【作者】 周勇；

【导师】 梁国华；

【作者基本信息】 扬州大学 ， 植物生物技术， 2009， 博士

【摘要】 水稻理想株型是高产形成的基础。继水稻矮化育种和杂种优势利用获得成功以来,直立穗、半直立穗粳稻的培育和大面积应用,被认为是我国水稻育种史上的第三个里程碑。直立穗品种表现出耐肥抗倒,较好的群体通风透光条件,较高的光合效率和增产潜力,因此,探明直立穗形成的分子机理,不仅能使我们更多地了解水稻穗部发育机制,而且便于我们通过分子设计改良株型。水稻粒型与产量密切相关,同时也决定稻米的品质。因此,水稻粒型的遗传和分子基础研究对水稻品种的改良至关重要。本研究通过选育直立穗基因qPE9-1与粒型基因qGS7-1的近等基因系,利用分子标记进行相关基因的精细定位和克隆研究,主要研究结果如下：一、水稻直立穗基因qPE9-1的图位克隆1、通过杂交回交和分子标记辅助选择,分别构建了qPE9-1位点在三个背景下(R6547、武育粳3和武运粳8)的近等基因系,系统分析该位点对穗型、株型和产量的影响。来源于弯曲穗品种的等位基因对穗弯曲度、穗长、粒长、千粒重、叶片长度和单株产量的增加有显著的正效应,表现出多效性,来源于直立穗品种的等位基因有显著的负效应；qPE9-1对主穗粒数、结实率、一次枝梗数和二次枝梗数没有影响；2、利用以R6547为背景的BC3F2分离群体对穗弯曲度、穗长、粒长和千粒重进行遗传分析。结果表明,穗弯曲度、穗长、粒长和千粒重受一个不完全显性基因控制；3、将qPE9-1基因精细定位在PAC克隆AP005419上的分子标记S919和S927之间约32-Kb的物理距离内。根据TIGR网站水稻基因注释系统,在此区间内共有三个注释的开放阅读框,分别为Os09g26999,Os09g27010和Os09g27020,由于对粒长有明显的遗传效应,把Os09g26999确定为qPE9-1的候选基因。通过互补试验、RNAi和过量表达试验证明了Os09g26999就是qPE9-1基因,而且来源于R6547和日本晴的等位基因qpE9-1是功能基因,来源于武运粳8的功能缺失基因qpe9-1导致了直立穗表型；4、来源于R6547的qPE9-1基因包含5个外显子和4个内含子,编码一个426氨基酸的蛋白质,该蛋白包含3个Von Willebrand Factor Type C (VWFC)结构域,1个跨膜结构域,1个4-disulfide-core结构域。与R6547的等位基因进行比较,发现武运粳8的等位基因存在17处变异,包括13个单核苷酸变异(SNPs)和4个插入缺失变异(InDels),分别命名为SNP1-SNP13和InDe11-InDe14。但SNP1-SNP12和InDe11-InDe13均位于内含子内,没有造成二者在氨基酸水平上的差异。SNP13和InDe14位于第5个外显子内,并且都造成氨基酸变异。SNP13导致了一个氨基酸的替换,InDe14使武运粳8在第5外显子缺失了637个核苷酸,并有12个核苷酸的插入,在突变位点后形成一个蛋白质翻译终止密码子,造成武运粳8的等位基因编码的蛋白缺失了C端231个氨基酸。通过序列分析和转基因试验证明InDel4造成了直立穗表型；5、qPE9-1基因在茎和茎节中表达最高,在根中表达最弱,在叶和穗中也能检测到表达；在近等基因系的相同组织中表达量没有差异,基因组水平的变异没有对表达水平造成影响。将qPE9-1-GFP融合蛋白在水稻原生质体中瞬时表达显示,qPE9-1蛋白是一个膜蛋白。二、水稻粒型主效QTL qGS7-1的鉴定和精细定位1、从132个以日本晴为供体和广陆矮4为受体构建的染色体片段代换系中鉴定了一个粒型与轮回亲本有很大差异的株系C1044。该株系表现为粒型细长,而且两年两地的粒长数据非常一致,受环境影响很小,性状表达稳定。另外,C1044和轮回亲本广陆矮4相比,在株高、主穗长和主穗粒数有显著差异,但单株穗数、结实率和抽穗期没有显著差异；2、将C1044和广陆矮4杂交自交,构建一个衍生的F2群体,利用该分离群体对粒型性状进行了遗传分析。结果表明,粒长、粒宽和长宽比表现为单个孟德尔因子的遗传模式,F3株系分析也支持这一结论。QTL分析表明,qGS7-1对粒长、粒宽、长宽比和粒厚的贡献率分别为40.80%、53.1%、60.2%和8.1%,均以加性效应为主,来源于日本晴的等位基因有使粒长和长宽比增大,使粒宽和粒厚减小的效应。这说明qGS7-1是一个对水稻粒长、粒宽和长宽比影响较大,而对粒厚和千粒重影响较小的QTL3、此外,粒长、长宽比和粒宽、粒厚之间分别存在极显著负相关,但粒长和长宽比呈极显著正相关,粒宽和粒厚呈极显著正相关。这说明籽粒越长,宽度越窄,长宽比越大,厚度越小；籽粒越短,宽度越宽,长宽比越小,厚度越大。这可能也是对籽粒千粒重影响较小的原因。4、利用C1044／广陆矮4衍生F2／F3分离群体中表现极端短粒的隐性个体,将qGS7-1精细定位在AP005198上标记S7-45和S7-115之间大约87.6-kb的物理距离内,与标记S7-179共分离。更多还原

