【作者】 胡茂龙；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2006， 博士

【摘要】 随着世界人口的不断增长，粮食短缺已成为本世纪全球最严重的问题之一。水稻提供了半数以上世界人口的食粮，其产量的提高必然是解决未来全球粮食危机的有效途径之一。光合作用及相关生理性状是决定产量的重要因素。叶色突变体是开展光合生理研究的理想材料。本研究利用3个定位群体为材料，检测控制水稻光合功能相关生理性状QTL。同时以转绿型白叶突变体玉兔S为材料，开展了光合生理研究，并自行开发了分子标记精细定位和图位克隆了突变基因，从而为水稻高光效生理育种的分子改良和转绿型白叶突变体在生产上应用奠定了理论基础。主要结果如下：1利用重组自交系群体检测水稻光合功能相关性状QTL利用粳稻Kinmaze／籼稻DV85杂交后代单粒传衍生的81个F₁₁家系所组成的重组自交系群体，研究水稻光合功能相关性状的数量性状基因座（QTL）。在水稻抽穗后7d测定叶片全氮含量（TLN）、叶绿素a／b比值（Chl．a：b）和叶绿素含量（Chl），共检测到6个QTL，各QTL的LOD值为2.66～4.81，贡献率为11.2～29.6％。其中，在第1、2和11染色体上检测到3个与全氮含量相关的QTL，相应贡献率为17.3％、15.3％、13.7％；在第3和4染色体上检测到2个与叶绿素a／b比值相关的QTL，贡献率为13.8％和29.6％；在第1染色体上检测到1个与叶绿素含量相关的QTL，贡献率为11.2％。4个QTL为本研究新检测的基因座。有趣的是，控制叶绿素含量的qCC-1，位于第1染色体上RFLP标记C122附近，与已报道的NADH-谷氨酸合成酶基因位置一致，而叶绿素合成始于谷氨酸，暗示该基因座与水稻光合功能关系极为密切。然而，对抽穗后30d叶绿素含量进行QTL分析，结果未检测到与其相关的QTL，表明控制叶绿素含量qCC-1效应随水稻叶片的衰老而降低。2利用回交重组自交系群体检测水稻光合功能相关性状QTL利用98个家系组成的日本晴（粳稻）／Kasalath（籼稻）∥日本晴回交重组自交系群体(BC₁F₁₀)，研究水稻光合功能相关性状的数量性状基因座（QTL）。在水稻抽穗后7d测定叶片全氮含量（TLN）、叶绿素a／b比值（Chl．a：b）和叶绿素含量（Chl），共检测到8个QTL，各QTL的LOD值为2.61～6.42，贡献率为9.7～21.0％。其中，在第1和6染色体上检测到2个与全氮含量相关的QTL，相应贡献率为9.7％、10.8％；在第2、3和12染色体上检测到3个与叶绿素a／b比值相关的QTL，贡献率为10％、10.5％、21％；在第1、4和8染色体上检测到3个与叶绿素含量相关的QTL，贡献率为13.7％、10.7％、10％。有趣的是，此群体检测的qCC-1，也位于第1染色体上RFLP标记C122附近，进一步表明该基因座对水稻光合功能的重要性。对抽穗后30 d叶绿素含量进行QTL分析，结果在第4和6染色体上检测到2个QTL与其相关，而位于标记C122附近的qCC-1也未被检测到，从而进一步暗示此位点的效应随水稻叶片的衰老而降低。3利用染色体片段置换系群体检测水稻光合作用及相关生理性状QTL利用由籼稻品种IR24和粳稻品种Asominori杂交衍生的Asominori染色体片段置换系群体为材料，研究了水稻光合作用及相关生理性状的QTL。在水稻抽穗后7 d测定叶片光合速率（PHO）、蒸腾速率（TR）、气孔导度（SCO）、胞间CO₂浓度（IC）、叶绿素含量（CHL）、全氮含量（TLN）。共检测到10个QTLs，分布于第1、3、4、5、7、8和10染色体上，LOD值在2.77～8.42之间，贡献率变幅为9.5～46.5％。其中仅有控制气孔导度的qSCO-8与控制叶绿素含量的gCHL-8以及第10染色体上控制气孔导度的gSCO-10与控制胞间CO₂浓度的gIC-10位置相同，分别位于第8染色体上标记R727和第10染色体上标记C1166附近。其它QTL在染色体上的位置不同，暗示水稻光合功能遗传机理的复杂性。4水稻转绿型白叶突变体基因的图位克隆利用一个由⁶⁰Co-γ射线诱变得到的水稻转绿型白叶（vwl）突变体玉兔S为材料，其在2叶1心期前表现白化，以后慢慢转绿，恢复正常生长。对突变体的光合色素、叶绿体显微结构及突变基因克隆进行了比较系统的研究，得到以下结果：（1）光合色素测定结果显示，转绿前突变体叶片中无法正常合成叶绿素a、叶绿素b、β-类胡萝卜素；转绿后上述3种光合色素合成正常。表明突变体苗期白化是由总的光合色素减少所致。（2）转绿前后的叶绿体电镜显微观察表明，转绿前突变体细胞内完全丧失了叶绿体结构，只有一些类似前质体的结构；转绿后突变体细胞内具有正常的叶绿体结构，含有丰富的类囊体。（3）遗传分析表明，该突变属于单基因隐性突变。（4）利用公共图谱上SSR分子标记和F₂群体中672个突变体单株对突变基因进行了初步定位。结果表明，突变基因位于第3染色体上标记RM411和RM6832之间，距离分别为1.1cM和1.2cM。（5）利用根据水稻基因组数据自行开发的SSR、CAPS以及dCAPS分子标记和F₂群体中2240个突变体单株，构建了FWL基因区域高密度遗传图谱和BAC重叠群，最终将突变基因定位在标记P45和D8之间的45kb范围内，与标记P50共分离。（6）对定位区域内所有潜在的基因进行分析，发现在预期的目标基因位置存在一个编码假定PPR（pentatricopeptide repeat）蛋白的基因（暂命名为OsPPR2）。已知PPR蛋白属于RNA结合蛋白一类，控制叶绿体mRNA的稳定或翻译。因此，这个基因应是vwl的一个候选基因。测序结果显示，突变体中该基因编码区有一个5bp的缺失。基于缺失设计的InDel标记I1分析F₂群体中的2240个突变体单株和20个正常叶色的水稻品种，结果表明标记I1与vwl共分离。由此可以初步认为，OsPPR2就是VWL。更多还原

【Abstract】 Food shortage is one of the most serious global problems with the increasing of the worldwide population in this century. Rice is the most important food crop in the world and feeds over half of the global population. To meet the expanding food demands of the rapidly growing world population, increases in rice yield and production will be required. Photosynthesis and its related physiological traits play an important role in yield determination. Leaf coloration mutant is one of the most ideal materials for photosynthesis research. In the present study, three mapping populations were utilized for quantitative trait loci （QTL） detection controlling photosynthesis and its related physiological traits. Meanwhile, a virescent white leaf mutant （tentatively designated as vwl）, a Indica variety named Yutu S, was used for photosynthetic physiological analysis. Fine mapping and map-based cloning of vwl gene, were carried out on the mutant using molecular marker developed based on the public sequence data. These results should be useful for marker-assisted selection in rice high photosynthetic