【作者】 周焘；

【导师】 郭三堆；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2008， 博士

【摘要】 棉花是具有战略意义的农作物,如何提高棉花产量、改善棉花品质,历来受到了研究者的高度重视。由于棉花的经济价值主要通过其生殖器官的发育好坏体现,因此对其生殖发育进行深入研究,将是实现培育高产优质棉花的重要突破口。本论文根据以往的研究,推断在紧密连锁且同在生殖器官中特异表达的arf1和nodulin-like基因附近存在着一个与生殖发育相关的基因簇。从这一推断着手,开展了相应的研究,以期能够深入认识棉花的生殖发育机理,为高产优质棉花的培育提供坚实的理论依据和物质基础。首先,根据已知的arf1和nodulin-like基因设计了三对PCR引物,通过PCR池筛法对棉花18R BAC文库进行了筛选,获得了两个包含这两个基因的BAC克隆G2-J-15和G128-K-15,通过酶切鉴定其插入片段大小分别为38kb和90kb。选取了插入片段较小的G2-J-15进行了全序列测序。通过对G2-J-15的全序列测序和相关生物信息学分析发现,在短短的38kb序列中一共分布了7个基因,平均每5kb就存在一个基因,说明该基因簇位于基因富集区。除了已知的arf1和nodulin-like基因外,通过基因结构预测和BLAST,还发现了未在棉花中克隆过的GhGolS(肌醇半乳糖苷合酶基因)、GhAPm(AP复合体μ亚基基因)、GhPsbP(PSII放氧复合体外周蛋白基因)和GhNeuF(中性β-呋喃果糖苷酶基因),此外,还发现了一个未在任何物种中发现过的未知基因。对这5个新基因进行了一系列的生物信息学分析,了解了它们的基因结构,对蛋白质的性质、结构和功能也有了一定的了解。对这些基因进行生物信息学分析,发现了一个非常有趣的现象,即GhGolS、GhAPm和GhNeuF在棉花基因组中的排列顺序和方向与其在拟南芥中同源基因的排列顺序和方向一模一样,甚至棉花GhAPm和GhNeuF基因之间间隔了两个基因,而拟南芥中与之同源的基因之间同样间隔了两个基因。该基因簇在结构上异常的保守性预示着该基因簇的功能可能也非常保守,对棉花的生长发育起着至关重要的作用。利用real-time PCR对这5个棉花中新发现的基因进行了时空表达研究,建立了各基因的时空表达模型。结果发现除GhPsbP基因由于是棉花PSII OEC的外周蛋白而在叶片中特异表达以外,其它四个基因都在棉花生殖相关组织中特异表达:GhGolS基因在花药和35dpa棉纤维中特异表达,GhAPm基因在30dpa棉纤维中呈优势表达,未知基因在花药中的表达量最高,GhNeuF基因在花药和25dpa棉纤维中的表达量最高。结合以前arf1和nodulin-like基因在棉花生殖器官中特异表达的研究结果,证实了该区域所存在的由7个基因组成的紧密功能基因簇正如前面所推论的那样与棉花生殖发育相关。通过反转录PCR克隆得到了GhGolS和GhAPm的cDNA,其全长分别为1816和1590bp。GhGolS前面的基因结构预测结果除了错将一段90bp的外显子预测为内含子外,其结果与GhGolS的cDNA基本一致,GhAPm的基因结构预测结果则和其cDNA克隆完全一致。针对GhGolS和未知基因构建了过表达载体,构建了沉默棉花GhGolS和未知基因的RNAi载体,构建了沉默拟南芥AtGolS2和中性β-呋喃果糖苷酶基因的RNAi载体,构建了同时沉默拟南芥针AtGolS2、AP复合体μ亚基基因和中性β-呋喃果糖苷酶基因的RNAi载体,将其分别转化棉花和拟南芥,获得了拟南芥的T1代转基因植株的种子,而针对T0代转基因棉花的筛选也在进行。各类转基因植株的获得为对该基因簇进行深入研究,了解其在棉花生殖发育过程中所起的作用,奠定了良好的基础。本论文发现了一段与棉花生殖发育相关的功能基因簇,该基因簇包含了7个基因且相互间紧密连锁,对其进行了一定的研究,为今后进一步深入研究棉花生殖发育的相关机理奠定了基础,也可能会为将来高产优质棉花的培育提供新的理论基础和解决方案。更多还原

