【作者】 杜周和；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 家蚕的起源与分化不仅是基础科学问题,也是有关蚕业发生历史和产业发展的重大实践问题,其研究不仅具有重要的理论价值,而且对家蚕的品种鉴定、基因资源利用、杂种育种等产业实践具有重要指导意义。自印度昆虫学家霍顿提出家蚕的种源问题后,逐渐引起各国学者的注意。自1915年日本学者青木清首先用血清沉淀反应研究家蚕与野蚕的亲缘关系始,近一个世纪以来,中外学者,特别是中日学者从文献学、气候学、古生物学、生态地理学、考古学、遗传学、生理生化学、细胞学、分子标记(RAPD、AFLP、SSR等)、DNA序列分析等多角度、多层次进行了广泛、深入的探索,取得重要研究成果。首先明确并达成一致共识:家蚕来源于野桑蚕,中国野蚕是家蚕的直系祖先,日本野蚕与中国野蚕存在较大的遗传分化,对家蚕的起源没有直接贡献;家蚕最早起源于中国,野蚕在中国被驯化成家蚕后向世界各地传播,各地地理气候和人文社会环境不同,经过长期的生殖隔离、自然选择和人工定向选育,逐渐形成了不同的地理系统和生态类型。但是,在家蚕的起源地域和分化模式上,不同学者使用不同的技术手段、实验材料和分析方法,得出不同的结论,影响较大的主要有三种观点:一是以日本学者吉武成美教授为代表,基于家蚕地方品种多种酶系的同工酶多态性提出的“一化性一中心学说”;二是以我国学者蒋猷龙教授为代表的“多化性多中心学说”,其学术依据主要是古生物学、古气候学和考古学资料:三是西南大学鲁成教授依据现代分子标记研究提出的“混化性起源学说”。DNA是直接的遗传物质,所提供的遗传信息比其它形态标记、生化标记更加准确可靠。DNA序列分析通过测定核酸一级结构中核苷酸组成和排列顺序来比较同源分子之间的相关性,其揭示的遗传信息比其它分子标记更加丰富和详细,在生物的起源进化研究特别是短期驯化物种的起源进化研究上具有明显的优势,是目前分子进化及系统发育研究最有效、最可靠的方法。家蚕驯化时间短,且研究的是物种内不同类型间的亲缘关系,特别需要一个快速进化的基因作为检测标记。淀粉酶在猪、绵羊、家蚕及果蝇等多个物种中表现出较高的同工酶谱多样性及核苷酸多态性,广泛用于进化研究。本研究通过生物信息学分析、文献研究和序列测定确证Bmamy2是一个快速进化、多态性丰富的基因,且具有合适的基因结构,便于同时利用外显子和内含子携带的遗传信息进行综合分析,结论更加全面、可靠。实验以大量典型家蚕地方品种和有代表性的野蚕地理类型为材料,以Bmamy2基因为标记,通过DNA序列多态性探索家蚕的起源与分化模式,获得的主要结果如下:1.α-淀粉酶基因的生物信息学分析α-淀粉酶是一个与生命活动紧密相关的多基因家族,在不同物种中具有不同的基因结构和分布特点。在三刺鱼、非洲爪蟾、鸡以及人等脊椎动物中,物种间和物种内不同基因拷贝间具有保守的基因结构,分布在一条染色体上,存在较多的重叠基因;物种内不同基因拷贝间氨基酸序列相似性极高,物种间序列相似性低。而在果蝇、蚊子和家蚕等昆虫中,α-淀粉酶基因没有类似的保守结构和重叠基因,不同拷贝以串连形式分布在多条染色体上:物种内和物种间不同基因拷贝间序列相似性较低。在秀丽隐杆线虫等低等生物基因组中只有一个α-淀粉酶基因拷贝,在昆虫、哺乳动物等高等生物中发生了显著的基因扩增,基因数量明显增加。脊椎动物中各淀粉酶基因严格按物种聚类,物种间没有交叉,推测基因扩增发生在物种分化之后。昆虫中,α-淀粉酶基因起源进化关系复杂,蚊子和果蝇同属双翅目,但基因拷贝数相差很多,表明两物种分化形成后蚊子中发生了显著的基因扩增;嗜凤梨果蝇和黑腹果蝇同属果蝇科,亲缘关系很近,在两物种分化形成后,嗜凤梨果蝇又发生了基因扩增,形成相对较多的基因拷贝。蚊子和家蚕虽然分属不同的目,但两者的α-淀粉酶基因却表现出较为相似的分化模式;各物种中α-淀粉酶基因都聚类成彼此间遗传差异较大的两族,其中一族与细菌具有较近的遗传距离;物种分化形成后发生了基因扩增事件,形成较多的基因拷贝。家蚕Bmamy2、Bmamy3、Bmamy4三个基因在同一染色体上紧邻着串连排列,且具有及其相似的基因结构,可能通过基因重复而产生,但三个基因的核苷酸序列差异显著,推测三基因经受了不同的进化选择。2.家蚕Bmamy2基因的克隆、表达及序列分析克隆测序了家蚕Bmamy2基因编码区序列,是家蚕基因组中第一个有实验证据的淀粉酶基因序列。家蚕Bmamy2基因CDS全长1752bp,除起始密码子外,编码583个氨基酸,有8个内含子,其中第一、第六、第七和第八内含子较长,分别为393bp、505bp、647bp和1828bp,其余均在100bp左右;除第四外显子较长外(582bp),其余外显子均小于100bp;外显子/内含子边界符合GT-AG规则。芯片数据和RT-PCR检测一致表明Bmamy2在家蚕幼虫中肠和脂肪体中表达,且中肠中表达量极高,脂肪体中表达量较低,在头部、表皮、血液、丝腺、精巢和卵巢等组织中不表达。功能结构域预测发现催化中心的两个关键氨基酸发生突变(D258Y,D423P),该酶可能已失去催化水解碳水化合物的功能。Bmamy2基因在细菌等微生物中存在直系同源基因,碱基变异丰富,家蚕地方品种中碱基突变率5.6%,野蚕中碱基突变率8.3%,遗传多样性高,没有明显的碱基使用偏好和特别长的内含子间隔,是家蚕多样性检测较好的标志基因。3.家蚕的种群结构及遗传分化虽然按地理系统和生态类型取样,但系统发育分析并未检测到按系统或生态类型聚类的结果,分子方差分析也未检测到按系统或生态类型归类的组群结构。90个家蚕地方品种聚类成三大簇群,每一簇群均由来自不同系统和生态类型的品种构成,归属同一地理系统或生态类型的品种分布在不同的聚类枝上。三大簇群间存在较大的遗传距离,簇群内不同品种间遗传差异小。推测三大簇群分别来自三个独立的驯化起源。4.野蚕的多样性及地理种群结构野蚕是一个在东南亚地区尤其是中国广泛分布的物种,具有一化性、二化性及多化性等多种生态类型,在眠性上有三眠蚕、四眠蚕及少数的五眠蚕。野蚕的多样性在形态特征、生态类型、发育生理、食性、抗性及多种分子标记等各个方面和层次得到充分体现。基于Bmamy2基因部分区段的序列分析,不同个体间碱基突变率高达8.3%,内含子区域更高达10.8%,单倍型多样度h=0.987±0.023,核苷酸多样性π=0.02281±0.0034。野蚕的生态学特征之一是群体发育极不整齐,典型的现象是越年卵孵化的长期化和蛹期发育的多样化,发育早的已完成一个世代,而发育迟的尚未出壳,造成世代重叠。