【作者】 何礼；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2002， 博士

【摘要】 豇豆(Vigna unguiculata (L.) Walp.)隶属蝶形花科(Fabaceae)菜豆族(Trib. Phasoleae DC.)菜豆亚族(Subtrib. Phaseolinae Benth.)豇豆属(Vigna Savi)，该属约有150个种。豇豆作为全球范围内最重要的豆类作物之一，广泛栽培于热带亚热带地区和部分温带地区如非洲、亚洲、南美洲、地中海盆地和美国南部。目前，全球豇豆种植面积至少在1250万公顷以上，年产量超过300万吨。豇豆是我国重要的常规蔬菜品种之一，常年栽培面积超过蔬菜种植面积的10％，达50万亩以上。 本文从GenBank中获取了豇豆属42个种的ITS序列，结合自己材料的测序结果，对豇豆属的系统学进行了初步研究。在此基础上，收集了分布于全国各地的76份我国栽培豇豆品种资源，采用RAPD、ITS序列分析对这些代表性的豇豆品种资源进行了遗传多样性分析。鉴于胰蛋白酶抑制剂在抗虫中的作用，还克隆了豇豆胰蛋白酶抑制剂基因。本文得到以下主要结果。 1、遗传多样性的研究方法应更多地基于序列分析。 在豇豆属，ITS序列分析用于种下的遗传多样性研究，分辨率较低；但ITS序列分析用于属下的遗传多样性和系统发育研究，是比较充分的。ITS这类基于序列测定的方法，最大的优点是完全可重复的，所获得数据具有可移植性，将是系统发育重建和遗传多样性研究的更加可靠的数据来源。 RAPD可以用于种下的遗传多样性研究。但基于随机引物的RAPD-PCR扩增，受实验中各种因素的影响较大，导致数据获得的重复性不是很好、且不可移植。因此只能作为一种辅助方法。从技术上讲，为保证实验条件的一 我国栽培虱豆的邀传多样性研究及其育种策略的探讨致性，应该一次PCR反应中对所有的研究对象进行扩增，并在扩增片段的电泳分离检测时尽量保持条件的一致。 2、虱豆属的遗传多样性较高。 42个种聚为三个类群。亚洲来源的聚为一支，它们的遗传距离较小，大多低于15％，遗传多样性较低，位于系统树的最上部，可能为后生起源的种；其中绿豆（U radthe）与黑吉豆（y m呷）间的距离最近，为 11％左右；赤豆（V angUleris）与赤小豆（V umbellata）间的距离最近，为 3．5％左右；而赤小豆与绿豆、黑吉豆间的距离为＊％左右，赤豆与绿豆、黑吉豆间的距离稍大，为12％左右；这四个种与盈豆种的距离较远，都在33％以上。美洲来源的聚为一类，它们的遗传距离较远，大多在 20％左右，遗传多样性较高，位于系统树的最基部，可能为较原始起源的种。另一分支位于树的中部，它们的遗传距离较远，大多在30％左右，遗传多样性较高，来源不明。 3、我国栽培更豆资源的遗传多样性很低。 所收集的76个我国栽培更豆品种资源，都属于直豆属直豆种下的同一个亚种。ITS序列分析表明，这些栽培更豆品种间的ITS序列变异很小，但同样显示出我国栽培觅豆遗传多样性低的特点。 对RAPD数据的聚类分析表明，76份虱豆材料中刀个聚为两大类，其中除去湿赤小豆外，所有短更豆品种聚在一起；而山豆、黑仁白豆角、去湿赤小豆分别聚为一类，它们与其他社豆品种的遗传距离较远。 基于RAPD数据和ITS序列分析所得到的遗传距离，表明我国栽培盈豆的遗传多样性很低。 4、去湿赤小豆和成更1号与更豆种的遗传距离较远。 ITS序列分析结果表明所收集的品种资源中去湿赤小豆不应该划入页豆种，划入赤小豆种（y umbellQta）可能更为合适。来自 RAPD的数据也支持将去湿赤小豆从觅豆种中划出的观点。 在CpTI基因的扩增过程中，发现在山豆、黑仁白豆角和去湿赤小豆三个觅豆品种中不能获得 CPTI扩增产物，这与上述结果一致；进一步支持将去湿赤小豆从虱豆种中划分出来的观点，同时表明山豆和黑仁白豆角的分类地位需要进一步深入、综合的研究来确定。 ITS序列分析表明，成觅1号与更豆种不能聚到一支。由于ITS序列分析在系统发育研究中非常可靠，关于成更1号的分类地位有待更进一步的深入研究来确定。 5、CpTI基因的序列分析和克隆。n 摘 要 对7个虱豆材料CpTI基因的序列分析表明，它们之间存在细微差异，但所编码的氨基酸序列变化很小。除饭豆外，其余6份材料的虹豆胰蛋白酶抑制剂的活性中心，即Lys仰26－Ser（）27及Afg（）53－Ser（S）5没有变化。 采用了基于生物信息学的基因克隆方法。首先利用GenBank数据库和生物信息学分析，对CpTI进行了分析，发现该基因无内含子；然后采用PCR方法直接从总DNA扩增得到唯一的扩增片段，测序表明该片段为CpTI基因的编码序列，据此直接将PCR扩增片段克隆：对克隆片段的进一步测序分析表明，该克隆片段确为CpTI基因。 基于上述结果，我们认为我国栽培虹豆的育种策略应该转移到扩大虱豆资源的遗传多样性上来。要大幅度提高我国栽培缸豆资源的遗传多样性，要采取以下措施。 第一，要采用各种遗传分析方法，尤其是要采用分子生物学和生物信息学的方法，对我国现有虹豆品种资源的遗传?更多还原

【Abstract】 Cotrpea, opa ungUicuIata (L.) Walp., belongs to genus nga in Fabaceae.There are about l50 species in genus nga. As one of the most pulse crops allover the world, it is currently cultivated in all tropical, subtropical areas and sometemPefate areas, such as Africa, Asia, South America, the Mediterranean Basinand the southem states of the USA. WOrid coWPea acreage under cuItivation isestimated to exceed l2l5 million hectares, with an annual production of over 3million tons world-wide. As one of normal imPortant vegetable in China, thepercentage of acreage under cultivation is over l0% of vegetable acreage everyyea p exceeding tO 33 thOusand hectares.In this study, the phylogeny of genus Vigna was investigated based on ITSsequence data of 42 species Which were obtained from GenBank and our practice.And then, the genetic diversity of 76 cultivated coWPea germplasm