Study Of Ficus Carica L. ACC Synthase Gene Cloning And Its Plant Expression Vector Construction

Fig(Ficus Carica L.) belongs to Ficus genus of mulberry family. Its fruit has the effect ofanti-cancer and anti-aging. But the fruit is extremely durable storage. Conventionalmethods can not fundamentally solve the problem of fig senescence and softening. Thisseriously affected the edible value and the economic value of fig.The breeding of softless varieties has an important practical value for fig. thetraditional preservation methods have heavy workload,and greatly increased the labor costsand management difficulties. It is one of the important goals of figs breeding to cultivatesoftening resistent of figs variety.Ethylene is the key factor of climacteric fruit senescence and softening. Usingtransgenic technology to inhibit ethylene biosynthesis of the fruits can delay fruit softeningprocess. ACC synthase and ACC oxidase are the key enzymes during the biosynthesis ofethylene. We can improve the storage capability of climacteric fruit by inhibiting theexpression of the two enzymes with transgenie method.In this paper, the ACS1gene was cloned from green fig fruit‘Qingpi’, and the plantantisense and RNAi expression vector were constructed. The conclusion was that thevector has been transformed into LBA4401, which make a found of further study ondelay-ripening of ficus carica.The main results are followed:1. A pair of specific primers were designed according to the published fig fruit ACS1gene conservative amino acid sequence. The sequencing data showed that the PCR productwas590bp encoding196predicted amino acid residues. Comparison with nucleotidesequence and amino acid sequence homology of ACC oxidase gene landed were99%and98%, which showed the successful cloning of the ACC synthase gene.2. Double digested by XbaⅠ and BamH Ⅰ,ficus carica ACS1gene nucleotidesequence was subcloned into XbaⅠ-BamH Ⅰsite of pBI221vector in reverse orientation,which resulted in plasmid pBI221-anti-ACS1. A fragment including ACS1antisensesequence and gusA gene and NOS terminater was cut from pBI221-anti-ACS1vector byXbaⅠ and EcoRⅠ. The fragment was directionally inserted into the same restriction enzymes digested pBI121plasmid, which resulted in pBI121-anti-ACS1.3. Double digested by BamHⅠ and SmaⅠ,fig fruit ACS1gene nucleotide sequence wassubcloned into BamHⅠ-SmaⅠ site of pBI221-anti-ACS1in sense orientation, whichresulted in plasmid pBI221-RNAi-ACS1. The plasmid pBI121and pBI221-RNAi-ACS1were double digested by XbaⅠ and EcoR Ⅰ, respectively. The sequence with ACS1sensefragment and antisense fragment was inserted into the double digested pBI121vector,which result in pBI121-RNAi-ACS1plasmid.4. The vector of pBI121-anti-ACS1and pBI121-RNAi-ACS1was transferred into A.fumefeciens strain LBA4404by the freezing-thaw method. The transformation wasconfirmed by PCR amplification with specific primers.