【作者】 贾海燕；

【导师】 吴琴生； 马正强；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 遗传转化是作物遗传改良和基因功能研究的重要工具。小麦是比较难转化的禾本科作物之一，农杆菌介导的遗传转化效率目前还没有超过10％，远远低于水稻等作物的转化效率。转化受体植株再生困难是限制小麦遗传转化效率的主要因素，而且受基因型影响很大。小麦遗传转化主要以幼胚或其愈伤组织作为受体。除此之外，成熟胚和花药也已被尝试用作转化受体．与幼胚相比较，成熟胚的取材不受季节限制，有充足的材料来源．然而，其组织培养体系仍不成熟，限制了它在遗传转化中的应用。本研究的目的是阐明影响小麦组织培养特性的遗传基础，并试图建立较高效率的农杆菌介导的小麦遗传转化体系．我们首先进行了小麦成熟胚组织培养的遗传研究。普通小麦（Triticum aestivum L．）品种南大2419的成熟胚具有较高的组织培养能力．本研究利用<南大2419×望水白>重组自交系群体，对小麦成熟胚组织培养相关性状（TCR）进行了QTL分析。检测到5个控制愈伤组织诱导率（PEFC），4个控制愈伤组织植株再生潜力（PCRP）和4个控制愈伤组织的植株再生效率（NPRC）的染色体区段。其中，QPefc.nau-2A和QPcrp.nau-2A是分别控制PEFC和PCRP的主效QTL，均位于染色体2A的长臂上，分别解释22.1％和19.7％的表型变异率。同时，在染色体2D和5D上分别检测到一个控制NPRC的QTL，两者一起可以解释51.6％的表型变异率。另外，分别在染色体2A、2D、5A、5B和5D的不同区段上检测到至少两个与TCR相关的QTL。其中，QPefc.nau-2A和QPcrp.nau-2A是分别控制PEFC和PCRP的主效QTL，均位于染色体2A的长臂上，分别解释22.1％和19.7％的表型变异率。同时，在染色体2D和5D上分别检测到一个控制NPRC的QTL，两者一起可以解释51.6％的表型变异率。为了研究小麦幼胚组织培养特性的遗传规律，我们分别于2005年在温室和2006年在大田取材，进行了两次试验，定位了控制小麦幼胚组织培养特性的QTLs。发现了11个控制幼胚胚性愈伤组织诱导（PEFEC）和2个控制胚性愈伤组织植株再生效率（PCRP）的QTLs。在两次试验中，都检测到主效位点QPefec.nau-3B.2、QPefec.nau-5B和QNppc.nau-3A。与这些QTLs位点紧密连锁的分子标记，一方面可用于筛选具有较高组织培养能力的基因型材料，另一方面有助于对小麦组织培养能力进行遗传改良。结合前人研究的结果，我们认为小麦的第2群、第3群和第5群染色体上存在控制小麦组织培养能力的相关基因，这些相关基因在禾谷类作物中是保守的。小麦幼胚、成熟胚和花药的组织培养能力由相同的基因或基因组合控制。为了提高农杆菌介导的小麦遗传转化的效率，研究了转化前预培养、农杆菌菌株和菌液浓度对小麦遗传转化效率影响。我们发现在转化前将愈伤组织小块置于含有低浓度的细胞分裂素的分化培养基上预培养3天，可以提高株系B31697的遗传转化效率。利用株系B31394进行的转化实验表明，农杆菌菌液浓度为OD₆₀₀=0.6～0.8比较合适。在AGLI、EHA101、EHA105和LBA4404这4种农杆菌菌株中，菌株LBA4404的转化效率最低。以上述研究为基础，对69个材料进行了遗传转化实验，获得了遗传转化效率较高的小麦基因型材料。这些材料的转化效率明显高于目前小麦遗传转化中普遍使用的扬麦158和Bobwhite。此外，我们还构建了小麦泛素融合降解蛋白基因（UFD）、小麦甜菜素-葡糖基转移酶基因（BoGT）、小麦钙网蛋白基因（Crt）和小麦胚胎后期发育积累蛋白基因（LEA）的RNA干扰载体以及LEA基因的过量表达载体。这些基因均是本实验室克隆的基因，这些载体的构建为研究这些基因的功能打下了基础。更多还原

【Abstract】 Genetic transformation is an important tool for studying genes function and breedingnew varieties. Wheat （Triticurn aestivum L） is one of the cereal crops recalcitrant totransformation, in literatures, the highest transformation efficiency obtained throughAgrobacterium tumefaciens- mediated was just over 10％, which is far below than that ofrice. One of the main factors limiting transformation is the callus regeneration ability,which is dependent of genotype. Immature embryos are currently the main source forgenetic transformation but mature embryos and anthers have also been used. Matureembryos are arguably one of the best explants for genetic transformation because of itsunlimitedness in sources and no growth season restriction. However, efficient regenerationsystem using the mature embryos as explants in wheat is still not available. This studyaimed at clarifying the genetic basis of wheat tissue culture response and to establish moreefficient Agrobacteriurn tumefaciens-mediated transformation system in wheat.We started with studying the genetic analysis of tissue culture response （TCR） of wheatmature embryos. We found that callus derived from mature embryos