【Abstract】 Cotton is a kind of strategic crop, it is always a focus that how to improve cotton yield and quality. The development of cotton reproductive organ directly affect the economic value of cotton, so studying cotton reproductive development in-depth is a very important breakout for breeding high yield good quality cotton. Base on the research ago, we know that arf1 and nodulin-like genes is very close to each other in genome, and they both express in cotton reproductive organ specificly, so we deduce that there is a gene cluster correlative with cotton reproductive development around arf1 and nodulin-like genes. Aiming at this problem, we carry on a series of researches, the target is understanding the mechanism of cotton reproductive development deeply, providing theory basis and physical foundation for breeding high yield good quality cotton.First of all, we designed three pairs PCR primers base on arf1 and nodulin-like genes, and use these primers to screen cotton 18R bacterial artificial chromosome library by PCR pool screen. At last, we got two BAC clones include this two genes, G2-J-15 and G128-K-15. Restriction endonuclease analysis shows that the inserting segments of these BAC clones are 38kb and 90kb respectively, and we finished the sequencing of G2-J-15 BAC clone.The sequencing and bioinformatics analysis of G2-J-15 show that there are seven genes in the inserting segment, average 5kb lies a gene, we can conclude this gene cluster locates in a gene rich area. Except for arf1 and nodulin-like genes, by gene structure prediction and BLAST, we found four gene never be cloned in cotton, include GhGolS(galactinol synthase gene), GhAPm(clathrin adaptor complexes medium subunit family gene), GhPsbP(photosystem II reaction center PsbP family gene) and GhNeuF (neutral beta-fructofuranosidase gene), and a new gene it never be cloned in any species. By a series of bioinformatics analysis, we had understood their gene structure and protein character, furthermore, we got some information about their protein structure and function. By these bioinformatics analysis, we found a very interesting phenomenon that the arrangement and direction of GhGolS, GhAPm and GhNeuF in cotton genome is the same with their homologous genes in Agrobacterium tumefaciens, even there are two genes between GhAPm with GhNeuF, and also there are two genes between their homologous genes in Agrobacterium tumefaciens. The conservative structure of this gene cluster maybe indicate the function of this gene cluster is conservative too, and the gene cluster is necessary for cotton development.We studied the spatio-temporal expression pattern of these gene by real-time PCR, the result indicates that except for GhPsbP expresses in leaf specifically, the other four genes all express in cotton reproductive tissues specifically: GhGolS expresses in anther and 35dpa fiber specifically, GhAPm is predominant in 30dpa fiber, the expression of unknown gene is highest in anther, and the expression of GhNeuF is highest in 25dpa fiber. Contacting the spatio-temporal expression pattern of arf1 and nodulin-like gene, we had proved our supposition is correct, this gene cluster is correlative with cotton reproductive development.We had gotten the cDNA of GhGolS and GhAPm by RT-PCR, the length are 1816bp and 1590bp respectively, the gene structure prediction of GhGolS is not exactly, the gene structure prediction software treated a 90bp exon as intron, however, the prediction result is the same with cDNA approximately, on the other hand, the gene structure prediction result of GhAPm is the same with cDNA exactly.We have constructed the overexpression vectors of GhGolS and unknown gene, the RNAi vectors of silencing cotton GhGolS and unknown gene, the RNAi vectors of silencing Agrobacterium tumefaciens AtGolS2 and neutral beta-fructofuranosidase gene, and the RNAi vector of silencing Agrobacterium tumefaciens AtGolS2, clathrin adaptor complexes medium subunit family gene and neutral beta-fructofuranosidase gene together. Transferred these vectors to cotton and Agrobacterium tumefaciens respectively, and got the Agrobacterium tumefaciens T1 seeds, and the cotton T0 seeds is being screened. All of these will help us to understand the function of this gene cluster in cotton reproductive development.This paper found a gene cluster is correlative with cotton reproductive development, this gene cluster includes seven gene, and this gene is close to each other, we carry on a series of research, all of these is the foundation for studying cotton reproductive development deeply, and maybe will providing new theory and technique for breeding excellent cotton varieties.更多还原