【作者】 蔚亦沛；

【导师】 温孚江；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 水稻是世界上最重要的粮食作物之一,全球约二分之一的人口以稻米为主食,水稻也是受病虫害最多的作物之一。由Xanthomonas oryzae pv. oryzae引起的水稻白叶枯病是世界范围的细菌性病害,严重影响水稻的产量和品质。水稻每年因鳞翅目害虫(二化螟、三化螟、稻纵卷叶螟、稻螟蛉、稻苞虫等)为害造成的损失占总产量的10%-30%。另外,杂交稻纯度和稻田杂草影响水稻的产量与品质,使用除草剂费工费力,增加了生产成本,已成为生产上的主要问题。抗病、抗虫、抗除草剂转基因水稻的培育为水稻的高产与稳产提供重要保证的同时,可以减少化学农药的使用,改善环境质量。实践证明,将多个抗性基因聚合在同一水稻品种中能有效提高水稻的抗性和保持抗性持久,分子标记辅助选择以其快速、准确的选择优势成为实现基因聚合育种的有效手段。对转基因水稻中外源转cry1Ab基因进行定位,构建转cry1Ab基因水稻的分子遗传图谱,可为转cry1Ab基因水稻的培育进行标记辅助选择提供更多的分子标记,同时为研究转cry1Ab基因水稻的基因组变化,进行转基因水稻中外源基因的位置效应奠定基础,为转cry1Ab基因水稻生物安全性评价提供更多的理论支撑。本论文主要开展了三个方面的研究:多抗性(抗白叶枯、螟虫、除草剂)转基因水稻恢复系的培育;水稻香味基因与抗白叶枯基因的聚合育种;转基因水稻中转cry1Ab基因的分子标记定位。主要研究结果如下:1.多抗性(抗白叶枯、螟虫、除草剂)转基因水稻恢复系的培育(1)将恢773与携有转cry1Ab和bar基因的水稻新品系中国91(cry1Ab)杂交,并以恢773作为轮回亲本,连续回交三代和自交一代,对各回交世代的单株进行cry1Ab和bar基因的PCR分析、田间抗虫性鉴定和除草剂Basta抗性鉴定,经过选择获得了转基因水稻恢复系T773-1,田间种植对螟虫及除草剂Basta表现很好的抗性。(2)将恢773与带有转Xa21基因的水稻新品系豫粳6(Xa21)杂交,并以恢773作为轮回亲本,连续回交三代和自交一代,经过PCR分析、GUS活性测定和白叶枯病菌接种鉴定,获得带有Xa21基因的并具有优良农艺性状的转基因水稻恢复系T773-2,田间种植表现很好的水稻白叶枯病抗性。(3)转cry1Ab和bar基因的水稻恢复系T773-1和转Xa21基因的恢复系T773-2进行杂交、自交,对自交世代单株进行cry1Ab、bar和Xa21基因的分子标记辅助选择,获得了带有三基因的恢复系T773,田间种植对水稻白叶枯病、鳞翅目害虫、除草剂具有很好的抗性。并利用恢复系T773与不育系早花2A进行杂交,进行配合力测定,获得了抗白叶枯病、螟虫与除草剂的多抗转基因水稻恢复系T773。T773及其杂交后代的抗谱和抗性水平达到其抗性基因供体亲本的水平,抗性改良效果明显。2.水稻香味基因与抗白叶枯基因的分子标记聚合育种通过分子标记辅助选择与传统的杂交育种技术有机结合,将香稻栽培品种香粳9407与抗白叶枯病多基因聚合系IRBB60(Xa4、xa5、xa13、Xa21)进行杂交、自交,采用田间病原菌接种鉴定和香味鉴定筛选,结合农艺性状考查进行选择,在自交F2代获得两个抗白叶枯基因和香味基因聚合系(2R+fgr)1株;在自交F3代获得四个抗白叶枯基因和香味基因聚合系(4R+fgr)1株,三个抗白叶枯基因和香味基因聚合系(3R+fgr)2株,两个抗白叶枯基因和香味基因聚合系(2R+fgr)7株,一个抗白叶枯基因和香味基因聚合系(1R+fgr)3株;在自交F4代获得四个抗白叶枯基因和香味基因聚合系(4R+fgr)10株,三个抗白叶枯基因和香味基因聚合系(3R+fgr)28株,两个抗白叶枯基因和香味基因聚合系(2R+fgr)20株。获得的多基因聚合系单株高抗水稻白叶枯病并且具有浓郁的香味,主要农艺性状优良,可直接应用于抗白叶枯病香稻育种,对水稻抗性育种和品质改良具有重要的实践意义。3.转基因水稻中转cry1Ab基因的分子标记定位(1)首先利用纯合稳定的转cry1Ab基因的中国91株系与IRBB60杂交,对其F2代分离群体的单株进行除草剂Basta抗性检测,结果显示,在F2代分离群体中cry1Ab基因的分离符合期望的3:1分离比例,遵循孟德尔分离规律,证明了cry1Ab基因在转基因株系中以单显性位点整合。(2)利用Tail-PCR技术获得转基因水稻中外源cry1Ab基因插入的侧翼序列,通过序列比较分析确定了转cry1Ab基因在水稻基因组插入的物理位置。根据插入位置附近的水稻基因组序列进行微卫星序列的搜索,并利用Primer Premier 5.0软件设计了24对SSR标记引物。(3)利用设计的24对SSR引物对亲本转cry1Ab基因的水稻中国91与IRBB60之间进行多态性筛选,获得了6对多态性引物,然后利用这6对引物以及公开引物RM1261对杂交的F2代分离群体240个单株进行分析。依据每个单株的基因型和表型数据,利用MAPMAKER/EXP Version 3.0软件进行分析,构建了外源转基因cry1Ab在水稻第12染色体的遗传图谱,外源cry1Ab基因被定位在标记CM22和RM1262之间,两个标记相距9.8cM。微卫星标记CM22、CM01、CM12、CM13位于cry1Ab基因的一侧,与cry1Ab基因的遗传距离分别为4.6cM、4.2cM、3.1cM和2.7cM;微卫星标记CM24、CM10和RM1261位于另一侧,与cry1Ab的遗传距离分别为3.1cM、3.5cM和5.2cM。更多还原

【Abstract】 Rice is one of the most important crops in the world, providing staple food for more than half of the world’s population, but also one crop tends to be infested by many kinds of diseases and pests. Bacterial blight caused by Xanthomonas oryzae pv. oryzae, a bacterial disease of rice worldwide, affects the yield and quality of rice seriously. The losses in rice yield caused by lepidopteran insects (Chilo suppressalis walker, Tryporyza incertulas, Cnaphalocrocis medinalis Guenee, Naranga aenescens Moore, Borbo cinnara Wallace) can account for 10-30% of total yield. In addition, purity of hybrid rice and field weeds affect the yield