【作者】 杜红毅；

【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， 2012， 博士

【摘要】 已经于2008年完成克隆的水稻杂种不育基因S5在对亚洲栽培稻群体的比较测序中发现了籼型、粳型和广亲和型三种等位基因型,其中籼型和粳型等位基因型的编码区段之间仅存在两个碱基的差异(从基因的5’到3’方向,籼型依次为C、C；粳型依次为A、T)。其对应编码的蛋白质水平分别为两个氨基酸的替换,而广亲和品种的S5等位基因型由于编码蛋白N端的缺失导致其功能丧失而被定义为“中性”类型。在本研究中,我们对S5基因对应在亚洲栽培稻O. sativa、一年生尼瓦拉野生稻O. nivara、多年生普通野生稻O. rufipogon三大类群体中共计138个水稻品系的基因组序列进行了测序分析,涵盖S5上游启动子区、编码区及下游旁侧序列4.7kb区域。PCR扩增比较测序的结果表明S5有19种单倍型(编码区的自然变异),并且可以进一步根据籼型、粳型和广亲和型等位基因型的特征将单倍型分为三类。其中籼类和广亲和类各有8种单倍型,而粳类S5等位基因只有3种单倍型的存在。对三类S5等位基因进化关系的分析表明,H19和H5分别是籼类和粳类的原始类型,这两种单倍型在对应等位基因类型中频率最高,且H19和H5仅在两个特征SNP,即C819A和C1412T处有区别。H17是广亲和类的原始类型,由其在单倍型网络结构所处位置可以看出该单倍型是由H19发生一次缺失突变后形成的。我们对进化选择压力的计算表明广亲和类的S5等位基因型受到正向选择,推测原因是携带这一类型等位基因的个体在杂种中的高亲和性而导致它们受到选择,频率得以上升。综合上述结果可以推断出,S5籼类、粳类和广亲和类等位基因的形成出现在O.sativa、O. nivara和O. rufipogon的分化之前,而籼类和粳类这两类不亲和的等位基因型存在于更加原始的祖先种中,说明生殖隔离不仅在栽培稻的籼粳两个亚种中存在,而且极有可能发生在更早的物种分化事件之前,即在未被人工驯化的野生稻群体中有可能已经建立起生殖隔离的系统。同样在这一系统内部,近似籼稻和近似粳稻的水稻群体在野生稻中可能已经分化出来,这一结果也支持籼稻和粳稻独立起源的假说。进一步研究还发现在所用的水稻自然种群中,区分籼型和粳型的两个特征SNP,即C819A和C1412T都是紧密关联的。这一结果表明,两个位点的同时存在对于行使其功能有关键作用。在已发表的利用籼型S5全长基因转化粳型受体使雌配子和小穗育性显著下降的结果基础上,我们通过构建S5在两个特征SNP发生重组的供体片段且分别由S5籼型和粳型启动子驱动,共计6种片段类型进行遗传转化,考察T0与T1代单拷贝植株小穗育性。转化结果表明只有C819和C1412同时存在才会导致籼粳杂交后的半不育表型,C819和C1412的关联是行使S5功能所必需的。同时我们也对无突变体支持反向遗传学研究的S5基因进行了粳稻受体表达量抑制的转化操作,转化后代与籼稻母本测交后观察是否使杂种育性得到恢复,结果真杂种阳性单株与阴性单株之间并无显著育性差异,表明该基因不存在剂量效应。此外我们也使用S5基因以正义转化和超量表达两种方式转化了拟南芥以探索其物种间的功能保守性,结果表明并未影响拟南芥相关育性指标,但植株出现的矮化表型与该基因所编码的天冬氨酸蛋白酶所属家族成员在拟南芥中已报道的功能相符,并且作为一个新的病原抗性性状改良基因源具有被深入利用的潜在价值。水稻籼粳亚种间杂种不育是一种典型的生殖隔离现象。生殖隔离在物种进化、分化和新物种的形成中起着十分重要的作用。水稻杂种不育基因S5在进化中表现出其特有的复杂性,且S5籼类、粳类和广亲和类等位基因在种群和进化的层面上也表现出相当重要的功能,因此对该基因自然变异和功能演化的深入研究将有助于我们更好地理解生殖隔离和物种分化所形成和发展的过程。更多还原

