Cloning And Expression Analysis Of β-galactosidase Gene CDNA In Peach

There are abundant beta-galactosidases in plant. The different content in everyperiod take a very important part in mature, flavor, colour and manufacture after plucked.In this paper, the complete cDNA sequence of beta-galactosidase was cloned in the peachleaf at the first time and it was expressed in E.coli. This contributed to make clear themechanism of the mature of fruits and vegetables as well as of the formation of flavor. Italso established the foundation that using biotechnology to produce the popular fruits andvegetables. The beta-galactosidase from plant can be applied to food, medicine, immunity,environment protection etc. Total RNA from young leaves of peach was extracted by chomczynsk' single stepmethod of RNA isolation, which was partially modified on the basis of the features ofbiochemistry composition in fresh peach leaves. The degenerate primers were designedand synthesized according to the highly conservative sequences among the knownbeta-galactosidase genes. A cDNA fragment of 387bp was amplified by RT-PCR, whichwas subsequently cloned into pMD18-T Vector for sequencing analysis. The result ofsequencing analysis showed that the nucleotide and deduced amino acids sequences hadhigh identity with the published corresponding parts of beta-galactosidase cDNAs of otherplants. The results indicated that the special PCR product was probably a cDNA fragmentof beta-galactosidase in peach. Two special primers of RACE were designed based on the above sequence of theRT-PCR product. Two fragments of 5'cDNA (840bp) and 3'cDNA (2343bp) wereamplified by RACE reaction. Two complete sequence primers were designed based on theresult of RACE. A 3285bp sequence was amplified by PCR. By the Blast programcomparison, the analysis of the product showed that the nucleotide shared more than 80%homologous to the corresponding parts of beta-galactosidase gene family of Pyraspyrifolia, Lycopersicon esculeutum, Arabidopsis thaliana, Cicer arietinum, Vigna radiateetc. The results indicate that 3285bp sequence is probably complete cDNA fragment ofbeta-galactosidase in peach. The nucleotide sequence was analyzed by the molecularbiology software, The results shows that the open reading frame is 2559bp which encodes853 amino acids(AA). The 3'UTR is 539bp. The translation is from the 185th base to the2746th base.The primers were designed according to the complete cDNA having been sequenced.The open reading frame cDNA was amplified by the methods of PCR. Then it wasconstructed into the vector pET-32a(+) designed for recombinant strain. After IPTGinduction, it was observed that there was a special band about 115kDa on SDS-PAGE. Butthere was no beta-galactosidase activity when assayed using ONPG as substrate.