【作者】 何余堂；

【导师】 傅廷栋； 涂金星；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 白菜型油菜原产我国，不仅是重要的油料作物，也是重要的蔬菜作物。我国白菜型油菜的遗传资源丰富，具有许多优良特性，如耐瘠薄，耐干旱，抗寒性强等。利用芸薹属基本种和复合种之间的杂交，有可能将长角果、黄籽、多室及高含油量等有利性状转育到甘蓝型油菜中，丰富甘蓝型油菜的遗传背景。 本研究选取我国172份白菜型油菜种质资源，利用RAPD技术研究其遗传多样性。根据形态和RAPD分析结果，剔除部分同类型资源；利用MEGA(Molecular Evolutionary Genetic Analysis)软件，分析保留下来的部分资源，研究其起源与进化。在资源鉴定中发现了特异种质：多室油菜和自交亲和油菜。取不同发育时期的多室角果，进行解剖学研究。利用同源序列法(Homologue sequencing)和PCR步行法(PCR Walking)克隆自交不亲和及自交亲和油菜的S-locus上的SLG和SRK基因；通过DNA及氨基酸序列比较，分析自交亲和性形成的原因。主要研究结果如下： 1 我国白菜型油菜的遗传多样性 1．1 我国白菜型油菜分为15个类群：6个类群为北方小油菜，8个类群为南方油白菜，还有1个混合类群。 1．2 我国白菜型油菜的遗传多样性与地理分布密切相关，且白菜型春油菜的多样性水平高于冬油菜。 1．3 来自云南、贵州和湖北省的地方品种，以及来自青海、甘肃和新疆的地方品种的遗传变异较大，比其它地方品种表现出更高的遗传多样性水平，这可能与白菜型油菜的起源有关。 2 我国白菜型油菜的起源与进化 2．1 结合形态标记与RAPD分子标记构建的系统进化树，揭示出白菜型油菜在我国的起源与进化关系，所得出的推论与前人的研究结果是一致的，并有所深入。而且，基于形态数据、RAPD数据及综合数据产生的三个进化树，所揭示出的白菜型油菜的系统发育关系基本上是吻合的。 2．2 北方小油菜的起源早于南方油白菜，冬油菜的起源早于春油菜。陕西可能是北方小油菜的起源地；而南方油白菜起源于云南、贵州、湖北、四川等地。南方油白菜的栽培和驯化较晚，其形成也晚于北方小油菜，但由于南方的地理和气候多样，形成了多种类型的油白菜品种。 3 白菜型多室油菜的遗传与解剖学研究 3．1 通过对多室油菜不同发育时期的雌蕊子房的横切切片观察，发现多室油菜的子房中有12～16个心皮维管束。子房在发育过程中，心皮束连成一个圆环，之后房室开始分化，胚珠和假隔膜同时发生，逐渐形成三室、四室和胚珠、胚囊；然而，一些子房虽然有3～4个隔膜，但其中部分却表现退化，最后发育为两室。两室油菜的子房中只有8个维管束，心皮束连成圆环，发育为两室，形成两室角果。 3．2 多室角果性状受一对隐性基因控制(mcmc)，并受环境因素的影响。 4 SLG和eSRK基因的克隆与比较研究华中农业大学博士学位论文20034.1利用同源序列法克隆s-locus上的eSRK和孔G基因。‘青海大黄’、‘黄籽沙逊’ 和‘关中油白菜’的SLG基因片段的大小均为882bP(GenaBank登陆号分别为: 灯445026，AY448028，AY44so3o);而e朋尤的大小分别为915bp(‘青海大黄’， AY448oZs)，914 bp(‘黄籽沙逊’，AY448027)和948bp(‘关中油白菜’， AY448029)。利用PcR~walking技术克隆eSRK和SLG基因的两翼序列。克隆到 的esRK的上游序列为468帅，下游序列分别为1 56bP(‘青海大黄’)，1 56 bp(‘黄 籽沙逊’)，358bP(‘关中油白菜’)。经过拼接，得到eSRK的全长片段分别为 1495 bp(‘青海大黄’)，1494 bp(‘黄籽沙逊’)和1727 bp(‘关中油白菜’)。没 有得到SLG基因的两翼序列。4.2经GENESCAN分析，发现‘青海大黄’、‘黄籽沙逊’和‘关中油白菜’的eSRK 的编码序列分别为一275 bp，999帅和1 3 1 1 bp。4.3‘青海大黄’和‘黄籽沙逊’的自交亲和性的遗传是一样的，自交不亲和对自交 亲和为显性。克隆的SLG和。占天犬基因分别与甘蓝和白菜型油菜的SLG和SRK 基因具有很高的同源性。SLG和eSRK蛋白中的保守区域与芸蓝属SLG和esRK 中保守区域的大小、位置均相同。说明本研究所克隆的SLG和eSRK基因位于 s-loeus上。4.4对白菜型油菜‘关中油白菜’，‘青海大黄’和‘黄籽沙逊’的SLG基因序列、氨 基酸序列、蛋白质结构及保守区域进行分析，结果表明:三个品种的SLG基因 的DNA序列、SLG蛋白的氨基酸序列、一级结构、二级结构和保守Domain的 差异较小:而eSRK基因及其编码的esRK胞外Domain(extra.celtular SRK，or eeto一SRK，eSRK)蛋白的氨基酸序列、结构却存在很大差异。4.5与自交不亲和的‘关中油白菜’相比，‘黄籽沙逊’在eSRK的DNA序列、esRK 的氨基酸序列上有很大差异，缺失了104个氨基酸残基，其中包括缺失了12个 保守的半肤氨酸中的8个，使eSRK失去了蛋白质结合域PAN一AP，无法识别 SC侧SPll蛋白，不能激活SRK激酶Domain，导致自交亲和。4.6青海大黄在eSRK的DNA序列、esRK的氨基酸序列上，与‘关中油白菜’比较 也有很大差异，其氨基酸序列上有几个小片段的缺失，分别在第299个氨基酸， 第30今308处，第32企334?更多还原

【Abstract】 Brassica campestris originated in China. It is well used as an oil crop and also a vegetable. There are rich germplasm of B.campestris in China. B.campestris has several elite traits such as tolerance to poor soil and drought, resistance to coldness etc. These good traits including long pod, yellow-seeded, multi-chamber and high oil content etc. can be transfered into B.napus by interspecific hybridization to improve target traits of B.napus and enrich its genetic background.In this study, 172 germplasm selected from the previous collection were used to study the genetic diversity of B.campestris in China by RAPD technology. Based on the results of morphological identification and RAPD analysis, a part of collection with similar genetic background was removed in the further study. The left 82 varieties were employed to study the origin