Construction Of ACC Oxidase Gene Over-expression Vector In Whangkumbae And Identification Of Arabidopsis Mutants

Whangkeumbae pear(Pyrus pyrifolia cv.Whangkeumbae) belongs to Pyrus pyrifolia Nakai system, and it is one of the main cultivars in China. It is well known for its excellent flavor. However, it is difficult to be stored and transported and its shelf life is very short. Whatever storage in room temperature or low temperature, it is easy for fruits to lose water and decay, which affects its post-harvested quality severely. In this paper, an over-expression vector named Ca MV35S::Pp ACO1 was constructed using Pp ACO1 gene cloned from Whangkeumbae fruit according to the Pp ACO1 gene sequence that had been published in NCBI(National Center of Biotechnology Information, Gene bank accession No. JN807390) as the targeted fragment and the commonly used p BI121 as the plant expression vector; In the meantime, Arabidopsis mutants with At ACO2 gene deleted by T-DNA insertion was identified using “three primers” method from materials purchased from ABRC(The Arabidopsis Biological Resource Center). And also, the phenotypic and physiological differences between wild-type Col-0 and the mutants identified were studied. Figuring out the function of ACO is important. How to control fruit ripening via regulating the ethylene bio-synthesis pathway by the method of genetic engineering can lay foundation for how to improve the post-harvest quality and prolonging shelf life of Whangkeumbae pear. Results are as follows:1 The full-length coding sequence of Pp ACO1 was amplified by RT-PCR from Whangkeumbae fruit c DNA using the primers ACO1-F and ACO1-R(Table 1).2 The targeted fragment was amplified using Pp ACO1 as templates by PCR amplification using the primers ACO1-EF and ACO1-ER(Table 1). The primers contain XbaⅠ and SacⅠ linker sites on the forward and reverse primer, respectively. This fragment was cloned into the p MD19-T vector by TA cloning. The insert was released from the vector by digestion with XbaⅠ and SacⅠ. The fragment was excised using the restriction enzyme sites in the linkers and ligated downstream of the Ca MV35 S promoter in the binary vector p BI121 with T4 DNA ligase, modified by removal of the GUS coding region by digestion with XbaⅠ and SacⅠ. The correctness of the construct was identified by PCR amplification and double digestion and the fidelity of the construct was confirmed by DNA sequencing.3 Homozygous Arabidopsis mutants with T-DNA insertion had been identified by “double primers” method and “three primers” method. Results showed that either of the methods could be used for the identification of mutants, but the “three primers” method was chosen in this study because of its Simplicity and efficiency.4 Results of survey into the prototypical differences between wild-type Col-0 and the mutants revealed that deletion of At ACO2 caused: a) germination delay b) weakened “triple response” phenomenon c) significantly reduced rosette leaves diameter d) flowering period delay and extension.5 Results of survey into the prototypical differences between wild-type Col-0 and the mutants showed that SOD activity was significantly enhanced and PPO activity significantly reduced in leaves; POD, CAT and LOX activity were all significantly reduced in flower; POD and CAT activity increased significantly while SOD and PPO activity decreased significantly in fruits. These would lay a foundation for further study of function of Pp ACO2.