【作者】 李明浩；

【导师】 王秀娥；

【作者基本信息】 南京农业大学 ， 作物遗传育种学， 2010， 硕士

【摘要】 为了建立和优化Alondra’s的高效再生及遗传转化体系,为小麦遗传转化提供更多的受体基因型。本研究以Alondra’s的幼胚为外植体,研究了培养基种类、不同激素等对其幼胚愈伤组织的诱导及其再生的影响。结果表明,在使用N6培养基时,添加3 mg.L-1的2,4-D并附加1000 mg.L-1的CH对其愈伤的诱导效果较好；添加4 mg.L-1的ZT、不附加IAA对其分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6,利用基因枪法将Hyg基因导入到Alondra’s幼胚愈伤组织,以探索Alondra’s的高效遗传转化体系。最终在含100 mg.L-1潮霉素选择培养基上筛选、分化,获得了30株抗性植株,经PCR检测,其中5株为阳性转基因植株,转化率为0.5%。Alondra’s遗传转化体系的建立丰富了小麦遗传转化的基因型,为小麦品种的转基因改良和在不同背景中研究基因的功能奠定了良好的基础。农杆菌介导遗传转化具有外源基因表达稳定、一般为单拷贝等优点,遗传转化效率低、体系不稳定限制了该转化方法在小麦中的广泛应用。为了优化农杆菌介导的小麦遗传转化条件,本研究以普通小麦品种扬麦15和Alondra’s的幼胚产生的愈伤组织为受体材料,通过检测Gus基因的瞬时表达情况,比较了农杆菌介导的主要影响因子的转化效率。结果表明,在相同条件下两个品种农杆菌侵染的最佳条件不尽相同。扬麦15的最佳优化条件为：先在不添加AS的培养基中预培养,然后在菌液浓度为OD600=1.0、AS浓度为100μmol/L条件下侵染10min,最后在25℃共培养Id; Alondra’s则在菌液浓度为OD600=0.5、共培养时间为2d时,表现出最佳的Gus基因瞬时表达率。植物在逆境胁迫及病原菌侵染下,体内活性氧会大量积累,从而破坏正常新陈代谢时清除平衡,活性氧的清除系统对于维持植物的正常生理功能及正常的新陈代谢具有重要的意义。因此,通过植物基因工程的方法,将能够清除植物体内活性氧类物质的过氧化物酶基因导入到综合性状优良的小麦品种中,对于提高小麦的抗氧化胁迫能力有着重要的意义。本实验室在扬麦5号及与其回交7代的小麦/簇毛麦6VS/6AL易位系为材料建立的白粉菌诱导的叶片SSH文库中,克隆了一个小麦抗坏血酸过氧化物酶基因(Ta-APX),并发现该基因在白粉菌侵染初期发挥一定作用。为了进一步研究该基因在抗白粉病中的功能及其作用机制,研究其抗氧化能力与白粉菌侵染应答的关系。本研究在构建植物表达载体pAHC-Ta-APX的基础上,利用基因枪法将其导入到小麦品种扬麦158的幼胚愈伤组织。在含除草剂的培养基上经过筛选和分化,最终获得38株抗性再生植株。根据bar基因、ubi启动子及目的基因序列设计引物,对38株再生植株进行PCR鉴定,最终共获得10株含目的基因的阳性植株。为了研究转基因植株的抗氧化胁迫性,分析了PEG6000诱导及白粉菌胁迫下T1代转基因植株内APX酶活性,结果表明,在两种胁迫诱导下转基因株系的APX酶活性均高于对照植株。在大田条件下对T1代转基因植株进行抗白粉病和赤霉病接种鉴定,结果表明,转基因植株的白粉病和赤霉病抗性均有不同程度的提高,推测转基因植株通过过量表达Ta-APX基因提高了其抗氧化胁迫性,进而提高了对植物病原菌侵染的抵御能力。对转基因T1代植株的主要农艺性状调查结果表明,除株高外转基因植株的其它主要农艺性状与对照相比差异不显著。更多还原