efficiency breeding and utilization of vwl mutant in rice production. The main conclusions are as follows:1 QTL detection for traits associated with photosynthetic functions in rice, using recombinant inbred linesA mapping population of 81 F₁₁ lines （Recombinant Inbred Lines, RILs）, derived from a cross between a japonica variety Kinmaze and an indica variety DV85 by single-seed descent method, was used to detect quantitative trait loci （QTL） for traits associated with photosynthetic functions. Total leaf nitrogen content （TLN）, chlorophyll a/b ratio （Chl.a:b） and chlorophyll content （Chl） were measured in leaves of rice （Oryza sativa L.） at seven days after heading, and a total of six putative QTLs were detected with percentage of variance explained （PVE） running between 11.2 - 29.6%, and LOD of QTLs 2.66 - 4.81. Of those putative QTLs, three for TLN were detected on chromosomes 1, 2 and 11, with PVE of 17.3%, 15.3% and 13.7%, respectively; Two controlling Chl.a:b on chromosomes 3 and 4, PVE 13.8% and 29.6%; One controlling Chl on chromosome 1, PVE 11.2%. Four of those detected QTLs were newly reported in this study. Interestingly, the QTL controlling chlorophyll content, namely qCC-1 reported here, was detected in the region of the RFLP marker C122 on chromosome 1, where harbored NADH-glutamate synthase structure gene according to the previous study. Because the biosynthesis of chlorophyll begins with glutamate, qCC-1 would play a vital role in photosynthetic functions. Whereas, no QTL controlling chlorophyll content were detected 30 days after heading, suggesting that the effect of the QTL controlling chlorophyll content decreased during leaf senescence.2 QTL detection for traits associated with photosynthetic functions in rice, using backcross inbred linesA mapping population, the backcross inbred lines derived from a cross of Nipponbare/Kasalath//Nipponbare by the single seed descent method, was used to detect quantitative trait loci （QTL） for traits associated with photosynthetic functions. Total leaf nitrogen content （TLN）, chlorophyll a/b ratio （Chl.a:b） and chlorophyll content （Chl） were measured in leaves of rice （Oryza sativa L.） at 7th day after heading, and a total of eight putative QTLs were detected with percentage of variance explained （PVE） running between 9.7～21% and LOD of QTLs 2.61～6.42. Of those putative QTLs, two for TLN were detected on chromosomes 1 and 6, with PVE of 9.7% and 10.8%, respectively; Three controlling Chl.a:b on chromosomes 2、3 and 12, PVE 10%、10.5% and 21%; Three controlling Chl on chromosome 1、4 and 8, PVE 13.7%、10.7% and 10%. More interestingly, the QTL controlling chlorophyll content, namely qCC-1 in this population, was also detected in the region of the RFLP marker C122 on chromosome 1. So qCC-1 would play a vital role in photosynthetic functions. Two QTLs controlling Chl were detected on chromosomes 4 and 8 at the 30th day after heading, however, no QTL were also detected in the vicinity of C122, further suggesting that the effect of the qCC-1 controlling Chl decreased during leaf senescence.3 QTL detection for photosynthesis and its related physiological traits in rice, using chromosome segment substitution linesSixty-five chromosome segment substitution lines （CSSLs）, derived from a cross between an indica （IR24） and a japonica variety （Asominori） of rice （Oryza sativa L.）, were utilized for quantitative trait loci （QTL） mapping for photosynthetic and related physiological traits. Net photosynthesis rate （PHO）, stomatal conductance （SCO）, transpiration rate （TR）, intercellular CO₂ concentration （IC）, leaf chlorophyll （CHL）, and total