and quality of rice,the use of herbicide to control field weeds increases the production cost, these have been the main problems in rice production. So breeding of transgenic rice with multiple resistance to diseases, insects and herbicide can ensure the quantity and quality of rice, decrease the use of pesticides and be beneficial to the environment. Many studies had indicated that pyramiding multiple resistant genes into one rice variety can significantly enhance the resistance and keep durable resistance of rice. Molecular marker-assisted selection (MAS) has been advocated as a highly efficient breeding method for pyramiding multiple resistant genes, because it can offer rapid and precise selection of the target gene.Molecular mapping of transgene cry1Ab and genetic linkage map construction of transgenic cry1Ab rice increases molecular markers used for marker-assisted selection in transgenic cry1Ab rice breeding. Furthermore, this research establishes the foundation to investigate the transgene location affection, and provides the theory theoretic support for transgenic rice penalty evaluation.The objective of this paper was to study from three aspects, respectively: Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide; Marker-assisted breeding of pyramiding rice fragrance gene and multiple bacterial blight resistance genes; Molecular mapping of transgene cry1Ab in transgenic rice. Main research results are as follows: 1. Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide(1) Transgenic rice line Zhongguo91 (carrying cry1Ab and bar gene) and elite restorer line Hui773 were crossed, then backcross was carried out three times using Hui773 as recurrent parent and self-cross was carried out one time. All plants were subjected to Basta painting and PCR analysis to verify presence of the cry1Ab and the bar genes. Through selection we obtained transgenic restorer line T773-1 with good agronomic traits and obvious resistance to striped stem borer and herbicide.(2) Transgenic rice line Yujing6 (carrying Xa21 gene) and elite restorer line Hui773, then backcross was carried out three times using Hui773 as recurrent parent and self-cross was carried out one time. All plants were subjected to GUS activity assay and PCR analysis to verify presence of the Xa21 gene. In BC3F2 population we obtained transgenic restorer line T773-2 with good agronomic traits, showing high resistance to bacterial blight disease under field condition.(3) Transgenic rice line Zhongguo91 (carrying cry1Ab and bar gene) and transgenic rice line Yujing6 (carrying Xa21 gene) were crossed. Through marker-assisted selection in self-cross populations we obtained transgenic restorer line T773 carrying three genes (cry1Ab, bar and Xa21) with resistance to bacterial blight, striped stem borer and herbicide. Transgenic restorer line T773 showed high resistance to bacterial blight disease, striped stem borer and herbicide under field condition. The hybrid F1 generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis.2. Marker-assisted