【作者】 张卫峰；

【导师】 孙传清；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2017， 博士

【摘要】 亚洲栽培稻(Oryza sativa L.)由其野生祖先种普通野生稻(Oryza rufipogon Griff.)驯化而来,在驯化过程中,诸多性状发生了改变,株型的改变尤为显著。野生稻大多表现匍匐生长,有利于其在复杂多变的自然条件下繁衍,在驯化为栽培稻过程中,PROG1基因突变,水稻变成直立生长,有利于适度密植,增加单位面积产量。然而,部分野生稻尽管PROG1基因功能丧失,依然有较大的分蘖角度,表现倾斜生长习性。鉴定控制倾斜生长习性基因,对进一步揭示水稻驯化过程中株型演变的分子机理具有重要意义。本研究室前期利用PROG1基因功能部分缺失、株型为倾斜生长的印度一年生野生稻W2014与籼稻品种9311构建了一套渗入系群体。本研究从中鉴定一个/倾斜生长的渗入系OIL31,利用OIL31与轮回亲本9311回交,构建F2分离群体,将控制倾斜生长基因ING1(Inclined Growth1)定位在第8染色体长臂9-kb的区间内,该区间有1个候选基因(LOC_Os08g33530),编码一种TCP家族的转录因子。遗传互补实验表明该基因就是控制野生稻倾斜生长的基因ING1。通过对931 1和9311为背景的ING1近等基因系NIL(ING1)进行分蘖角度、负向重力性生长的观察,发现9311和NIL(ING1)的响应重力能力没有差异,说明9311和NIL(ING1)分蘖角度的差异不是由于向重力性生长的差异引起的。组织学分析发现,9311的近地侧的细胞显著大于远地侧,而NIL(ING1)两侧的细胞的大小差异较小,表明近地面和远地面细胞相对大小影响了9311和NIL(ING1)分蘖角度的差异。通过9311和OIL31的2660bp 长的ING1基因组序列(1360bp启动子+800bp CDS+500bp 3’UTR)进行测序比对,发现两者的CDS 区仅存在一个单碱基的同义替换(C/T),而在启动子区则存在3个Indel和12个SNP。基因表达模式分析表明,ING1在茎基部的表达量最高,9311和NIL(ING1)之间的表达量存在显著的差异,而且在NIL(ING1)的远地侧的表达量是近地侧的数倍。RNA-seq分析发现ING1对于茎基部细胞生长的调控可能涉及到诸多激素通路、细胞分裂、细胞膨胀及细胞壁重新构建过程。ChIP-qPCR结果显示,ING1可能直接结合在TAC1、EXPB5等基因的启动子区域,调控这些下游基因的表达。核苷酸多样性分析发现,栽培稻中ING1的编码区受到人工选择,其周围区域的核苷酸多态性降低。ING1基因的克隆不仅为揭示水稻分蘖角度的调控机制提供了重要线索,而且为深入阐释栽培稻驯化过程中株型演变的分子机理提供了新的认识。更多还原

【Abstract】 Asian cultivated rice(Oryza sativa L.)is domesticated from common wild rice(Oryza rufipogon Griff.).Compared with the wild ancestor,many traits of the cultivated rice changed,and the change of plant architecture is particularly striking.Typical common wild rice tends to have a prostrate growth habit,which is favourable to its survival in natural habitats.During the domestication,mutations of PROG1 in cultivated rice disrupt the progl function,leading to erect growth,increasing plant density and enhancing grain yield.However,some accessions of wild rice harborring no functional PROG1 gene still show a larger tiller angle and display the inclined growth habits.To identify the gene controlling inclined growth habit is very important to uncover the molecular mechanism of plant architecture evolution in rice domestication.In an attempt to identify the gene responsible for inclined growth habit,a set of introgression lines was constructed using an indica cultivar 9311 and an asccession of annual wild rice W2014 from India which displays an inclined growth habit and was characterized with functional partial missing PROG1 gene.In this study,an introgression line OIL31 showing inclined growth was identified.Using a F2 mapping population derived from OIL31 and 9311,Inclined Growth 1 {ING1)controlling the inclined growth was delimited within a 9-kb region on the long arm of chromosome 8.Within this region,there is only one hypothetical gene(LOC_Os08g33530)in the nipponbare genome.This gene encoded a transcription factor of the TCP family.The transgenic complementary experiment verified that LOC_Os08g33530 in the O.rufipogon genome was a key gene(ING1)controlling inclined growth habit.Observation of the tiller angle,negative gravitational growth of 9311 and ING1 near isogenic line NIL(ING1)with background of 9311 showed there was no difference in the gravity response ability of 9311 and NIL(ING1),indicating that the difference of the tiller angle between 9311 and NIL(ING1)was not due to the difference of negative gravity growth.Histological analysis of the tiller base showed that near-land cells of 9311 were significantly larger than that of the far-land cells,however there was no significant difference in cell size between the far-land and the near-land tissue in NIL(ING1)indicating that the relative size of near-land and far-land cells affected the tiller angle difference between 931 1 and NIL(ING1).Sequencing analysis including 1360bp promotor region,800bp the coding sequences(CDS)and 500bp 3’ UTR region revealed that one synonymous substitution in CDS region,3 insertion/deletions(indels)and 12 SNPs were detected in the ING1 sequence between 9311 and W2104.Expression pattern analysis showed that the expression of ING1 was tissue-specific and had the highest expression at the base of rice,and the ING1 expression level in NIL(ING1)was significantly higher than that in 9311.Moreover,the ING1 expression level on the far land side of NIL(ING1)was several folds of near land side.RNA-seq analysis showed that the mechanism of ING1 regulating the growth of tiller base cells might involve hormone pathways,cell division,cell expansion,and cell wall reconstruction.ChIP-qPCR results showed that ING1 may directly bind to the promoter sequences of TAC1,EXPB5 and other genes,and regulate the expression of these downstream genes.Analysis of the nucleotide diversity revealed that ING1 in cultivated rice was artificially selected,and the nucleotide polymorphism in the surrounding area decreased.Identification of the ING1 gene not only provides a useful paradigm for revealling the mechanism for genetic regulation of rice tiller angle,but also offers a new insight on elucidating the molecular mechanism of plant architecture evolution during rice domestication.更多还原