Cloning Of Vacuolar Processing Enzyme Gene, MhVPE, In Malus Hupehensis (Pamp) Rehd. And Its Bioinformatics Analysis

Vacuolar processing enzymes (VPEs) have been proved to function in processing and activating functional proteins. Otherwise, VPEs also take an important role in regulating plant programmed cell death (PCD) and senescence. Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang (Chinese name is PingYiTianCha, PYTC) is the special resources of our country, which is usually used for common rootstock of Malus and Ornamental crabapple. In this study, the full-length cDNA of MhVPE gene is isolated with the methods of RT-PCR and rapid-amplification of cDNA ends (RACE) based on the materials of PYTC roots. Otherwise, we analyze the sequence of MhVPE gene and predict the properties as well as functions of protein that it encodes by bioinformatics analysis. The main results are as follows:1. A rapid and effective method for RNA extraction from different tissues of fruit plants which contain rich polysaccharides and polyphenols, such as apple and grapevine, had been established and ameliorated.2. According to the homologous sequences from other plants, specific primers, which were based on the EST database of apples were designed to amplify specific DNA fragment using cDNA. By the methods of RT-PCR and RACE, we isolated a VPE gene from PYTC roots, which was named MhVPE, with the accession number of GeneBank FJ891065. The full-length cDNA of MhVPE was 1,770bp, and the integrated ORF was 1,485bp, which encoded a 494-amino acid polypeptide. The number of methylated loci was 47, and it had a preference to the condons of UGA, AGA and GGA.3. Software analysis showed that the molecular weight of MhVPE was 54325.2Da and the isoelectric point was 5.60. MhVPE was a hydrophobic protein, which shared the typical structures of VPE, containing 11 conserved subregions. MhVPE was mainly consisted ofα-helixes and random coils, which contained one strong transmembrane helices between the region of 7 to 25 amino acid. Sequence comparison showed that MhVPE protein was closer to that of Citrus and Vitis vinifera.4. In order to predict the subcellular localization of MhVPE, we took use of different softwares to indicate that MhVPE was localized in vacuolar. Further analysis indicated that there was a cleavage site of signal peptide at the N-terminal region and there were 19 phosphorylation sites. We also got that MhVPE acted as an unstable hydrolase, belonging to C13 peptidase superfamily, which had the same catalysis subregion as human caspase-1.5. In order to study the function of endogenous MhVPE in plants, we constructed vectors of pBI121-MhVPE containing full-length cDNA in sense and pBI121-3'segment containing 3'segment in antisence orientation. Both vectors were assembled by the constitutive cauliflower mosaic virus 35S promoter and introduced into tobacco, and gained transgene plants.