【作者】 斯钦巴特尔；

【导师】 哈斯阿古拉； 张辉；

【作者基本信息】 内蒙古大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 亚麻(Linium usitatissimum L.)是我国北方地区重要的油料作物和纤维作物或油纤兼用作物。亚麻籽和亚麻籽油具有保健和药用功效。亚油酸具有降低血液中的胆固醇的作用,而α-亚麻酸是生成二十碳五烯酸(eicosapentaenoic acid,EPA)和二十二碳六烯酸(docosahexaenoic acid,DHA)的前提物质,EPA和DHA的适当的混合物具有降低血脂和减少血栓形成的作用。亚麻木酚素具有调节人体激素平衡、抑制癌症等功效。近年来,亚麻的保健、医药功能不断得到开发,对优质品种的需求不断提高。但由于亚麻是自花授粉作物,其遗传变异的扩大和杂种优势的利用却受到限制。因此,亚麻花药、花粉发育过程中有关基因及其启动子的研究对获得基因工程亚麻雄性不育系及亚麻杂种优势的利用具有重要的意义。本文采用同源序列克隆法,从亚麻花蕾中克隆出与拟南芥雄性不育基因-MS2同源的亚麻MS2-F基因并对其进行了序列和表达分析以及蛋白质结构和功能预测。构建MS2-F基因RNAi表达载体,通过农杆菌介导法进行亚麻的遗传转化。同时,开展了显性雄性核不育亚麻可育、不育花蕾mRNA差异表达以及差异表达基因片段序列分析和功能预测等研究,为揭示显性雄性核不育亚麻不育分子机理提供了有价值的线索。研究结果如下:(1)根据拟南芥等其他植物中雄性不育相关基因氨基酸序列的保守性,应用同源序列克隆法,首先在亚麻花蕾中扩增出258bp的一段序列,然后以该序列为基础,通过RACE技术扩增了基因的5′、3′端序列,其大小分别为975bp和869bp。拼接后获得了1911bp的cDNA序列。(2)根据该cDNA序列,设计基因特异引物,扩增基因的全长cDNA和gDNA。测序结果表明,cDNA为1709bp,gDNA为2696bp,序列分析结果显示,该基因含有8个内含子和9个外显。(3)该cDNA中包含一个1608 bp的ORF,编码535个氨基酸,5′非编码区70bp,3′非编码区31bp。推导的蛋白质序列中包含两个雄性不育保守区:NAD结合区域和雄性不育C-末端区域。该基因与油菜、拟南芥雄性不育基因的一致性分别为59.65%和59.16%,为花蕾特异表达基因,推测在亚麻花粉发育过程中与脂酰辅酶A还原酶有相似功能。因此,成功获得了亚麻雄性不育相关基因,将其命名为MS2-F。其cDNA序列及其对应的gDNA序列的基因库登录号分别为EU363493和EU365361。(4)构建了两个MS2-F基因的RNA干扰载体,一个含有该基因非雄性不育保守区序列;另一个含该基因雄性不育保守区序列。并利用农杆菌介导法,将构建的MS2-F基因的RNA干扰载体导入亚麻中,获得了卡那霉素抗性再生苗,现在正在进行生根诱导培养,有望获得抑制MS2-F表达的转基因亚麻植株,为研究MS2-F基因的功能奠定基础。(5)在显性雄性核不育亚麻不育花蕾和可育花蕾mRNA差异显示研究中,共获得有或无差异片段42条。其中29条为不育差异片段,其余的13条为可育差异片段。对这42条差异片段进行反向Northern杂交验证,最终获得了5条不育花蕾阳性片段:G—E07—100、G—E07—330、G—E07—830、G—S273—500、A-S267—300;2条可育花蕾阳性片段G—S274—250、G—S274—450。(6)这些阳性片段经克隆、测序和BLAST序列分析,结果显示:G-E07-100与植物GTP结合蛋白高度同源,在细胞信号转导过程中有调节作用;G-E07-330与已公布的亚麻花蕾的一段cDNA有较高的同源性;G-E07-830与β—木糖苷酶同源性很高,它可能影响了亚麻花药、花粉中碳水化合物的代谢;G-S273-500与NAD+ADP核糖转移酶有很高的同源性;A-S267-300与分泌蛋白高度同源,推测它可能参与了核不育亚麻信号传导途径、形态发生、细胞凋亡等过程,最终影响了雄性不育的发生;G-S274-250和G-S274-450与已知植物的核苷酸碱基序列和氨基酸序列同源性很低,推测它们可能是与育性有关的新基因。(7)将G-E07-100、G-E07-330和G+E07+830阳性差异片选定为3个候选基因,并经过Northern杂交表达分析,进一步证明,这3个基因在不育花蕾中特异表达。更多还原