and evolution of B.campestris in China using MEGA software (Molecular Evolutionary Genetic Analysis) . Multi-chamber and self-compatible B.campestris were discovered during the identification of germplasm. Buds were sampled from different developmental stages to carry out the anatomic study. Homologue sequence and PCR walking were employed to clone SZ-G and eSRK. The reason of self-compatibility was analyzed based on comparison of DNA and arnino acid sequences predicted between self-compatible and self-incompatible B.campestris. The main results are as follows:1 Genetic diversity of B.campestris in China1.15. campestris were separated into 15 groups: 6 groups from northern origins, 8 groups with southern origins and one mixture group.1.2 The origins of B. campestris in China were significantl related to geographical distribution. The diversity level of spring-type is higher than that of winter-type.1.3 The genetic variation of landraces from Yunnan-Guizhou-Hubei and Qinghai-Gansu-Xinjiang provinces is more than from other provinces because the genetic diversity of them is much higher than landraces from other provinces. This might be related to origin of B.campestris.2 Origins and evolution of Brassica campestris in China2.1 Based on morphological, RAPD and RAPD-morphological combined data, three phylogenetic trees were constructed and revealed origins and evolution of B.campestris in China. The results are in correspondence with previous study. The phylogenetic relationships of B.campestris revealed by three phylogenetic trees are coincident.2.2 It was showed that B.campestris var. oleifera originated earlier than B.chinensis var. oleifera. In B.campestris var. oleifera, winter type originated earlier than spring type. B.chinensis var. oleifera might originate in the area of Yunnan, Guizhou, Sichuan and Hubei. The evolution of B.chinensis var. oleifera was later than B.campestris var. oleifera, but different landraces formed because of southern geographical diversity.3 Anatomic study on multichamber character in B. campestris3.1 Anatomic structure was studied by investigation of ovary crossing sections in different developmental stages. In multichamber ovary, there are 12-16 carpel vascular bundles which link into a circle during carpel development. Afterwards, carpel chamber begin to differentiate. Ovule and pseudo-septum generate simultaneously separating the ovary chamber into tri-chamber or tetra-chamber. Some ovarys develop into bi-chamber because of degeneration of pseudo-septum. In contrast, there are 8 carpel vascular bundles in bi-chamber ovary. Ovule and pseudo-septum generate simultaneously separating the ovary chamber into bi-chamber, developing intobi-chamber pods. 3.2 The multichamber trait was controlled by one pair of recessive genes (memo) andaffected by environmental factors. 4 Cloning of SLG and eSRK genes and comparative analysis4.1 Homologue sequence method was used to clone S-locus gene: eSRK (extracellular SRK) and SLG. The fragment size of SLG of qinhuang, yellow sarson and guanzhong were 882 bp (GenaBank ID: AY448026, AY448028, AY448030). The eSRK of qinhuang, yellow sarson and guanzhong were 915 bp, 914 bp and 94更多还原