【Abstract】 Three allelic types, indica-like, japonica-like and WCG (wide compatibility gene)-like were found during the comparative sequencing of a hybrid sterility gene S5recently cloned in2008within Asian cultivated rice population. The indica-like alleles differ from japonica-like alleles by two nucleotides in the coding region (from5’to3’, indica-like are C and C, japonica-like are A and T). The protein level can be distinguished by two AA replacements, and the WCG-like S5alleles are defined as "neutral" type due to the deletion in N-terminal and further loss of function. In our study, we analyzed the genomic sequences of S5alleles within138rice accessions including Asian cultivated rice O. sativa, annual wild rice O. nivara and perennial common wild rice O. rufipogon, covering upstream promoter, coding region and downstream flanking sequence totally4.7kb in length. Results from PCR-amplified comparative sequencing showed19haplotypes (natural variation in coding region) and could be further classified into three groups as indica, japonica and WCG-like alleles. There were8haplotypes in indica-like and WCG-like group separately, and only3haplotypes in japonica-like group.Analysis on evolutionary relationship among the three allelic groups indicated haplotype H19and H5were primitive to indica-like and japonica-like groups separately, with the highest frequency in each group and could only be distinguished by SNP C819A and C1412T. H17was primitive to WCG-like group resulting from a deletion mutation in H19. Calculation on evolutionary selective force suggested the WCG-like group was favored by positive selection due to the high compatibility in hybrids from individuals carrying correspondant alleles to inflict increased frequency. To conclude all, the formation of indica, japonica and WCG-like alleles were earlier than speciation of O. sativa, O. nivara and O. rufipogon, and the incompatible allelic types indica-like and japonica-like already existed in more ancestral species, suggesting that reproductive isolation exists not only within indica and japonica cultivated rice but also in earlier speciation events. The reproductive isolation might be established within the untamed wild rice populations. Also in this isolation system, the similar indica and japonica rice populations might had already differentiated out and the result also supported the independent origination hypothesis of indica and japonica subspecies.Further study found the two characteristic SNPs between indica and japonica in the sampled rice germplasm, C819A and C1412T were tightly associated, suggesting the coexistence of these two sites played an essential role of gene function. On basis of the published data from transformation of indica full length S5into japonica recipient, both female gametic and spikelet fertility were decreased, we constructed6donor-fragments from all recombinations of these two SNPs and driven by S5indica and japonica native promoters for genetic transformation to evaluate the spikelet fertility of single copy positive individuals in To and T1generations. Results indicated only the coexistence of C819and C1412could repeat the semi-sterile phenotype as indica-japonica hybrids, and the association between these two sites was necessary for S5to act functionally. In the same time we commenced expression-repressing transformation of S5into japonica rice recipient due to the lack of allelic mutant, and to observe possible hybrid fertility restoration after test-crossed with indica. The result showed no significant fertility difference between hybrid-positive and hybrid-negative individuals and no dosage effect of S5. We also used complementary and over-expression transformation into Arabidopsis to estimate the functional conservation, and results indicated no fertility-related traits were influenced but the dwarfing phenotype still matched the reported functions of aspartic protease gene family members in Arabidopsis, granting S5the potential to be further utilized as a new pathogen resistance genetic source in trait improvement.The intersubspecific hybrid sterility between indica and japonica rice is a typical reproductive isolation which plays an extremely important role in allelic evolution, differentiation and speciation. The hybrid sterility gene S5in rice demonstrated its specific complexity in evolution. The indica, japonica and WCG-like alleles of S5also showed very important functions on speciation and evolution, further study into natural variation and functional evolution of this gene will be helpful for better understanding of reproductive isolation and speciation procedures from formational and developmental level.更多还原