Cloning And Functional Characterization Ofα-Galactosidase Gene From Chimonanthus Praecox (L.) Link

a-Galactosidase (EC3.2.1.22), Which is distributed widely in microorganisms, plants and animals, can catalyze hydrolysis of a-1,6linked a-Galactosidase residues. Acid a-galactosidase and alkaline a-galactosidase can be classified according to its optimal pH. The research suggests that it plays an important role in promoting seed germination and maturation, development and aging of leaves, fruit development and ripening process. In addition, a-Galactosidase has been widely used in food, sugar, feed, pharmaceutical industry and other fields. Commercial production of a-galactosidase is less scarce and expensive in country. High activity, pH and heat stability of a-galactosidase was obtained through genetic engineering, which is an effective way for large-scale production and application of the enzyme.Based on the cDNA library from Chimonanthus praecox flowers and EST analysis, a new member of heat shock proteins(HSPs) was cloned by5’RACE, named as CpGAL (GenBank accession No. JQ217380), and the bioinfonnatics, prokarvotic expression, plant expression and real-time quantitative PCR were used to analyze its potential biological functions. The main results are as follows:1. Molecular characteristics of CpGALThe full length of CpGAL cDNA sequence was1376bp, with an open reading frame (ORF) of1239bp encoding a putative polypeptide of412amino acid residues. Sequences analysis indicated that there was no intron within the genomic region Sequence alignments and phylogenetic analysis revealed CpGAL to contain signal peptide of28amino acid residues in the amino-terminal (N-terminal) domain, which located between28and29. CpGAL protein secondary structure was composed of a-helix (33.98%), and random coil (48.54%), extended strand (17.48%). The encoded protein had CK-2(casein kinase Ⅱ) domains which encoded by the gene having glycosyl transferase family conservative sequence DYLKYDNC and R***D.2. Prokaryotic expression of CpGALCpGAL was inserted into an prokaryotic expression vector pET28a(+) with Resection of N_terminal signal region. Then the expression vector was transformed and expressed Escherichia coli BL21. We got the recombinant protein finally. The SDS-PAGE electrophoresis analysis showed that the recombinant protein was only present in the inclusion body. We broke cell by lysozyme and ultrasonic disruption.The purity and recovery of inclusion body were significantly increased by this method. We used dissolution buffer contained8M Urea dissolved the inclusion body. Then the supernatant was dialysis refolded after high speed centrifugal in4℃. The enzymatic activation analysis showed that the refolded protein had higher activation. The gradient test of temperature showed that the chitinase activation was highest in40℃, kept activity stable below35-45℃, and largely decreased beyond45℃. The gradient test of PH showed that the chitinase activation were highest in pH6.0.3. Transcriptional Expression of CpGALAccording to expression studies by real-time quantitative PCR, CpGAL was constitutively expressed in vegetative and reproductive organs at defined developmental stages under normal conditions, which was highly expressed in roots than other organs (stems, cotyledons, young leaves, mature leaves, flowers in full bloom). CpGAL transcripts were detected during flower opening and more abundant in middle petals and stamens. CpGAL were significantly reduced in Chimonanthus praecox flowers, with the transcripts decreasing from the bud sprouting to bud stage, the lowest rise of exposed valve to the initial period, a rapid decline in full bloom, in the aging period increasing significantly to reach the peak. After6-BA treatment, CpGAL expression was significantly reduced, which delayed the senescence of flowers. All these transcriptional expression analysis results suggested that CpGAL may play a significant role in plant development.4. Plant expression of CpGALThe target gene CpGAL was linked with plant expression vector pCAMBIA2301g by restriction enzyme digestion, ligation, transformation, and then transformed into Agrobacterium tumefaciens GV3101. It showed that the above plant Expression vector with the target gene was successfully transformed in Agrobacterium tumefaciens GV3101by using BamH Ⅰ and Sac Ⅰ double restriction enzyme digestion and PCR confirmation. The plant expression vector containing limonoids glycosyltransferase gene CpGAL was transformed into wild tobacco plants through employing Agrobacterium-mediated leaf disc method, and twenty-five strains of transgenic tobacco were obtained by means of resistance screening, GUS staining and PCR amplification, and the real-time quantitative PCR test. Transgenic tobacco was observed, whose leaves become larger than. Phenotypic observation found that the transgenic tobacco, with bigger and thicker leaf, growed significantly faster than the non-transgenic tobacco.