利用Cre/lox重组系统建立番茄基因工程雄性不育恢复系

 【目的】利用Cre/lox重组系统具有的重组删除特性建立番茄工程恢复系,特异删除F1中的雄性不育基因恢复其育性。【方法】将TA29-Barnase雄性不育基因表达盒置于两个同向lox位点之间并与NPTⅡ基因、Bar基因融合后获得植物表达载体pBinBarloxTABn,转化番茄获得雄性不育转基因植株。Cre基因在 CaMV35S启动子的驱动下转入番茄获得工程恢复系。两者在开花时进行杂交,利用Cre重组酶删除F1中的TA29-Barnase不育基因表达盒使育性恢复。【结果】利用NPTⅡ作为转化筛选标记基因获得了番茄雄性不育转基因植株。转基因植株中的Bar基因能正常表达,真叶叶盘在含PPT 3 mg?L-1（phosphinothricin）分化培养基上能分化愈伤组织及芽;真叶具有抗PPT 20 mg?L-1浓度以上的能力。获得的TA29-Barnase转基因植株表现雄蕊退化、无花粉产生或产生少量形状畸形且无生活力的花粉。雄性不育植株自花授粉不能坐果,用非转基因保持系花粉授粉后,果实正常膨大结籽,杂交后代对除草剂Basta的抗性按1﹕1分离。不育植株与Cre转基因工程恢复系杂交后,果实也正常膨大结籽。对不育植株与Cre转基因工程恢复系杂交后代进行分子检测,结果发现同时含Bar 基因和Cre基因F1植株中的TA29-Barnase雄性不育基因被精确删除。TA29-Barnase基因删除植株育性被恢复,能正常开花结果。【结论】利用Cre/lox重组系统建立的Cre工程恢复系成功将番茄F1代中的不育基因删除,恢复了 F1的育性。该研究结果为植物基因工程雄性不育系的育性恢复提供了一条新的途径。

Construction the Engineered Restoring Line of Tomato Engineered Male Sterile Line bV Cre/lox Site-Specific Recombination System

【Objective】 An engineered restoring line of tomato engineered male sterile line was constructed using Cre/lox site-specific recombination system, which restore the fertility by deleting the male sterile gene directly in F1. 【Method】 The expression vector of pBinBarloxTABn, contained a TA29-Barnase cassette flanked by two lox sites in a directed orientation, a NPTⅡ gene expression cassette and a Bar gene expression cassette was introduced into the tomato genome to bring male sterile line. The Cre gene, under the control of CaMV 35S promoter, was transformed into tomato to construct the engineered restoring line. The Cre gene was introduced into the F1 generation by pollinating the tomato male sterile plants with pollen from Cre-expression plants, expression of the Cre in the hybrid leads to the removal of the TA29-Barnase gene, then the plants was fertile. 【Result】 The tomato male sterile plants were obtained by Agrobacterium tumefaciens-mediated transformation, using the NPTⅡ gene as the transformation maker gene. The Bar gene in the male sterile plants expressed well, and these plants showed considerable resistance to herbicide Basta, the leaf disc differentiated callus and shoots on the medium supplemented with PPT 3 mg?L-1, and kept fresh in the solution containing PPT 20 mg?L-1 in 6-7 days. The male sterile plants characterized stamen degenerate, no pollen or tiny innormal pollens- without vigor. No normally expanded fruits and formed seeds were observed in the transgenic plant after self-pollination of the male sterile plants. However, the normally expanded fruits and seeds were observed after cross-pollination of the male sterile plants using pollens from wild-type plant, and the progenies from the hybrid showed a 1﹕1 ratio of Basta resistant to Basta susceptive. Cross-pollinated the male sterile plants (T0) using pollens from Cre-expression plants, the normally expanded fruits and seeds were observed. The molecular analysis on the progenies from the hybrid was performed, the results showed that those progenies inherited both the Cre gene and Bar gene had lost the TA29-Barnase gene without exception, and those progenies could flower and fruit normally, indicated that the male sterility had been restored. 【Conclusion】 Using the Cre/lox site-specific recombination system, the male sterility-causing gene integrated into the tomato genome was successfully eliminated by crossing with pollen from a engineered restoring line expressing the Cre recombinase, yielding hybrid fruits and seeds. This results have provided an alternative method for the fertility restoration of plant engineered male sterility.