【作者】 薛为亚；

【导师】 张启发； 邢永忠；

【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】 高产稳产历来是作物科学研究中最重要的目标之一,水稻产量相关基因的研究一直是水稻育种和水稻分子生物学的重点研究内容。水稻单位面积产量由每穗粒数,有效穗数,千粒重和结实率等因素构成。同时,株高,生育期对水稻株形,适应性等性状影响巨大。目前,水稻中控制抽穗期,株高,分蘖,每穗粒数和粒重的基因都被相继克隆。在本研究中,我们克隆了位于水稻第7染色体着丝粒区QTL,Ghd7,它同时控制水稻的株高,每穗粒数,生育期,来自明恢63的等位基因为不完全显性基因。通过对Ghd7的表达分析和植株的显微结构的比较研究,初步阐明了Ghd7的作用机理,这是在水稻中首次成功克隆这样的一因多效基因。通过回交的方法,将含有来自明恢63的Ghd7的片段导入到珍汕97中,获得了珍汕97背景的近等基因系（NIL）。NIL与轮回亲本珍汕97相比,抽穗期大约晚了21.2天,株高增加了33厘米,主茎颖花从130粒增加到216粒,单株产量也提高了50%。通过经典的图位克隆的方法,利用3个NIL-F₂大群体,把Ghd7精细定到0.28cM,它对应于大约2000 kb的范围,显然,此区间位于着丝粒区,重组交换受到明显抑制,无法把基因区间进一步缩小。通过对该区间序列的分析,发现一个与成花发育相关的基因,该基因含有一个CCT结构域,将含有来自明恢63的基因区段遗传转化合江19,牡丹江8号,日本晴等小穗品种进行功能互补试验,转基因阳性单株都表现抽穗延迟,株高增高,每穗颖花数增加,验证了基因的功能。同时发现Ghd7基因功能效应的大小还受到遗传背景的影响。从Ghd7的表达分布来看,主要在幼嫩的叶片,顶端分生组织,二次枝梗分化原基,成熟叶片的维管束的韧皮部表达。从Ghd7的表达部位的显微结构来看,细胞的数目明显增多,推测Ghd7加速了细胞的分裂速度,在幼穗发育中,分化出了更多的二次枝梗,成为Ghd7提高穗粒数的一个解剖学基础,同时茎秆发育的增粗,也更有利于整个植株保持较好的株形,有利于稳产。Ghd7是受光周期控制的基因,mRNA呈昼夜节律性表达,短日下表达受到抑制;长日下,Ghd7抑制Hd3a和Ehd1的表达,这种控制开花的途径跟拟南芥相比,可能是水稻所特有的。Ghd7属于CCT基因家族的成员,在植物中CCT类基因是一类快速进化的基因,从基因的进化关系来看,Ghd7跟其他基因的分化距离较远,而与温带长日照植物新笾锌刂拼夯幕騐RN2亲缘关系相近。对于热带亚热带起源的短日照植物水稻来说,没有春化过程,但这两种基因都具有抑制开花的类似功能。从表达模式来看,这类基因也比较相近,都是长日下促进表达,而在短日下表达受到抑制。通过对这类基因的进化关系的深入研究,将为理解温带作物的进化提供有益的帮助。通过对分布在亚洲的19个品种的Ghd7等位基因的比较测序发现,Ghd7各种等位基因编码的蛋白共有5种基因型:第一种基因型以明恢63为代表,这种基因型的等位基因具有较强的功能,含有这种等位基因的品种,多分布在我国南方和热带亚热带的水稻生产区,生长时间长;第二种基因型以日本晴为代表,此种等位基因的功能减弱,含有这种等位基因的品种多分布于我国华北及其同纬度的地区;第三种基因型以合江19和牡丹江8号为代表,这种等位基因发生了终止突变,基因完全失去了功能,分布在我国北方的黑龙江省,夏季较短,水稻生育期也与这种气候相适应;第四种基因型只在特青的品种中发现,这种基因型的功能很强,分布的地理区域与第一种类似;还有一种就是Ghd7完全发生了缺失,含有这种基因型的品种,为分布在我国的双季稻区的早稻品种。以上的发现说明Ghd7不同的基因型与水稻品种区域分布相关,这类基因除了参与发育调控外,也可能跟植物的地理分布相关联。对Ghd7在90个品种中的序列进行了比较序列,发现了74个SNP位点,将这些位点和表现型结合群体结构作了初步的关联分析,发现了一些关键的SNP位点,其中有的SNP位点与株高关联比较紧密,有的与抽穗期关联比较紧密,而有的与每穗粒数关联紧密,有的与两个性状关联紧密。而大多数SNP位点则与性状没有关联。芯片数据的分析表明,Ghd7不仅仅参与了开花的调控,而且参与了植物激素,细胞分裂素的代谢调控。在水稻中,CO类基因参与激素调控是一个全新的领域,还未见到公开的文献报道,通过对这方面的深入研究,将可能全面揭开Ghd7的分子调控机理,为水稻的分子育种提供有力的工具。更多还原