breeding of pyramiding rice fragrance gene and resistance gene to bacterial blightThrough the combination of marker-assisted selection and conventional cross breeding technology, we crossed Xiangjing9407 and transgenic pyramiding rice line IRBB60 (carrying Xa4, xa5, xa13, Xa21), screened the self-cross population using inoculation appraisal technology and fragrance identification to obtained the multiple resistance gene and fragrance gene pyramiding plant, supplemented by investigation of agronomic traits. We obtained 1 individual plant of (2R+fgr) pyramiding in F2 self-cross population. And we obtained 1 individual plant of (4R+fgr) pyramiding, 2 individual plant of (3R+fgr) pyramiding, 7 individual plant of (2R+fgr) pyramiding, 3 individual plant of (1R+fgr) pyramiding in F3 self-cross population. Finally, we obtained 10 individual plant of (4R+fgr) pyramiding, 28 individual plant of (3R+fgr) pyramiding, 20 individual plant of (2R+fgr) pyramiding in F4 self-cross population. All the plants selected had rich aroma and high resistance to bacterial blight, with excellent agronomic traits, can be used to breed aroma rice with resistance to bacterial blight. This study has important significance of rice resistance breeding and quality improvement practice.3. Molecular mapping of transgene cry1Ab in transgenic rice(1) First, the F2 segregation population plants of transgenic Zhongguo91 (cry1Ab)×IRBB60 were subjected to herbicide Basta resistance detection. The result demonstrated that the segregation of cry1Ab gene was the ratio of 3:1, following Mendelian segregation law. The result proved that the cry1Ab gene was single dominant locus integrated in transgenic rice.(2) The flanking sequences of cry1Ab gene insertions in transgenic rice genome were rescued by Tail-PCR. Then using sequence blast analysis on NCBI website we confirmed the cry1Ab gene insertions location in chromosome 12 of rice. Using software“simple sequence repeats identification tool”and“Primer Premier 5.0”, 24 pairs of primers were designed on the basis of the rice genome sequence close to the transgene cry1Ab insertions location. (3) Polymorphism screen using parent Zhongguo91 (cry1Ab) and IRBB60 we obtained 6 pairs of primers with polymorphism, then using the 6 pairs of primers and primer RM1261 we analyze the genotype of 240 plants in F2 segregation population. By“MAPMAKER/EXP Version 3.0”software we construct the molecular genetic linkage map of exogenous cry1Ab gene in 12 chromosome of rice genome according to their genotype and phenotype. The result indicated that exogenous cry1Ab gene is located between the SSR markers CM22 and RM1261 in 12 chromosome of rice genome. SSR marker CM22, CM01, CM12, CM13 were found to be linked to cry1Ab gene with genetics distances of 4.6, 4.2, 3.1 and 2.7 respectively, close to the short arm side; SSR marker CM24, CM10, RM1261 were found to be linked to cry1Ab gene with genetics distances of 3.1, 3.5 and 5.2 respectively, close to the other side.更多还原