分子方差分析显示,野蚕没有形成稳定的地理组群结构,彼此间亲缘关系的疏密与空间距离的远近没有必然联系。聚类分析中,来自不同地区的野蚕同聚在相同的聚类枝上,表现较近的亲缘关系;而同一地区的个体却分布于不同聚类枝上,亲缘关系较远。各地野蚕是多种眠性和生态类型混栖的混杂群体。5.家蚕的起源与分化基于Bmamy2基因的序列多态性,应用系统发育分析、Network分析、AMOVA分析等多种分析方法,检测到一致的系统发育模式。90个家蚕和野蚕样本混合聚集成不同的聚类簇群,每一簇群都同时由家蚕和野蚕组成,家蚕来自不同的地理系统和生态类型;簇群间具有较大的遗传距离,簇群内的不同品种间遗传差异较小。不同系统或生态类型的家蚕品种具有相似的分化程度,与野蚕间具有相似的遗传距离,应当具有相似的驯化起源时间,经历了相似的分化历程,而各地野蚕又是多种类型的混杂群体,推测从野蚕到家蚕的驯化当是由多种类型的野蚕同时开始的。聚类簇群间较大的遗传距离表明不同簇群可能具有不同的起源背景和进化经历,来源于不同的地理区域。即,家蚕很可能是在几个不同地域由多种生态类型混杂的野蚕驯化而成的,起源之初就有多种化性的遗传背景,家蚕的起源为“混化性多地域”模式。多种化性的家蚕向世界各地传播,适应当地的自然气候和人工选择,逐渐分化形成不同的地理系统和生态类型。更多还原

【Abstract】 With important theory value and practice guidance to variety identification,resource using and hybrid vigour application,it is an important basic science subject about the sericulture history and sericultural industry production to study the origin and differentiation of silkworm,Bombyx mori.A lot of researchers from many countries have been attracted by the subject of the origin of silkworm since Indian entomologist,Hutton,pointed out it.1915,Japanese scholar,Qing mu qing,studied the relationship between B.mori.and Bombyx mandarina with blood serum-sediment reaction method, after that in about a centry,many researchers from internal and foreign,especially from China and Japan had made many efforts and obtained a lot of results by using various methods,such as bibliography,climatology,palaeobiology,bioecology,geography,archaeology,genetics,physiology, biochemics,cytology,molecular markers(RAPD,AFLP,SSR,etc),DNA sequence analysis,etc. They made a general approval that B.mori originated from Chinese Bombyx mandarina and were bred into different geography systems and ecological types by nature and artificial selection in the other world after spread from China,but there has long been dispute about where and how B.mori originated at the first time.There are three main hypotheses.The first is "univoltine and one centre" hypothesis pointed out by Jiwu based on the isoenzyme polymorphism of several enzymes.The second is the "multi-voltine and multi-centre" theory pointed out by Prof.Jiang based on the information of palaeobiology,climatology,and archaeology.The third is the "mix-voltine" theory pointed out by Prof.Lu based on the research using molecule markers.DNA is true genetic material which can provide more accurate genetic information than morphological and biochemical markers.Molecular phylogenetic analysis based on DNA sequences is the best effective and reliable method for systematics of organisms especially for those domesticated species.However,to investigate phylogenetic relationships among closely related species,it is necessary to look for a rapidly evolved gene.In this study,I sequencedα-amylase gene of different silkworm