【作者】 施利利；

【导师】 郭玉华； 王松文；

【作者基本信息】 沈阳农业大学 ， 作物遗传育种， 2003， 硕士

【摘要】 水稻是世界上重要的粮食作物，并已成为分子生物学研究的模式作物之一。本研究利用农杆菌介导法将Bar基因导入北方粳型恢复系津稻162和津稻18中。构建了阿特拉津氯水解酶基因植物表达载体，并将其转入粳稻保持系滓稻107中。本研究的主要结果： 1．利用农杆菌介导法将pDsBar1300/EHA105转入水稻恢复系津稻162和津稻18中，分别获得77株和55株独立转化株系，并对转基因T0代进行了Basta涂布试验，其抗性植株率分别为92％和78％。对抗性转基因株系做PCR检测，均能扩增出400bp的Bar基因特异条带，证明外源基因已整合到水稻基因组中。对转基因T1代进行潮霉素抗性鉴定，除个别株系分离比异常外，大多数转基因株系表现3：1的分离。 2．构建了阿特拉津氯水解酶基因植物表达载体p1301-atzA。构建方法为：第一步将用EcoRⅠ和HindⅢ双酶切得到的atzA基因与也经过这两个酶双酶切的pBluescript Ⅱ SK(+/-)载体连接，构建成pBS-SK-atzA，用EcoRⅠ和HindⅢ双酶切鉴定重组子插入片段大小为1.5kb；第二步将pBS-SK-atzA用SalⅠ和SacⅠ双酶切，同也经过这两个酶双酶切的pCambia1301载体连接，用SalⅠ-SacⅠ双酶切鉴定重组子插入片段大小为1.5kb，从而构建成p1301～atzA。通过冻融法将p1301-atzA导入农杆菌EHA105，完成了atzA基因植物转化系统的构建。 3．利用构建完成的atzA基因植物转化系统，对水稻保持系津稻107进行了转化，共得到249株独立的转基因株系，经PCR和PCR—Southern检测证明atzA基因已整合到水稻基因组中。 4．对转抗除草剂基因atzA水稻做除草剂抗性检测，结果表明，不同转基因水稻株系对除草剂阿特拉津普遍具有抗性，但抗性表达水平存在差异。更多还原

【Abstract】 Rice is the most important food crop in the world and has become one of the model crop for the research of molecular biology. Exogenous Bar gene and atzA gene were introduced into rice restoring lines and maintaining lines respectively. The main results are as follows:1. Two japonica varieties were used for the genetic transformation (Oryza saliva L.) by using Agrobacterium tumefaciences EHA105 harboring binary vector containing Bar gene and selectable marker gene of HPT. 77 and 55 transgenic rice lines were obtained respectively. PCR analysis showed that Bar gene had been integrated into the rice genome. The segregation of HPT gene in most of T1 progeny corresponded to the Mendelian ratio.2 . An Agrobacterium binary vector carrying chlorohydrolase gene was successfully constructed based on pCambia1301. The stragety of construction plant binary vector is as follows: first, the target gene atzA has been prepared by digestion with EcoRI/HindⅢ, then the fragment can be ligated with vector pBluescript SK(+/-) cut with the two same enzyme. The recombinant plasmid pBS-SK-atzA was digested by EcoRI/HindⅢ, which can separate the 1.5kb atzA gene fragment ; Second, plasmid pBS-SK-atzA and pCambiaBOl were digested by EcoRI/HindⅢ at the same time, then ligating the two fragment into a new plasmid vector p1301-atzA.3 . The target gene was successfully introduced into mature embryos of a Japonica rice cultivar Jindaol07 by Agrobacterium-mediated transformation. 249 transgenic plants were obtained. The exogenous atzA gene in those transgenic plants that expressing resistance to herbicide atrazine was confirmed with PCR and PCR-Southern blot analysis .4. The results of herbicide resistance of transgenic rice showed that most of transgenic rice plants have behaved common herbicide resistance to atrazine , but the expression level of herbicide resistance is different.更多还原