【作者】 陆广文；

【导师】 万建民；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 水稻品种的抽穗期主要由其感光性、感温性和基本营养生长性决定。感光性、感温性和基本营养生长性组合的多样性,使得抽穗期呈现多种多样的变化；这使得抽穗期的遗传表现异常复杂,呈现典型的质量/数量性状(QTL)遗传特征。因此,发掘和鉴定新的水稻抽穗期基因/QTLs,开展抽穗期基因精细定位、克隆等方面的研究,深入探讨水稻抽穗期基因的网络调控对于阐明植物开花的分子机理与合适生育期水稻品种的选育具有重要的理论和实践意义。利用Asominori遗传背景携带IR24插入片段的染色体片段置换系,并进一步构建近等基因系,我们成功地鉴定了一个感光基因dth2和一个控制低温生长的感温基因LTGl。本文中,我们还对LTG1的功能进行了分析,发现它的C端发生单个氨基酸替换后,使得水稻变得对低温极其敏感,影响了水稻的生长发育进程,进而影响水稻产量。LTG1还可能是一个驯化相关基因。主要结果如下：1.水稻抽穗期QTL定位及感光基因dth2的分离、鉴定与精细定位。利用源于籼稻品种IR24和粳稻品种Asominori的重组自交系(RILs)群体,检测到3个抽穗期QTLs,dth2,dth6和dth8,分别位于第2,6和8染色体,LOD值分别为3.82,5.23和5.46,贡献率分别为14.64%,20.03%和27.35%。这三个QTL分别和前人报道的Hd7,Hd1以及Hd5等位,鉴于Hdl和Hd5均已被克隆而Hd7仅仅被初步定位于相距20cM以上的标记C1221与C1408之间,我们选择dth2进行更深入、细致的研究。为了精细定位QTL dth2,利用以Asominori为背景亲本,IR24为供体亲本的染色体片段置换系(CSSLs)群体,鉴定出晚抽穗的携带dth2插入片段的家系CSSL23.从CSSL23与Asominori杂交构建的次级F2群体中,再次检测到dth2(LOD值24.70,贡献率26.22%)。此外,还检测到另一个QTL dth3(LOD值13.12,贡献率14.91%),该QTL和前人报道的Hd8等位,并被限定在3.2cM以内。利用南京自然长日照环境、人工遮光短日环境以及海南冬季自然短日等多个环境处理发现,dth2和dth3都具有在长日照条件下延迟抽穗的功能并且这两个QTL是独立遗传的,这两个感光性质相同的QTL之间的联合作用显著延迟了CSSL23的抽穗期。进一步利用分子标记辅助选择的方法(MAS),构建了仅携带dth2的近等基因系-NIL(dth2)并利用NIL(dth2)×Asominori的次级F2、F2：3以及F3：4群体,最终将dth2限定在标记RM13910与ind8之间约56.8kb的区间范围内,共有13个候选基因。序列分析发现第8个编码Zn-finger蛋白且与Hdl同源的基因在两亲本之间存在序列差异,导致氨基酸序列改变,暗示着该基因可能是目的基因。这些结果为对dth2的进一步图位克隆奠定了基础。2. LTG1的图位克隆与功能分析在本研究中,我们在携带IR24插入片段的Asominori染色体片段置换系(共66个家系)中,发现一个对温度极其敏感的家系CSSL13。利用构建的次级分离群体,将该基因定位于标记RM3762和RM263之间,并命名为LTG1。随后利用初定位的结果,通过回交以及分子标记辅助选择的方法,构建了微目的片段的近等基因系NIL(ltg1),将该基因进一步定位于标记dcapsl和ind14之间约25.4-kb的区间范围内,和标记L264、dcaps2共分离。该区间内有4个假定的ORFs,从序列比较和转基因的结果判断：ORF3是目的基因,它编码一个Casein kinase I亚家族的蛋白。尽管LTG1无论在高温还是低温条件下,在两个亲本之间的酶活都无显著差异,但在低温条件下,来自IR24位点的Ug1表达量明显减少。亚细胞定位表明,LTG1主要定位在细胞核和细胞质,并且不随温度的改变而改变。RT-PCR、Real-time PCR以及GUS染色结果表明LTG1是一个组成性表达的基因。为了探明LTG1的作用途径,利用基因芯片以及Real-time PCR分析了LTG1的可能参与途径,结果表明LTG1可能参与ABA作用途径,并且位于光周期基因Hd3口以及RFT1的上游。最后我们分析了LTG1各种基因型的地理分布以及感温性变化,并且探讨了LTG1在北方种植时对产量的影响以及其在稳定水稻抽穗期方面的潜在育种价值。更多还原

【Abstract】 Heading date (HD) is a key determinant for adaptation to different cultivation areas and cropping seasons in rice. Therefore, high-yielding potential combined with moderate heading date is a major target in rice breeding. Heading date is mainly affected by three factors, i.e. duration of the basic vegetative growth (BVG), photoperiod-sensitivity (PS) and temperature-sensitivity (TS). Diversification of heading date resulted from the complexity of the genetic mechanism of HD provides abundant resources for rice breeding in different habitats. However, it also synchronously enhances difficulty in breeding of rice cultivars with optimum growth duration. Therefore, identification, isolation as well as map-based cloning of novel genes/QTLs will be helpful for disclosing the molecular mechanism of flowering regulation pathway and provide guidance for breeding in rice.In this study, by using a set of chromosome segment substitution lines (CSSL), we successfully isolated a photoperiod sensitivity QTL dth2and a temperature sensitivity gene LTG1which controls low temperature growth. Then we mainly focused on LTG1and analyzed the function of LTG1.Our results suggested that LTG1is a domestication-related gene and the substitution of artificial selection in the Itgl region during domestication led to the transition from low temperature sensitive to insensitive. On the other hand, we found that LTG1controls yield when planted in a low temperature duration condition. Moreover, LTG1also plays a very important role in stabilizing rice HD, which is crucial for rice breeding. Taken together, the main founding are as follows:1. QTL analysis of heading date in rice and fine mapping of a photoperiod sensitivity allele, dth2, located on chromosome2In this study, the genetic basis of HD in rice was dissected into three quantitative trait loci (QTL, denoted dth2, dth6, and dth8) using71F7recombinant inbred lines derived from a single cross between Asominori (Japonica) and IR24(Indica). These three QTLs were located on chromosome2,6and8, respectively. The loci of these three QTLs overlap with previously reported heading date QTL Hd7, Hdl and Hd5, respectively. As Hdl and Hd5had been cloned and Hd7was only roughly mapped to about20cM region between markers C1221and C1408, we selected dth2for fine mapping. The genetic effect of dth2was first confirmed in the chromosome segment substitution line23(CSSL23) through its extremely late heading and transgressive phenotype. Then the QTL was remapped using the CSSL23/Asominori BC4F2population. The QTL dth3, corresponding to the previously reported hd8, was also detected in this population. The LOD score was13.12and the phenotypic variance explained was14.91%. Two nearly isogenic lines (NILs), NIL (dth2) and NIL (dth3), were developed from the BC4F2population using a marker-assisted selection strategy. Further analysis showed that dth2and dth3were independently involved in photoperiod sensitivity, resulting in the transgressive phenotype of CSSL23. Finally, based on the segregation of the HD phenotype and molecular marker genotype in NIL (dth2)/Asominori F2, F3, and F4populations, dth2was dissected into a single recessive gene located in a56.8-kb genomic region with13predicted open reading frames (ORFs). We speculated that ORF8was the candidate gene. This study provides a basis for map-based cloning of the dth2gene and guidance for controlling HD in rice breeding.2. Map-based cloning and functional analysis of the LTG1gene controlling low temperature growth in riceIn this study, by using CSSLs, we found a line CSSL13which was more sensitive to low temperature condition than Asominori. Further, we mapped this gene (named as LTG1) between markers RM3762and RM263by using the secondary segregation population. Then we constructed the near isogenic line (NIL) which harbored a very narrow target segment. By using this NIL and the advanced segregation population, LTG1was delimited to a25.4kb region located between markers dcapsl and ind14and co-segregated with markers L264and dcaps2. According to the genome annotation by TIGR, this region has four putative ORFs. Based on the sequence polymorphism beween the two parents and expression information, we speculate the ORF1and ORF3were the most probable target genes. We then confirmed that ORF3was the authentic target gene by genetic transformation experiments. Subsequently, Blast analysis demonstrated that ORF3encodes a putative casein kinase I subfamily protein. In order to clarify the potential reason of the phenotype differences between the two parents, we measured and compared the kinase activity of the LTG1protein encoded by the two parents under different temperature conditions. Moreover, we also analyzed the expression differences under different temperature conditions. Our results suggested that proteins encoded by the LTG1and Itgl allele may target different substrates to produce the phenotype differences under low temperature condition. The decreased expression of Itgl in NIL (Itgl) under low temperature condition may further enhance the low temperature sensitivity of Itgl. In addition, the LTG1protein was localized in the nuclear and cytoplasm and this subcellular location pattern did not change with the variation of the temperature. On the other hand, RT-PCR, real-time PCR and GUS staining analysis indicated that LTG1was a constitutively expressed gene. By using the microarray and real-time PCR analysis, we found that LTG1may participate in the ABA regulation pathway and HD pathway. LTG1may locate upstream of the HD genes Hd3a and RFT1. Finally, we also analyzed the geographical distribution and the temperature sensitivity variation of rice cultivars with different haplotypes of the LTG1gene. The potential breeding value of LTG1with regard to controlling yield and stabilizing HD was discussed.更多还原