collecting fromall over the China was evaluated based on RAPD and ITS sequence data.Moreover, coWPea trypsin inhibitor(CpTI) gene was cloned from Chinesecultivated coWPea. The main results were shown as following.l. Low resolution was obtained in genetic variation investigation of differentcoWPea cultivated germplasm of nga unguicuIata based on ITS sequence data,While good results could be obtained in genetic diversity and phylogeny ofdifferem species of genus nga.The ITS sequences contain many variable sites and phylogenetical informativesites among related species of genus nga and phylogenetic relationships amongecies could be established succesfully. The topology of the strict consensus treeand Neighbordoining tree inferred from ITS sequence data shows that:42 species were clustered to three groups. The species from Asia clustered toone grouP and lied in the uppermost of the systematic tree Which suggested these; lvspecies may be speceis of origining later. The bootstraP value is l00%. The geneticdistance betWeen them was below l5% in most cases, which indicated the geneticdiversity among them was low Among them, the distance between U radtata andY mungo, between V angularItr and V umbellata, between V umbellata and Vradtata V mungo, bctween Y anguleris and V radha, Y mungo was abollt l1%,3.5%, 1 l%, l2% respectively Fndermore, the distance between the 4 species andY unguiCulata was faf, reaching to above 33%.The species from America clustered to one group and located in the base of thesystematic tree which suggested these species may be speceis of originating earlier-The bootstraP value is less than 50%. The genetic distance between tl1em wasabout 15% in most cases, Which indicated the genetic diversity level among themwas high.The other species, which origin were unknown, clustered to another group andlocated in the middIe of the systematic tree. The bootstraP value is 63%. Thegenetic distance between them was about 30% in most cases, which indicated thegenetic diversity level among them was higl1 too.All the 76 cultivated coWPea germplasm cbllected from China belong tongaunguiculata ssp. unguiculata. Because the variation level of ITS sequence withinthe 76 cultivars was very low ITS was not suitable for phylogenetic analysis at thelevel of cultivars in nga unguiculata ssp. unguiculata. But, it indicated that thegenetic diversity 1evel was very low among Chinese cultivated coWPea.In our collected germplasm, cultivar Qushichixiaodou must not be member ofmpna unguiculata, maybe it was more reasonable that cultivar Qushichixiaodoubelong to nga umbeIlata based on ITS sequence data. Furtheomore, cultivarChengiiang Nol was 11ot clustered to grouP nga unguiculala, whiIe clustered toY umbellata. But, it must be member of Y unguiculata based on morPhology. So,the taxonomic category of Chengiiang No. l need to be researched further.2. RAPD can be used ’in the study on genetic diversity at the level of specie.Among 76 coWPea cultivars, 73 of which were clustered to two large grouPs basedon RAPD data.更多还原