【作者】 崔洪志；

【导师】 郭三堆；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2004， 博士

【摘要】 常规的栽培及育种技术使粮食产量再上一个大的台阶难度很大。面对人多地少的矛盾，解决未来我国粮食安全问题必须依靠一次新的农业科技革命，也就是转基因农作物的产业化。在转基因农作物的研究中，人们至今仍对外源基因与植物基因组整合的机制所知有限，尤其针对具有我国特色的花粉管通道法遗传转化机理的相关研究更少。本研究在我国转基因抗虫棉研究的基础上，一方面，进行了透明颤菌血红蛋白基因转基因植物的研究，分析了其在增产转基因作物研究方面的作用；另一方面，对抗虫棉GK12中杀虫基因的整合进行了初步分析，对于进一步揭示花粉管通道遗传转化的机理、改进并提高转化效率以及尝试通过同源重组实现定位整合的研究等方面都具有积极的意义。 根据透明颤菌血红蛋白的氨基酸序列，按照植物偏爱密码子，人工设计合成了vgbM基因，并成功构建了植物表达载体pGBI4ASVHB。在对通过农杆菌介导法获得的转基因烟草的研究中发现：该基因的表达可增加烟草的生长量，并促进提早开花和增加花量，叶绿素含量增加23.4％。进一步构建了vgbM和GFM Cry1A Bt双价基因植物表达载体pGBI4ABV，通过花粉管通道法导入棉花，获得了转双基因的7个株系。转基因棉花在表现对棉铃虫显著抗性的同时，还发现其萌发速度较快，叶绿素含量平均增加13％左右，花期明显提前，结铃数显著增加。本研究首次报道了转透明颤菌血红蛋白基因的棉花在增产和抗涝性提高两方面具有作用，并注意到了叶绿素含量测定可作为检测vgbM基因表达的指标，为进一步获得增产转基因农作物提供了研究基础。 本研究还通过改进的基于单链及巢式PCR的染色体步移方法，对通过花粉管通道法转化获得的抗虫棉GK12中杀虫基因整合的旁侧序列进行了分析。结果发现：整合在棉花基因组中的杀虫基因表达盒和其它外源DNA片段发生了串联重组，支持了外源基因整合的“两步整合机制”模型。并发现了整合过程有拓扑异构酶和转座作用参与的分子证据；同时证明了构建在普通质粒上的外源基因可以通过花粉管通道法成功转化植物。暗示了不包括载体骨架的外源基因表达盒片段直接转化作物是可行的。这对于提高转基因植物的安全性具有一定的意义。更重要的是，没有载体骨架序列的干扰对于通过同源重组实现外源基因在植物中的定位整合(基因打靶)也是有利的。利用整合旁侧序列分析的结果，结合影响外源基因表达的已知相关因素，直接将外源目的基因定位整合到‘稳定高表达’位置，可简化转基因农作物研究中的筛选鉴定程序，建立无载体参与的外源基因片段植物转化新模式。无疑对于促进转基因农作物的研究和产业化具有重要意义。更多还原

【Abstract】 To increase the yield of crops in a large scale, the potentiality of traditional breeding and cultivation technology is rather limited so far. Therefore, a new agricultural revolution namely genetic modified crop is indispensable to solve the problems of the future food security in China. However, the integration mechanism between foreign gene and the plant genomic DNA is still an enigma about the transgenic plant. Especially few endevours had been done to discover the mechanism of the pollen tube pathway transformation method, which was developed by Chinese scientist. On the basis of the achievement of the Bt cotton, this study, on the one hand, had developed vgbM (modified Vitreoscilla hemoglobin gene) transgenic plants and evaluated the effect of the GM crop on yield improvement. On the other hand, the integration loci of the insecticidal gene on GK12 Bt cotton were analyzed. The result will be helpful to the studies in understanding the transformation mechanism, improving gene transfer efficient, and the site-specific integration by homologous recombination.According to the amino acid sequence and plant preference codon usage, vgbM gene was designed and synthesized and the plant expression vector pGBI4ASVHB was constructed successfully. Studies found that the transgenic tobacco plants obtained by agrobacterium-mediated transformation exhibited enhanced growth and showed more flowering rates. Meanwhile, the bloom date moved up. Furthermore, transgenic plants contained, on average, 23.4% more chlorophyll in leaves. A bivalent plant expression vector pGBRABV carrying both vgbM gene and Bt insecticidal gene was constructed in further. Seven transgenic lines were obtained by pollen tube pathway transformation. The transgenic cotton plants had showed obvious cotton bollworm resistance. Simultaneously, we also found their geminating speeded up, leave chlorophyll content improved by 13%, flowering date moved up and the boll number increased significantly. This work had proved that the transgenic cotton expressing Vitreoscilla hemoglobin showed both yield improvement and water logging resistance. We also noticed that chlorophyll content was a good mark to judge the expression of Vitreoscilla hemoglobin in transgenic plant. This work had laid a research foundation for developing yield improvement transgenic crops in further.This study also developed a genome walking method based on single chain PCR and nested PCR. Utilizing the method, the integration flanking sequence of the insecticidal gene in GK12 cotton was analyzed. We found the CrylA expression box had recombined with other foreign DNA fragment. The result had supported the ’Two phase integration mode’. Meanwhile, we found some evidence for that topoishomerase and transposon may involve with foreign DNA integration process. We also proved foreign gene harbored in a normal small plasmid can be transformed successfully by pollen tube pathway transformation. This suggested that a vector-free transformation is feasible. So it is beneficial for GMO safety evaluation. More important, it is favorable for site-specific integration (gene targeting) through homologous recombination without the vector impediment. According the integration flanking sequence and considering the know factors that may influence foreign gene expression, if the foreign gene can be targeted on a ’stable-expression’ position directly, the tedious screening procedures during GM crop development could be simplified. The setup of a vector-free new plant transformation mode will undoubtedly promote the studies and the commercialization of the GM crops.更多还原