【Abstract】 Developing high yielding variety is one of the most important goals in crop science.Cloning and characterizing the genes related to yield traits is a major object in rice breeding and rice molecular biology.Yield potential,plant height and heading date are three distinct classes of traits that determine the productivity and adaption of many crop plants.Here we report the story that a quantitative trait locus Ghd7,with major effects on an array of traits in rice including number of grains per panicle, plant height and heading date,has been isolated from an elite rice hybrid by map-based cloning.In our previous study,one QTL here named Ghd7 with pleiotropic effects on heading date,plant height and spikelets per panicle was repeatedly detected in the cross Zhenshan 97 and Minghui 63 derived populations.A recombinant line 50, whose 74%genetic background is from Zhenshan 97 was used to consecutively backcross with the recurrent parent Zhenshan 97.Consequently,near isogenic line for Ghd7(NIL(mh7)) was obtained.As compared with recurrent parent,Zhensan 97,the NIL(mh7) plants delayed heading by 21.2 days,increased plant height by 33.3 cm,and produced 86 more spikelets on the main culm.Further examination showed that NIL(mh7) out-yielded NIL(zs7) by 8.9 g(50.9%) per plant,primarily due to an increase in number of grains per panicle,although NIL(mh7) had fewer tillers per plant than NIL(zs7).Three separate populations derived from NILs/zhenshan 97 were used to fine mapping Ghd7.Ghd7 was located between the markers RM5436 and C39 in the centromere region of chromosome 7 corresponded to a DNA fragment approximately 2284 kb according to Nipponbare sequence.A candidate gene with CCT motif was transformated into rice cultivars Hejiang 19,Mudanjiang 8 and Nipponbare,all of which have relatively small panicle,short stature and early heading.All the transgenic plants showed multivariation of the three target traits and confirmed the candidate gene underlying the genetic basis of trait variation. Meanwhile we found the genetic background also affected the effort of the Ghd7. And Ghd7 could promote cell division in the LD conditions.RNA in situ hybridization and Ghd7 promoter-driven GFP transformation were brought to examine sites of Ghd7 expression.The result indicated that Ghd7 mRNA expressed in the shoot apical meristem,whole young leaves,young panicle primordia,culm,phloem of leaves,and roots.The diurnal expression pattern of Ghd7 was investigated by quantifying the relative abundance of the mRNA in young leaves of NIL(mh7) using quantitative RT-PCR.The transcripts of Ghd7 oscillate with diurnal rhythm.Ghd7 expression was enhanced in the day time under LD conditions and was repressed in SD conditions.Ghd7 can suppress the expression of Ehd1 and Hd3a in the LD conditions,and slightly promote Ehd1 expression in the dark period under SD conditions.The result suggested that Ghd7 together with Ehd1 and Hd3a were involved in a novel rice flowering pathway,which does not exist in Arabidopsis.Phenogenetic analysis of CCT family showed that Ghd7 and VRN2 shared the same clade.They had a very similar expression pattern.These suggests that tropical SD plants that do not use vernalization as a flowering cue,and temperate LD plants that do use vernalization,actually use related genes to repress flowering in LDs. Such similarity in flowering control provides important clues as to how the temperate cereals might have evolved.Five alleles of Ghd7 were identified by comparison of Ghd7 sequences in 19 varieties including a common wild rice,representing a wide geographic range of Asia. The varieties with normal functional alleles like Minghui 63 and common wild rice have a long lifecycle and are distributed in Southern China and Southeast Asia.The allele from Teqing is unique and has a strong function.The varieties with weak functional alleles like Nipponbare have an intermediate lifecycle and are distributed in northern China and Japan.Two types of alleles are nonfunctional,one like Hejiang19 has a premature termination in the first exon;another one has a complete deletion of Ghd7.The varieties carrying these two alleles have a short lifecycle. These results indicate that Ghd7 locus has contributed greatly to both productivity and adaptability of the cultivated rice in a global scale.Diversity and association analysis of Ghd7 was carried out in a natural population containing 90 accessions,74 SNP points were found in the sequence of Ghd7.Some SNPs were highly associated with the phenotype.Microarray analysis shows that Ghd7 was involved in phytohormones, cytokinins metabolism.The OsCKX2 is repressed in the transgenic plants.It is a new finding that CCT family genes related to the plant hormones in rice.The molecular function of Ghd7 will be comprehensive clarified through further studying the relationship between Ghd7 and phytohormones.更多还原