT content is 51.61%. The whole gene consists of five exons and four introns. The mean AT values for all the exons and all the introns are 49.35% and 62.23% respectively.There is an entire non-LTR retrotransposon in Intron II, which encodes a reverse transcriptase; also a segment in intron II is highly homologous with another retrotransposon.2. Sequence comparison of alpha-amylase gene of Bombyx mori and Bombyx mandarinaThe alpha-amylase gene structures of Bombyx mori and Bombyx mandarina are very similar, bothcomprised of five exons and four introns, with comparable length. The mean AT contents of exons and introns are not much different. The nucleotide sequence similarity of alpha-amylase gene of Bombyx mori and Bombyx mandarina is 90% and the putative amino acid sequence similarity is as high as 98%.In the alpha-amylase gene, the length of the non-LTR retrotransposon, located in Intron II, is equal in Bombyx mori and Bombyx mandarina, with a nucleotide sequence similarity of 98%; The length of the coded reverse transcriptase is equal in both nucleotide and amino acid sequence with a similarity of 98% also.Compared with Bombyx. mori, the exon regions of alpha-amylase gene of Bombyx mandarina have 13 transitions, 4 transversions and a 12bp insertions, while the intron regions have 362 transitions, 343 transversions, a 94bp insertions and a 56bp deletions. The whole alpha-amylase gene (including the 5’- and 3’-UTRs) of Bombyx mandarina has 380 transitions, 350 transversions, 113bp insertions and 57bp deletions.Statistics on code bias of alpha-amylase gene of Bombyx mori and Bombyx mandarina show that little difference exists between them.3. Sequence comparison of alpha-amylase gene of five insects.The nucleotide sequence similarity and the putative amino acid sequence similarity of alpha-amylase gene of Bombyx mandarina to those of Bombyx mori, Spodotera fruiperda, Ostrinia nubilalis on 25 and Drosophila subobscwa are 90%, 12%, 12%, 5% and 98%, 80%, 78%, 58% respectively. Those results indicate that Bombyx mandarina and Bombyx mori are closely related, while Bombyx mandarina and Drosophila subobscwa are relatively far related.4. Base composition comparison of codons of the alpha-amylase gene of Bombyx mori and Bombyx mandarinaIt was shown by base composition comparison of the codons that it is very similar in both Bombyx mori and Bombyx mandarina. The first base of codons clearly shows a bias for purines (59.90% for Bombyx mandarina and 5更多还原