【Abstract】 Flax ( Linium usitatissimum L.) is one of the important crops in the northern region of China for oil and fiber. Flaxseed and flaxseed oil have health function and medicinal effects. Flaxseed linoleic acid can reduce the cholesterol level in the blood andα-linolenic acid(ALA), an essential fatty acid that generates eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has a function of lowering blood lipids and reducing the formation of the thrombosis. Lignan in flaxseed plays a role in the prevention of cancer. In recent years, more and more wealth and medical function of flaxseed has been developed, demanding more quality varieties. But the expansion of genetic variation and the use of heterosis have been restricted, as flax is a crop of self-pollinaton. So Researching genes and promoters related to pollen and anther development in flax has become significantly important for creating genetically modified male sterile flax line and heterosis utilization in production. In this study, male sterility related gene MS2-F, homologous to Male Sterility 2 (MS2) in Arabidopsis thaliana was isolated by sequence homology cloning method, and its sequence and expression analysis were carried out. Structure and function of the deduced protein were also predicted. Two plant expressing vectors of RNA interference (RNAi) were constructed, using different fragments of MS2-F, and were used in genetic transformation of flax, resulting in Kanamycin resistant regenerated plants. The mRNA differential expression was compared by mRNA differential display technology between male sterile and fertile flower buds of dominant genomic male sterile flax, providing some valuable information for the revealing of the molecular mechanism of male sterility in flax. The results are as follows:1. A gene conserved fragment of 258 bp was amplified in flax flower buds according to the male sterility related genes in other plants including Arabidopsis thaliana by sequence homologous cloning method. Then, 5’and 3’ ends of this gene were amplified based on the nucleotide sequence of the gene conserved fragment, by rapid amplification of cDNA ends technology. They were 975bp and 869bp respectively in size and a cDNA sequence of 1911 bp was obtained after splicing the sequences.2. A pair of gene specific primers was designed according to the cDNA sequence, and was used to amplify full length of cDNA and gDNA corresponding to the cDNA, The sequencing results proved that the complete cDNA and gDNA were 1709bp and 2696bp respectively. The results of sequence analysis revealed that the gene consisted of 8 introns and 9 exons.3. The complete cDNA contained an ORF of 1608 bp, which encoded a protein of 535 amino acids including two male sterile conserved domains: NAD-binding domain and male sterile C-terminal domain. MS2-F amino acid sequence identities to MS2Bnap in Brassica napus and MS2 in Arabidopsis thaliana were 59.65% and 59.16% respectively. The gene was exclusively expressed in flower buds and may act as an acyl CoA reductase during flax pollen development.In conclution, a male-sterility related gene, named MS2-F, was isolated in flax. The GenBank Accession Numbers of the cDNA and the gDNA are EU363493 and EU365361.4. Two RNAi vectors of MS2-F gene were constructed, one using a gene fragment that does not contain the male sterile conserved domains in the gene and the other using a gene fragment containing a piece of the male sterile conserved domain in the gene. By agrobacterium-mediated method, several kanamycin resistant regenerated plants were obtained. The construction of RNAi vectors and subsequent genetic transformation of flax were carried out in the hope of producing transformed flax in which the expression of MS2-F gene is interfered, and thus providing some useful clues for revealing the function of MS2-F gene during pollen development in flax.5. In the study of mRNA differential expression in male and fertile flower buds, seven positive differential fragments were confirmed by Reverse Northern Dot Blotting, and then sequence analysis of these fragments was carried out: 5 fragments were specific to male sterile flower buds: G-E07-100, G-E07-330, G-E07-830, G-S273-500, A-S267-300; the other 2 fragments were specific to male fertile flower buds: G-S274-250, G-S274-450.6. The positive fragments were sequenced and analyzed with BLAST. G-E07-100, homologous to GTP-binding protein of plant, may play an important role in signal transduction. G-E07-330 has a high identity with a known cDNA sequence that is specifically expressed in flax flower buds. G-E07-830, which is most homologous to the sequence of Beta-D-xylosidase, may influence carbon hydrate metabolism in flax anther and pollen, resulting in pollen abortion. G-S273 -500 is homologous to NAD+ ADP-ribosyltransferase. A-S267-300, homologous to a secreted protein, may play roles in such process as signal transduction, Morphogenesis and Apoptosis in male sterility flax and finally lead to male sterility. G-S274-250 and G-S274-450 had no significant similarity with any known genes in plants, so they may be newly found genes related to fertility in flax.7. G-E07-100, G-E07-330, G-E07-830 were selected as candidate genes. Expression analysis by Northern hybridization confirmed that they were exclusively expressed in the male sterile young flower buds of flax.更多还原