leaf nitrogen content （TLN） were measured in flag leaves 7 d after heading. The CSSLs showed transgressive segregation for many of the traits, and significant correlations were observed for most of the traits. In total, ten QTLs were detected on chromosomes 1, 3, 4, 5, 7, 8, and 10 with a range of percentages of variance explained （PVE） stomatal conductance （qSCO-10） and intercellular CO₂ concentration （qIC-10） in the vicinity of C1166 on chromosome 10. No other overlapping loci associated with different traits were detected. These results suggested that the genetic mechanism of photosynthesis was very complex in rice.4 Map-based cloning of a virescent white leaf gene in riceThe virescent white leaf mutant （tentatively designated as vwl） was screened from a PA64S M₂ population mutagenized with ⁶⁰Co gamma rays. The mutant （named Yutu S, a Indica rice） leaves exhibit albino but finally turn to normal green after 3rd leaf stage. However, no difference in other agriculture traits was observed between the wild type and the mutant. Systematical research including photosynthetic pigment, chloroplast microscopic observation and gene cloning was carried out on the vwl mutant. The results are as follows:（1） The measurement of photosynthetic pigment in the leaves showed that the mutant had no neither chlorophyll a and chlorophyll b, norβ-carotenoid before the leaves turned to normal green, however, no difference in photosynthetic pigment was observed between the mutant and the wild-type leaves after the leaves turned to normal green. These results suggested that the deficient in the total photosynthetic pigment led to the albinism at the stage office seedling.（2） Transmission electron microscopic examination revealed that the mesophyll cells of the mutant contained some proplastid-like structures instead of the normal chloroplasts before the leaves turned to normal green, however, had plenty of normal chloroplasts which contain thylakoid membranes after the leaves turned to normal green.（3） The genetic analysis of the vwl mutant indicated that the phynotype of virescent white leaf was controlled by a recessive gene. （4） The vwl locus is primarily located between markers RM411 and RM6832, at the distances of 1.1 and 1.2 cM, respectively, on chromosome 3, using available SSR molecular markers and 672 mutant individuals of the F₂ population.（5） For fine mapping of the vwl gene, more PCR-based markers, such as SSR, CAPS and dCAPS, were designed on basis of public rice genome sequence data. Using these markers and over 2240 mutant plants of the F2 population, the high-resolution genetic map and BAC eontig of the vwl locus were constructed. Eventually, the vwl locus was narrowed to a 45-kb genomic DNA region between two markers, P45 and D8, and cosegregated with marker P50.（6） In the 45-kb target gene region, a gene （tentatively designated as OsPPR2） encoding putative pentatricopeptide repeat （PPR） protein was discovered at the expected position of vwl. PPR proteins belong to RNA-binding protein family, which have shown to influence stability or translation of specific chloroplast mRNA. We took OsPPR2 as the possible candidate gene of vwl. Sequencing the OsPPR2 and comparing the counterpart DNA region between the vwl mutant and the wild-type revealed that the mutant had 5-bp deletions within the coding region. The InDel marker I1 developed on basis of the deletion was used to survey over 2240 mutant plants of the F2 population and 20 different wild-type rice varieties. The results showed that the I1 was cosegregated with the vwl lucos, suggesting that the gene OsPPR2 is very likely VWL.更多还原