strains and different B.mandarina populations to study the origin and differentiation of domesticated silkworm,B.mori.The major results are as follows1.The bioinformatics analysis of theα-amylase geneα-amylase gene is a multi-gene family with improtant function for animals.Different species have differentα-amylase gene structures and corresponding distribution patterns in respective genomes.In some vertebrate,such as Gasterosteus aculeatus,Xenopus tropicalis,Gallus gallus and Homo sapiens,there are many overlapping genes and the a-amylase genes from different species have conservative structure,the different gene copies intra-species resemble in amino acid sequence extremely.But in some insects,such as Drosophilidae,Culicidae and B.mori,there is no overlapping gene and the genes have no conservative structure,the different gene copies within-species differ in amino acid sequence greatly.Theα-amylase genes differentiate extremely and display rich polymorphism in Culicidae and B.mori.There is only one copy ofα-amylase gene in C.elegans genome,while the gene family enlarged greatly in higher animal such as insect and mammal.The different gene copies intra-spesies cluster together respectively suggesting the gene copied after the species split in vertebrate,but in insect the clusters consist of gene copies from different species suggesting the gene copied before the species split.Further,theα-amylase gene families enlarged greatly after species split in Culicidae and B.mori.The gene copise from Culicidae and B.mori cluster into two clustes,one of which keeps close relationship with bacteria.Three of B.moriα-amylase genes with similar gene structure distribute in the same chromosome implying the copy relation.2.The cloning,expression pattern and sequence analysis of Bmamy2 gene in silkwom,B.mori.Bmamy2 CDS is 1752bp long and codes for 583 amino acids.There are 8 introns following the "GT-AG" rule in Bmamy2 gene and the first intron is 393bp long,the sixth 505 bp,the seventh 647bp,the eighth 1828bp,the others about 100bp;and the fourth exon is the longest,582bp,the others are short than 100bp.The Bmamy2 gene is expressed highly in midgut,lowly in fat body while not in head,epidermis,hemolymph,silk gland,testes,ovary inspecting by RT-PCR and microarray.It may lose the function to catalyze hydrolysis carbohydrate caused by the two key amino acid mutations in the catalysis centre(D258Y,D423P).The Bmamy2,which has orthologous gene in bacterium,is a fine marker to investigate polymorphism in silkworm.Our results suggested that nucleotide mutation rate is 5.6%in B.mori and 8.3%in B.mandarina.3.The population structure and genetic differentiation in B.mori.Although we initially sampled the silkworm according to