【作者】 刘东；

【导师】 朱利泉；

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

【摘要】 芸薹属植物(Brassica)是我国栽培面积最大，产量最高的一类蔬菜与油料作物，在我国蔬菜和油料生产和供应中占有极其重要的地位，芸薹属作物也是杂种优势利用最为普遍的一类作物，其自交不亲和性的分子机理和雄性不育系的选育及其应用基础的研究深受人们重视。本文通过PCR和RT-PCR等一系列分子生物学方法克隆了芸薹属植物中的甘蓝和油菜自交不亲和信号传导过程中SRK底物蛋白基因ARC1、THL1和THL2，并使用各种相关生物信息学软件对SRK底物蛋白基因序列进行了分析，然后在Internet网上利用在线软件对蛋白质的结构和功能进行了预测和探讨，以期为芸薹属植物自交不亲和性的分子机理的研究提供新的内容。主要研究结果如下： 1．以甘蓝和油菜为材料，通过各种提取方法的比较分析，我们对提取植物基因组DNA和总RNA的CTAB法进行了改良，并取得了很理想的效果。 2．以甘蓝(200110197)和油菜(Y578-2)自交不亲和系的植株为材料，提取幼苗中的gDNA及花蕾期的柱头、子房、花瓣、叶片、根的mRNA，并对mRNA进行反转录合成cDNA，然后分别以gDNA与cDNA为模板扩增ARC1蛋白编码序列。将从gDNA与柱头cDNA中扩增出的产物测序。序列同源分析表明：在甘蓝中克隆的ARC1Boler基因与油菜的ARC1基因序列的同源性为94％。gDNA和cDNA的扩增产物的测序结果表明，在甘蓝中，ARC1蛋白编码序列只有一个单一的开放阅读框，无内含子存在。氨基酸序列的同源性分析表明，甘蓝的ARC1Boler基因编码的氨基酸序列与油菜和拟南芥的同源性分别为85％和52％。序列分析还表明在ARC1Boler、ARC1和ARC1At基因编码的蛋白质中均存在两种结构，Ubox结构和臂重复结构。这种Ubox结构均位于氨基酸序列的中间区域，这个区域有可能是蛋白质发生二聚化的区域或下一级组分的结合的位点。在ARCIBoler和ARC1基因编码的蛋白质C端存在5个臂重复区段，而在ARC1At基因编码的蛋白质中只有1个。这种差异的存在，也可能是ARC1At与ARCIBoler和ARC1功能不同的根本原因。酶切分析也表明在甘蓝和油菜中ARC1蛋白的C端区域有可能是ARC1蛋白的保守结构域。同时在ARC1蛋白质中还发现了拉链结构和多个磷酸化位点，包括cAMP和cGMP依赖的蛋白激酶磷酸化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、酪氨酸激酶磷酸化位点、糖基化位点等，拉链结构为ARC1蛋白之间及与其它蛋白的相互作用提供了可能，而磷酸化位点是ARC1参与信号传导过程所必需的。在预测的ARC1蛋白质高级结构中，发现的大沟和小沟可能为其它蛋白质的结合提供了结合位点。 3．采用PCR和RT-PCR技术，从甘蓝和油菜基因组DNA和柱头总RNA中扩增获得了719bp的THL1基因和430bp的cDNA序列。序列分析首次表明，克隆的THL1基因有两个内含子，大小分别为207bp和82bp，碱基完全相同，cDNA序列编码117个氨基酸。酶切分析表明在THL1基因序列中各有一个BamHⅠ、HindⅢ、SacⅠ和SalⅠ酶切位点。甘蓝和油菜THL1基因序列有5个碱基的差异，而氨基酸仅有2个氨基酸的变异，同源性达99％。在THL1蛋白的氨基酸序列中发现了一个硫氧还蛋白家族的活性位点(CPPC)和多个磷酸化位点。尽管在自交不亲和中磷酸化位点可能没有发挥作用，但它们有可能参与其 西南农业大学硕士学位论文 摘 要它的生物学反应。 4．采用阿R和RTPCR技术，从甘蓝和油菜基因组和柱头总则A中扩增获得了THLZ基因，基因组中得到的基因大约为900hP和850hP，CDNA大约为477hP。在THLZ基因中首次发现了两个内含于，并且甘蓝和油菜THLZ基因的内含于序列同源性低于ICh，并且大小也有差异。甘蓝和油菜THLZ蛋白质中有5个氨基酸的差异，同源性为97％。酶切分析发现只在油菜 THLZ的第151个碱基处有一个Bgl酶切位点，而在甘蓝中没有发现一个常见的DNA限制性内切酶位点。在THLZ蛋白的氨基酸序列中也发现了一个疏氧还蛋白家族的活性位点和多个磷酸化位点。 5．以 2．5 u g的总 RNA为模板，进行定量 RT－PCR，结果分析表明 AM基因在甘蓝中的表达特异性和油菜一样，均是在花柱柱头表达的。THLI＄1 THLZ基因一样，在甘蓝和油菜各组织中均是组成性表达的。 本文对上述结果进行了必要的分析与讨论，阐明了它们在植物信号传导研究中的意义，并初步提出了下一步深入研究的方向与内容。更多还原

