【作者】 唐为江；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 抽穗期、株高和每穗颖花数都是水稻生长发育过程中重要农艺性状,在以前研究中我们在第7染色体着丝粒区段鉴定到一个同时影响抽穗期、株高和每穗颖花数的QTL。为了弄清这3个性状是由一个基因还是多个紧密连锁的基因控制和分离克隆该QTL,我们构建了一个以珍汕97为背景的近等基因系Hd1。在Hd1中包含4个来自供体亲本的片段,其中仅第7染色体上来自供体亲本的区段与3个目标性状相关。用Hd1与珍汕97杂交构建用于精细定位的F₂大群体。F₂群体单株的表型可分为2类:一类为具有早抽穗、株高较矮和每穗颖花数较少等特征,与珍汕97表型类似;另一类具有晚抽穗、株高较高和每穗颖花数较多等特征,与明恢63表型类似。它们之间比例为1:3（随机挑选190个单株统计结果,x²=0.11,P=0.740）。在具有8400个单株的F₂群体中,挑选1080个极端早抽穗的单株用于精细定位。这些极端早抽穗的单株都具有株高矮和每穗颖花数少的特征。通过对标记与目标性状之间的重组单株分析,将控制目标性状的基因定位在标记RM3859和C39之间,与标记RM5436和RM5499共分离。标记RM3859和C39之间物理距离约为2.5 Mb,标记RM5436和RM5499之间物理距离为912.4 kb。由于目标基因位于着丝粒区段,发生重组抑制,所以无法通过扩大群体来进一步缩小目标区间。直链淀粉含量、胶稠度、糊化温度是评价水稻蒸煮食味品质的重要指标。本课题组以前的研究结果将控制这三个性状的基因定位在第6染色体短臂末端与Wx基因紧密连锁的区段。为了弄清这3个性状是由一个多效基因还是三个紧密连锁的基因控制,我们利用本课题组构建好的近等基因系珍汕97（Wx^b）与珍汕97回交,得到5280个株系的F₂大群体。从大群体中挑选1084株性状与明恢63一致的单株用于图位克隆。通过分子标记分析将目标基因定位在一个明恢63 BAC克隆35M22和对应的珍汕97 BAC克隆2N1上,利用鸟枪法对这两个BAC克隆进行测序。对目标基因与在两个亲本之间有多态性的亚克隆标记进行重组事件分析,将目标基因定位在标记C6与P80之间,并与标记Mx4和C4共分离。将C6和P80的序列与35M22的序列比较显示这两个标记间的物理距离是14 kb,把目标基因限定在14kb的片段上,对14 kb片段的序列进行分析,其中仅包含一个完整的基因Wx基因和一个不完整的反转录转座子。结果表明,这3个性状均由Wx基因控制。在以前的研究中对认为,Wx基因编码合成颗粒淀粉合成酶,控制直链淀粉合成,而糊化温度主要由编码可溶性淀粉合成酶的Alk基因控制。但很少有报道关于Wx基因对糊化温度和胶稠度的影响。为了分析Wx基因与糊化温度及胶稠度的关系,本研究中分析了97个不同地方品种的Wx基因和Alk基因的多样性及其它们与这3个性状的关系。结果发现Wx基因对糊化温度有约7%的贡献率,Alk基因则解释了糊化温度约91%的变异,Wx基因和Alk基因对胶稠度都有贡献,Wx基因对胶稠度的影响大于Alk基因的影响（Wx基因解释了胶稠度约53%的变异,而Alk基因解释了胶稠度约37%的变异）,而Alk基因与直链淀粉含量完全不相关。这两个基因可能都是通过改变胚乳淀粉组成来改变淀粉结构,从而影响淀粉理化性状。在对Wx基因序列多态性的分析中,发现一个新的突变位点。位于Wx基因ATG上游1126位置的碱基由A突变为G,导致这些材料的直链淀粉含量上升,由中等直链淀粉含量类型提升为高直链淀粉含量类型。本研究中还分析了淀粉合成相关23个重要基因的表达谱。这23个基因分为5个家族:ADP-葡萄糖焦磷酸化酶、可溶性淀粉合成酶、颗粒淀粉酶、淀粉分支酶和淀粉脱分支酶。ADP-葡萄糖焦磷酸化酶主要负责提供糖链延伸的原料:ADP-葡萄糖,在直链、支链淀粉合成中均起作用。颗粒淀粉酶主要参与直链淀粉和长链支链淀粉合成。可溶性淀粉合成酶,淀粉分支酶和淀粉脱分支酶则主要参与支链淀粉合成。这23个基因主要有3种表达模式:在胚乳中特异表达,在光合作用组织中特异表达,在这两类组织中都有表达。在胚乳中表达的基因主要是参与胚乳贮藏型淀粉合成。在光合作用组织中表达的基因主要是将光合作用固定下来的碳源转化为淀粉,保持细胞内渗透压平衡。还发现很多基因在幼穗发育早期表达,主要功能可能是将从光合作用组织中运输过来的碳源转化为临时淀粉,或者是参与雄蕊内淀粉颗粒的合成。在光合作用组织中表达的基因受到光调控,而在胚乳中特异表达的基因对光不敏感。通过扫描电镜观察珍汕97和珍汕97（Wx^b）成熟胚乳的细胞精细结构,发现珍汕97（Wx^b）胚乳细胞较珍汕97胚乳细胞更容易破裂,但淀粉颗粒却比珍汕97更为紧密。这可能导致珍汕97（Wx^b）较难糊化和延伸,从而使得珍汕97（Wx^b）较珍汕97有更高的糊化温度和较短的胶稠度。更多还原

【Abstract】 Heading date, plant height and number of spikelets per panicle are 3 important agricultural traits in rice. In our previous studies, one putative QTL affecting heading date, plant height and number of spikelets per panicle were identified in the pericentromeric region of rice chromosome 7 using a recombinant inbred population. In order to determine that whether the 3 traits were controlled by a single locus with pleiotropic effects or by 3 tightly linked genes, we construct a near isogenic lines Hd1, with the background of Zhenshan 97 and 4 region from Minghui 63. Among them, only the region of chromosome 7 was relation with the 3 traits. In order to fine mapping the QTL, an F₂ population between Zhenshan 97 and the NIL containing 8400 individuals was developed. The phenotype of F₂ population was divided into two types: the type with shorter heading date, shorter plant height and much number of spikelets per panicle and the type with longer heading date, longer plant height and less number of spikelets per panicle, the ratio is 1:3（χ²=0.11,P=0.740）.1082 plants with extremely short heading date of the F₂ population were selected to fine mapping the QTL. All the extremely short heading date plants were with short plant height and less number of spikelets per panicle. No recombination were detected among the 3 traits in the extremely population. By analyzing the recombination of marker and the trait, the QTL were narrowed down to an interval between marker RM3859 and C39, and cosegregated with RM5436 and RM5499. The marker RM3859 and C39 spanning a physical distance