ecological type classification,we failed to identify ecological type-specific clades in the study.Three clades were identified in 72 local strains,each of which consists of strains from different ecological types while the same ecology type strains distribute in different clades.There is great genetic diversity among clades and small diversity within clade among strains.It is presumed that several domestication events took place at different sites independently to make mix-voltine B.mandarina into mix-voltine B.mori.4.The geographic population structure and polymorphism in B.mandarinaThe B.mandarina is whidespread in Southeast Asia especially in China with multiple voltine of univoltine,divoltine and multi-voltine,and multiple molts of three molts,four molts and fewer five molts.B.mandarina has big polymorphism in morphological and ecological traits,development, food habit,resistance and molecular marker.Based on partial sequence of Bmamy2 gene,the nucleotide mutation rate is 8.3%in entire sequence and 10.8%in intron sequence,the haplotype diversity is 0.987±0.023,the nucleotide diversity is 0.02281±0.0034.Overlapping generation is one of the most popular ecology characteristic to B.mandarina for eggs hatching chronically and pupa development diversify,some have completed a generation while some did not hatch.It is a method to survive hard condition and keep up hybrid vigour avoiding sib mating.AMOVA indicates that being of mix-populiation of several ecological types and molts;there is no geographic population structure in B.mandarina anywhere.The genetic distance is not correlated with space distance.On the phylogenetics tree,the individuals from different regions clustered in the same clade,while the individuals from the same region distribute in different clades.5.The origin and differentiation of silkworm,B.mori.The same phylogenetic pattern is revealed by several inspections such as phylogenetics analysis, network analysis,AVOVA analysis,based on the nucleotide sequence polymorphism of Bmamy2 gene.90 samples of B.mori and B.mandarina clustered into four clades,each of which consists of B. mori from different ecological types and B.mandarina from different regions,with great genetic diversity between clades and small diversity between strains within clades.The domestication should start at multi-type B.mandarina because the B.mandarina are mix-typed populations anywhere and the silkworm strains in different ecological types might experience the same evolutionary process.The great genetic diversity between clades reveals the different geographic origin of silkworm in different clades.In summary,our results suggest that the silkworms originated from mix-typed B.mandarina in several different regions.更多还原