of wheat cultivar’Nanda2419’ has good regeneration ability. Using a RIL population derived from cross of’Wangshuibai’ with ’Nanda2419’, QTLs for callus induction from mature embryos andcallus regeneration were mapped. Through whole genome scanning, we identified five, fourand four chromosome regions conditioning, respectively, percent embryos forming callus（PEFC）, percent calli regenerating plantlets （PCRP）, and number of plantlets perregenerated callus （NPRC）. The major QTLs QPefc.nau-2A and QPcrp.nau-2A weremapped to the long arm of chromosome 2A, explaining up to 22.8％and 17.7％of therespective phenotypic variation. Moreover, two major QTLs for NPRC were detected onchromosomes 2D and 5D, which together explained 51.6％of the phenotypic variation.To study the genetic mechanism underlying the TCR of immature embryos of wheat, callus induction and regeneration using the above-mentioned population were conductedtwice. In the first trial, the immature embryos were sampled from plants grown in thegreenhouse in the winter of 2005; in the second trial, the immature embryos were sampledfrom plants grown in the field in the summer of 2006. Through whole genome screening,eleven chromosome regions conditioning percent embryos forming embryogenic callus（PEFEC） and two conditioning the percent caUus-let regenerating plant （PCRP） wereidentified. The main loci QPefec.nau-3B.2、QPefec.nau-5B and QPcrp.nau-3A weredetected in both experiments.Based on this study and other reports, group 2, 3 and 5 chromosomes of wheat areimportant for tissue culture, and the corresponding genes were conserved among cereals.TCR of immature embryos, mature embryos and anthers of wheat might be under control ofsome common or tightly linked genes. The PCR-markers linked to TCR QTLs wouldfacilitate germplasm identification and genetic improvement of wheat tissue culture.To improve the efficiency of Agrobacterium-mediated genetic transformation in wheat,pre-culture times, Agrobacteria strains and cell densities were studied. As a result, 3dpre-culture on the regeneration medium we used is beneficial to transformation of lineB31697. For line B31394, OD₆₀₀=0.6～0.8 was the optimal cell density of theAgrobacterium strain used in transformation. Among the four Agrobacterium strain usedin transformation, including AGL I, EHA101, EHA105 and LBA4404, LBA4404produced the lowest transformation efficiency. Based on these results, 69 genotypes weretransformed and genotypes with transformation efficiency higher than that of controlcultivars Yangmai158 and Bobwhite were obtained.In addition, we constructed the RNAi vectors of wheat ubiquitin fusion degradationprotein gene （UFD）, Betalain Glucosyl transferases gene （BoGT）, calreticulin （Crt） and lateembriogenesis abundant protein gene （LEA）. We also constructed an over-expression vectorfor the LEA gene.更多还原