【作者】 吴季荣；

【导师】 庄杰云；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 硅在水稻正常生长发育过程中起着重要作用。已经有3个与水稻硅代谢相关的基因得到克隆。随着分子数量遗传学的发展,通过数量遗传学途径寻找控制相关性状的QTL已经成为研究的重要方向,与水稻硅性状相关的QTL研究在过去的4年中也逐步得到开展。前期研究中,以珍汕97B×密阳46重组自交系(recombinant inbred line, RIL)群体为材料,在第6染色体短臂检测到一个控制水稻谷壳硅含量的QTL qHUS-6,其贡献率达17.4%。在此基础上,利用1套衍生于1个水稻剩余杂合体的F2:3群体RHL6对该QTL进行验证和分解。该群体含221株系,在第6染色体短臂RM587-RM19784区间分离、其他区间基本纯合。再从该群体中筛选出杂合区间更小的单株,用于QTL的验证、分解和精细定位,并对所得候选基因进行基因序列分析,得到以下结果:1.2004年冬在海南种植RHL6 F2:3群体的221个株系。应用该群体在第6染色体短臂分离区间RM587-RM19784内共检测到两个控制水稻谷壳硅含量的QTL qHUS6-1和qHUS6-2,分别位于区间RM510-RM204和RM19715-RM19784内,增效等位基因分别来源于父本密阳46和母本珍汕97B,加性效应分别为1.50和-1.12,显性效应分别为1.17和0.03,贡献率分别为20.23%和23.75%,分别表现为正向显性和加性作用;背景中在第2染色体的一小段分离区间中检测到一个微效QTL,贡献率为4.1%,增效等位基因来自母本珍汕97B。2.从RHL6 F2:3群体中筛选出3个杂合区间更小的单株其中,其中2株覆盖qHUS6-1区间,各自交产生了1个F2群体,分别从两个群体中筛选出目标区间重组和非重组的单株衍生F3群体,称为FM3 F2:3群体和FM4 F2:3群体,分别含62个株系和40个株系。于2006年夏在浙江种植这两个群体,分别应用这2个群体对qHUS6-1进行定位,然后对两群体的结果进行比较,将qHUS6-1定位于RM510和RM19417之间约147.0 kb的区间内,增效等位基因来自父本密阳46,贡献率达65.26%和76.30%;另1株覆盖qHUS6-2区间,自交产生了1个F2:3群体,称为FM12 F2:3群体,从中挑选出在qHUS6-2区间具有不同基因型组成的5套F3株系于2006年夏种植于浙江,通过不同基因型组成的株系之间的方差分析比较将qHUS6-2分解为一个效应较大的QTL qHUS6-2a和效应较小的QTL qHUS6-2b,分别位于RM19706-RM19795和RM314-RM19665区间,增效等位基因均来自父本密阳46。3.从FM4 F2:3群体中挑选出杂合区间更小的两个单株TF6-15和TF6-17并衍生F2群体,分别从两个F2群体中筛选出10株目标区间母本基因型的植株、10株父本基因型的植株和20株杂合型的植株,于2007年夏在浙江种植F3株系。在一套近等基因系内进行母本纯合型株系与父本纯合型株系表型的方差分析,然后再进行两套近等基因系之间的比较。由TF6-15衍生的成对近等基因系之间有极显著差异,说明在分离区间有控制谷壳硅含量的QTL,而由TF6-17衍生的成对近等基因系之间无显著差异,说明在分离区间不存在控制谷壳硅含量的QTL。通过比较最终将qHUS6-1定位于第6染色体短臂RM3414-Si2944约62.4 kb的区间内,其加性效应为0.67,显性效应为-0.19,贡献率为26.20%,增效等位基因来自父本密阳46。4.在上述精细定位的区间内,共搜索到10个开放阅读框。经生物信息学分析,确定了目的基因。对其进行基因序列分析,发现该基因的编码区在两亲本间无碱基差异,编码区全长2,592bp;对该基因的cDNA序列分析发现,该基因无内含子,编码一个全长2,592bp的cDNA,编码产物为一个具有863个氨基酸的蛋白。两亲本间序列比对发现珍汕97B的第104个碱基发生RNA编辑(A→G),结果导致亲本间编码产物在第35个氨基酸改变,珍汕97B产物在该位点为半胱氨酸,而密阳46在该位点为酪氨酸。更多还原

【Abstract】 Silicon plays an important role during the process of rice growth and development. Three genes controlling silicon transportation and accumulation in rice have been cloned. With the advance of molecular quantitative genetics, gene identification through QTL mapping approach has been performed. Studies about QTL mapping for rice silicon traits have also been performed in the last 4 years.In our previous study, a QTL named qHUS6 controlling hull silicon content was located on the short arm of rice chromosome 6 by using the RIL population crossed by zhenshan97B and Milyang46 and it can explain 17.4% of the phenotypic variance. In present study, an F2:3 population containing 221 lines derived from an individual, which was selected from the RIL population, was used for QTL validation and dissection. This population has a segregate region RM587-RM19784 on the short arm of chromosome 6 but homozygous genetic background. A series of individuals containing smaller heterozygous target region were selected from this population for QTL dissection, validation and fine mapping and then the candidate gene analysis was performed. The results were summarized as follow:1. In the winter of 2004, the RHL6 F2:3 population containing 221 lines was planted in Hainan Province. With this population, two QTLs controlling hull silicon content were detected in the region RM587-RM19784 on the short arm of rice chromosome, of which qHUS6-1 was located in the region RM510-RM204 with the additive effect of 1.50 and dominance effects of 1.17 and qHUS6-2 was located in the region RM19715-RM19784 with the