Abstract:

【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.

Key words: Cre/lox site-specific recombination system, engineered male sterile line, Cre engineered restoring line, tomato