【Abstract】 The ideotype is the foundation for rice high-yielding. The development and cultivation of varieties with erect or semierect panicles is considered as the third landmark after dwarfing breeding and hybrid rice in the history of Chinese rice breeding. PE (Panicle erectness) varieties show increased lodging and fertilizer resistance, benefit ventilation and light penetration of population, higher photosynthetic rates and grain yield. Therefore, the nature of panicle erect not only makes us deeply understand the mechanisms of panicle development, but also facilitate the plant architecture improvement by molecular design breeding.Grain shape is closely connected with rice yield and directly determines rice quality. The genetics and molecular basis is essential for the improvement of rice varieties.In this study, the genetic effects of panicle erectness gene (qPE9-1) and grain shape gene (qGS7-1) were analyzed with near-isogenic lines. Map-based cloning of qPE9-1 and fine mapping of qGS7-1 was conducted. The main results are as follows:Part 1:Map-based cloning of qPE9-1 for rice panicle erectness.1. Three pairs of near-isogenic lines (NILs) with different backgrounds (R6547, Wuyujing 3 and Wuyunjing 8) were constructed by cross, backcross and MAS. An array of panicle architecture, plant architecture and yield traits were systematically analyzed using the NILs. The allele from panicle drooping variety had positive effects on panicle curvature, panicle length, grain length, length of leaf,1,000-grain weight and grain yield per plant, but had no effect on the number of spikelets on the main panicle, number of primary branch and secondary branch. The allele from PE varieties had negative effects.2. The bimodal distributions of the panicle curvature, panicle length, and grain length and 1,000-grain weight in the BC3F2 population with R6547 background suggested that these traits were likely controlled by a semi-dominant QTL.3. Fine mapping allowed us to delimit qPE9-1 within a～32-kb window defined by the markers S919 and S927 on PAC AP005419. Three genes, Os09g26999, Os09g27010 and Os09g27020, located in this region according to the TIGR Rice genome Annotation Database. And Os09g26999 was considered as the strongest candidate gene for qPE9-1 because of its large genetic effect on grain length. The hypothesis that Os09g26999 equaled to qPE9-1 was further confirmed by complementary test, RNAi and overexpression experiments. The allele from R6547 (qPE9-1) is functional, and the loss-of-function allele in Wuyunjing 8 (qpe9-1) resulted in the PE trait.4. The qPE9-1 allele from R6547 contains five exons and four introns and encodes a protein of 426 amino acid residues. The qPE9-1 protein contains three Von Willebrand Factor Type C (VWFC) domains, a transmembrane domain and a one 4-disulfide-core domain (http://www.ebi.ac.uk/InterProScan/). Thirteen single nucleotide polymorphisms (SNP1-SNP13) and four insertion-deletion polymorphisms (InDel1-InDe14) were found on qPE9-1 locus between R6547 and Wuyunjing 8. Except for SNP13 and InDe14, all the sequence polymorphisms were found in non-coding regions. SNP13 results in a Cystine to Tyrosine substitution at the site 105 (C105Y). The Wuyunjing 8 allele, due to its InDe14 (637-bp deletion and 12-bp insertion) in exon 5, encodes a presumably truncated protein that lacks 231 C-terminal residues. The recessive allele at qPE9-1 locus is a loss-of-function mutation that leads to the PE trait.5. GUS activity was detected mainly in elongating and dividing tissues, including the shoot apical meristem, the divisional and elongating zones of stem and knot. Real-time PCR analysis was consistent with GUS staining and suggested that the genomic sequence changes did not affect expression level.Part 2:Identification and fine mapping of a major quantitative trait loci, qGS7-1, controlling grain shape in rice. 1. C1044 is a chromosome segment substitution line derived from 132 introgression lines by introgressing chromosomal segments from a japonica doner cultivar, Nipponbare, into an indica recurrent cultivar, Guangluai 4. This line displayed obviously large grain length than Guangluai 4 based on phenotypic examination at two sites in two years. There were significant differences in plant height, main panicle length and grains per main panicle, while no significant difference in number of panicles per plant, seed set rate and days to heading between them.2. An F2 population was generated for genetic analysis by selfing the F1 plants of C1044 and Guangluai 4. Grain length, grain width and grain length-width ratio simultaneously showed as a single locus Mendelian segregation. F3 family analysis also supported this conclusion. QTL analysis indicated that qGS7-1 had effects on grain length, grain width, grain length-width ratio and grain thickness, and contributed 40.8%, 53.1%,60.2% and 8.1%of the phenotypic variation to these traits, respectively. The Nipponbare allele increases grain length and length-width ratio, but decreases grain width and grain thickness. Thus, QTL qGS7-1 was a major QTL for grain length, width and length-width ratio but a minor QTL for grain thickness and weight.3. In addition, grain length and length-width ratio showed significantly negative correlation with grain width and thickness, respectively. But grain length-width ratio showed positive correlation with grain length. Grain width and grain thickness was also highly significant positive correlation. Long grains were more narrow and thinner than short grains, or vice visa. That is reason why the grain weight did not change with grain shape.4. Using excessive plants with extreme short grains in F2/F3 populations, the qGS7-1 was finally delimited to a 87.6-Kb genomic DNA region between S7-45 and S7-115 and co-segregated with S7-179.更多还原