additive effects of -1.12 and dominance effects of 0.03. They can explain 20.23% and 23.75% of phenotypic variance, respectively. The alleles from the Milyang46 and Zhenshan97B increased hull silicon content, respectively. qHUS6-1 acted as a positive dominant QTL but qHUS6-2 as an additive QTL; in the background, a QTL with minor effect was detected on the chromosome 2. The allele from Zhenshan97B increased hull silicon content and it can explain 4.1% of the phenotypic variance.2. Three individuals carrying smaller heterozygous segments were selected from RHL6 F2:3 population, of which two individuals covered the qHUS6-1 region and another covered the qHUS6-2 region. Two F2:3 populations were derived from the selfed seeds of the two individuals covered the qHUS6-1. QTL mapping was performed by using these two populations which were planted in Zhejiang Province in the summer of 2006 and qHUS6-1 was delimitated to a 147.0-kb region flanked by RM510 and RM19417. It can explain as high as 65.26% and 76.3% of the phenotypic variance in the two populations respectively and the allele from Milyang46 increased hull silicon content in both of these two populations. Five groups of F3 lines with different genotypic compositions in the qHUS6-2 region were selected from the other F2:3 population and were planted in Zhejiang Province in the summer of 2006. Two QTLs were separated with two-way ANOVA, of which qHUS6-2a was located in interval RM19706-RM19795 and qHUS6-2b in interval RM314-RM19665. The allele from Milyang46 increased hull silicon content for both of these two QTLs. 3. Two individuals named TF6-15 and TF6-17 carrying smaller heterozygous segments covering the qHUS6-1 region were selected from FM4 F3 population and two F2 populations was derived from these two individuals, respectively. 10, 10 and 20 plants that were maternal homozygous, paternal homozygous and heterozygous in the target interval were selected from one of the F2 populations respectively. And another F2 population was also performed as above. An F3 population was derived with these materials and was planted in Zhejiang Province in the summer of 2007. Two–way ANOVA was performed for the lines from the same individual in order to detect whether there was QTL in the target interval and then compared the result from two series of NILs. There was significant difference between the NILs derived from TF6-15 and no difference between NILs from TF6-17. The qHUS6-1 was delimitated to a 62.4-kb region flanked by RM3414 and Si2944. The allele from Milyang46 increased hull silicon content.4. Based on the result of fine mapping of qHUS6-1, candidate genes search was performed and 10 ORFs were found in the fine mapping region. Through bioinformatic analysis, one of the candidate genes was thought to be the target gene. By comparing the sequences of the genomic DNA of the target gene from the two parents, there was no difference in the coding region between two parents and the full length of the gene was 2,592bp; by comparing the sequences of the cDNA of the target gene from the two parents, we found that there was no intron in this gene and the cDNA was 2,592-bp long and can produce a protein of 863 amino acid long. There was a RNA editing occurred at 104th nucleotide in the cDNA from Zhenshan 97B, resulting a change from tyrosine of Milyang46 to cysteine of Zhenshan97B at the 35th amino acid.更多还原