of more than 2500kb, and the distance of RM5436 and RM5499 is 912.4kb. As the recombination suppression occurred in the region, it is difficult to narrow down the region by enlarge the population.Amylose content, gel consistency and gelatinization temperature are the three most important traits determining the cooking and eating quality of rice. Using populations derived from Shanyou 63, an elite rice hybrid, we previously mapped the major genes for these three traits to a region tightly linked to the Wx locus on the short arm of rice chromosome 6. In order to determine that whether the 3 traits were controlled by a single locus with pleiotropic effects or by 3 tightly linked genes, an F₂ population between Zhenshan 97 and the NIL containing 5280 individuals was developed. A total of 1080 plants with the trait combination of Minghui 63 were identified from this population. Molecular marker analysis located the gene（s） for the 3 traits to a single BAC clone, 35M22, isolated from a Minghui 63 BAC library, and got a corresponding Zhenshan 97 BAC, 2N1. The two BAC clones were then sequenced using the shotgun strategy. Further recombinant analysis using polymorphism between the parents identified by the subclones localized the genes（s） for the three traits to an interval between two polymorphic markers, C6 and P80. Comparing the sequences of C6 and P80 with 35M22 indicated that the distance between these two subclones is approximately 14kb. In the 14kb region,there are two ORFs. One is the Wx gene, encoding granule bound starch synthase. The other one is an uncomplete retrotransposon. The result show that the three traits were controlled by the Wx gene in the population.We identified the 3 traits to Wx gene, but some research thought the Alk gene has effect on GT and GC. So here we selected 97 cultivars to check the correction of the 2 genes and the 3 traits. Univariate analyzed of the traits of AC, GT and GC by two genes Wx and Alk by software of SPSS 15.0, respectively. The result showed that AC only is controlled by Wx gene and Alk gene no effect on AC and 91.8% of the total variation of GT is explained by Alk gene and 7.1% of variation is explained by Wx gene and 36.9% of variation of GC if explained by Alk gene and 55.9% of it is explained by Wx gene. And no epistatic effect was detected between the two genes.We sequenced a 6.2 kb DNA fragment including Wx gene of 33 cultivars. 43 single nucleotide polymorphisms （SNPs） and 8 insertion/deletions （InDels） were identified. Of them 2 SNPs and 2 InDels can explained all the variation of AC. A new SNP mutation of G substituted A at site of-1126 were detected, result in the AC increase from intermediate AC to high AC.We analyzed the expression of genes in the progress of starch synthesis, including 5 families 23 genes, by Affymetrix chip. The result showed the 23 genes were divided into 3 groups: the type expressing in endosperm tissue, the type expressing in photosynthesis tissue and the type expressing in both these tissues. The starch, synthesized in photosynthesis tissue, is the temporary starch, as source for plant development. And the starch, synthesized in endosporm cell, is storing starch, as the nutrition for seed pullulation.We checked the difference of Zhenshan 97 and Zhenshan 97 （Wx^b） in microstructure by transmission electron microscope, the result showed that the endosporm cell of Zhenshan 97 （Wx^b） was more easily broken but the starch graunle was tighter than Zhenshan 97. It maybe result to that Zhenshan 97 （Wx^b） more difficult to gelatinize and extend. Thus, it maybe is the reason of Zhenshan 97 （Wx^b） with higher GT and shorter GC.更多还原