【Abstract】 In order to establish an optimized system for callus induction, regeneration and high-efficient genetic transformation of wheat variety Alondra’s, the effects of culture medium, hormones on the callus induction and plant regeneration of immature embryo were studied. It is found that when using the N6 medium, the use of 3 mg.L-12,4-D together with 1000 mg.L-1 CH got the best results for callus induction, and use of 4 mg.L-1ZT without any IAA had the highest regeneration frequency. The Vector pCAMBIA1301-220.6 was constructed and transformed into Alondra’s by particle bombardment. Thirty plants with hygromycin-resistance were selected and regenerated. Among them,5 were identified to be positive by PCR, with a transformation frequency as 0.5%. The establishment of the system further enriched the wheat genotype for transformation, and this will be helpful for both the genetic improvement and gene function analysis in various background by genetic transformation.Agrobacterium-mediated transformation system has advantages for its singly copy insertion and stable expression of the transgene. However, in wheat, this system is low repeatable and the transformation efficiency is relatively low compared with other methods. This limits the its widely used in wheat transformation. In order to optimize the agrobacterium-mediated genetic transformation in wheat, using the callus induced from immature embryo of wheat varieties Yangmai 15 and Alondra’s as materials and using the transient expression array of the Gus gene, main factors affecting the transformation efficiency was studied. The results showed that the optimized condition for the above two varieties were different under the same treatment. The best condition for Agrobacterium-mediated transformation in Yangmai 15 are:Firstly, pre-cultured without acetosyringone, then infected for 10 min with Agrobacterium tumefaciens bacterial at a concentration of OD600=0.2 and acetosyringone at a concentration of 100μmol/L, finally co-cultured for 1 day at the temperature of 25℃; Under the similar condition, the most transient expression frequency of Gus gene was obtained in Alondra’s when infecting with Agrobacterim tumefaciens bacterial at a concentration of OD600=0.5 and then co-culturing for 2 days.Oxidative stress is one of the major factors causing injury to plants exposed to environmental stresses. Cells of plants will produce reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion radical, and hydroxyl radicals when encountering injurious environments. The sweep off ROS is an mechanism in plant for the release of hurt from injuries. Genetic transformation is an important alternative for improving ROS resistance level, and the cloning of related genes is critical for this approach. In our lab, using a isogenic line with powdery mildew resistance and its recurrent parent Yangmai 5, a SSH library induced by Erysiphe graminis DC.f.sp. tritici was constrcted. A full-length cDNA gene coding the ascorbate peroxidase was cloned and designated as Ta-APX.To characterize the function of this gene in ROS and powdery mildew resistance, a expression vector pAHC-Ta-APX was constructed and Ta-APX was transformed into wheat variety Yangmai 158 by microproject-bombardment. After two rounds of herbicide bialaphos selection and regeneration, we obtained 38 regenerated plants. These herbicide-resistant plants were further identified by PCR using primers of the Bar gene and the target gene, and 10 transgenic plants were identified. The To derived T1 progenies were identified by PCR using the primer of the target gene, and the result showed that all the T1 lines segregated with different segregation ratios. This indicated that the To plants were heterozygous for the Ta-APX, and the copy number were different from one line to another. The T1 progenies were evaluated for powdery mildew and scab resistance by artificial inoculation under the field condition, and all the T1 lines showed improved resistance at different level. However, the resistance level of different T1 lines showed significant difference, and there was also segregation for resistance with the same T1 lines, which is in accordance with the PCR results. The APX enzyme activity of two lines. T0-8 and T0-1, were further analyzed, the results showed that the enzyme activity was higher in the transgenic plants than that in the non-transgenic plants at all the analyzed time points in both treatments of powdery mildew and PEG6000,indicating that the transgenic plants improved their ability to regulate the enzyme activity under the environmental stresses, and this might contribute to their improved plant pathogenic resistance. The major agronomic traits of the T1 transgenic plants were also investigated. Compared with the control, the transgenic lines showed no significant difference for all the investigated traits except plant height.更多还原