Cloning Of Fruit Specific Promoter 2A12 In Peach And Construction Of Antisense Expression Vector Of ACO Gene

Peach belongs to Rosaceae, perennial deciduous fruit tree, which is a typically climacteric fruit, When the fruit is physiologically mature, a climacteric peak appears soon after the post harvest period, which leads to softening rapidly and seriously impacts on their sales and logistics. Ethylene is a ripening hormone of mature climacteric fruits. A large amount of ethylene is produced after climacteric fruits starting the self-catalytic ethylene synthesis. ACC oxidase (ACO also called ethylene-forming enzyme EFE) which is the last enzyme in the formation of ethylene plays a key role. Fruit-specific promoters can activate the high-level expression of foreign gene in the fruit of receptors plants. Then, researching and cloning tomato fruit-specific promoter and ACC oxidase gene, combining with antisense RNA gene technology inhibits the expression of ACC oxidase gene in order to reduce the ethylene biosynthesis of peach. In the end, the purpose of delaying fruit softening can be achieved. It is hopeful to fundamentally change the Characteristic of peach fruit ripening rapidly, and nurture new types of anti-softening, which substantially increase the storage time and the economic benefit of peache. It is very important for theoretical and practical significance to promote the faster industry development of peach in our country.The study, with mature Baifen peach as the materials, took advantage of Cloning technology to get the fruit specific promoter 2A12 in tomato and ACO gene in peach, and then constructed the antisense ACO gene expression vector regulated by promoter 2A12, laying the foundation for establishing an efficient system of genetic transformation of peach. The results of this study were as follows:1. High-quality RNA was extracted successfully by useing plant total RNA extraction kit, reverse transcripted into the first strand cDNA, according to ACO gene sequence of peach (accession number: AF319166) published in GenBank, a pair of specific primers were designed for RT-PCR amplification to get the fragment. Sequence analysis showed that ACO gene is a 1246bp full-length sequence. Compared with GenBank landed ACO gene (AF319166) nucleotide sequence and amino acid sequence, the homology were separately 99% and 98%, indicating that ACO gene of peach was successfully cloned, named as pMD-ACO.2.Tomato genomic DNA was extracted by the method of CTAB. According to 2A11 promoter (accession number: AF319166) published in GenBank, a pair of specific primers were designed for PCR amplification, and 869bp of 2A12 fragment was obtained. Sequence analysis showed that the compared with the published sequence of the 2A11 promoter (No:M87659.1), the corresponding nucleotide sequence homology was up to 100%, which indicated 2A12 promoter successfully was cloned, and named as pMD-2A.3. pMD-ACO cloning vector and the pBI121 expression vector were digestioned respectively by Sacâ… and BamHâ… restriction endonucleases. GUS A fragment of the pBI121 vector was cut down, and then ACO gene fragment was reversely inserted into pBI121 vector to construct intermediate expression vector pBI-anti-ACO. pMD-2A cloning vector and the pBI121 expression vector were digestioned respectively by Hindâ…¢and BamHâ… restriction endonucleases. When CaMV 35S fragment was cut down from pBI121 vector, 2A11 promoter fragment was inserted into the pBI121 vector, and constructed the plant expression vector pBI-2A. With Sacâ… a nd BamHâ… digesting intermediate vector pBI-anti-ACO and the plant expression vector pBI-2A, ACO fragment cut down was reversely inserted into pBI-2A, and constructed the plant expression vector pBI-2A-ACO after PCR and digestion identification, which proved the vectors were constructed successfully, and the vectors can be used to transfer into the plant of peach.