【Abstract】 Brassica crops occupy the largest cultivated areas and produce the highest yields among the vegetables and oil crops in our countryside. It is one of the most prevalent crops in the utilization of heterosis and have been regarded as model plants in the process of researches on their molecular mechanism of self-incompatibility and breeding and application of male sterility.ARC1, THL1 and THL2, the substrate protein genes of S receptor kinase, were cloned through a series of methods of molecular biology such as PCR, RT-PCR, DNA cloning and sequencing. The resultings sequences were highly analysed by using the related biosoftwares on Internet, providing new insights in the field of the molecular mechanism of self-incompatibility in plants. The major results are as followings:1 . The method of CTAB was specifically improved for the extraction of genomic DNA and total RNAs from Brassica oleracea L. and Brassica napus L.2. ARClBoler gene was amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea L.(200110197) and Brassica napus L.(Y578-2). The DNA sequencing shows that ARClBoler gene shows 94% identity to the sequence of ARC1 gene of Brassica napus L.. There is only an 1983 bp ORF in ARClBoler gene that encodes 661 amino acids. The amino acid sequence shows 85% and 52% identity to those of ARClBnap gene and ARC1At gene respectively. These three proteins contain two common clif domains, Ubox and Arm Repeats. Ubox is putative domain of dimeration of ARC1 protein or the binding domain of next element. In the C-terminal of ARClBoler protein and ARClBnap protein, there are five arm repeats, in contrast to only one in ARClAt protein. This difference potentially determines the discrepancy of function of ARClBoler, ARClBnap and ARClAt. The enzyme digest analysis shows that the arm repeats of C-terminal are conceivably conservative domain. In ARC1 protein, there are some active sites including N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, Tyrosine kinase phosphorylation sites, N-myristoylation sites, amidation sites and Leucine zipper pattern. It probably take part in the signaling process of self-incompatibility. From the three dimensional structure of ARC1 can be found a wide groove and a narrow groove, which may be the binding sites of other proteins.3. THL1 gene was also amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea L.(200110197) and Brassica napus L.(Y578-2). The DNA sequencing shows that THL1 gene occupies a fragment of 719 bp in gDNA, which occurs in cDNA with a length of only 430 bp. The alignment analysis between gDNA sequence and cDNA sequence of THL1 gene shows that THL1 gene contains two introns of 207 bp and 82 bp respectively. Its cDNA encodes 117 amino acids. In THL1 gene, there are some famililar enzyme digest sites including BamHI, Hind , Sac I and Sal I .The sequence of THL1l from Brassica oleracea L. shows 99% identity to the sequence of THLl from Brassica napus L. There are some phosphorylation sites and an active site of thoiredoxin h members consisting of CPPC in THLl protein. Although these phosphorylation sites are silent in the signaling of self-incompatibility, it possibly play roles in other biological processes.4. THL2 gene was finally amplified by PCR with template genomic DNA and cDNA from roots, leaves, petals, stigmas and ovaries during bud stage in Brassica oleracea l.(200110197) and Brassica napus L.(Y578-2).The DNA sequencing result shows that the genomic gene of THL2 is approximately 900 bp from Brassica oleracea L. in contrast to that of 850 bp from Brassica napus L.. The cDNA of THL2 from Brassica napus L. is 447 bp. THL2 gene contains two introns with wide divergences between Brassica oleracea L. and Brassica napus L.. However, the deduced amino acid sequence of THL2 protein f更多还原