山葡萄类黄酮-5-O-葡萄糖基转移酶基因(5GT)的克隆表达及遗传转化分析

Cloning and expression and genetic transformation analysis of Vitis amurensis flavonoid-5-O-glucosyltransferase gene (5GT)

关键词:  山葡萄  克隆  类黄酮-5-O-葡萄糖基转移酶  原核表达  遗传转化

Key words:  Vitis amurensis  cloning  flavonoid-5-o-glucosyltransferase  prokaryotic expression  genetic transformation

摘要:

为探究5GT基因在山葡萄花色苷合成过程中的表达规律及作用，以山葡萄（Vitis amurensis）为材料，利用RT-PCR技术克隆山葡萄5GT基因的全长cDNA序列（GenBank登录号为GU237133），将该基因命名为Vam5GT并对该蛋白进行生物信息学分析预测其功能；利用实时荧光定量PCR检测Vam5GT基因在山葡萄8个不同转色时期的表达规律，将克隆获得的Vam5GT基因完整的ORF连接到原核表达载体pET28a上，转化到大肠杆菌E.coli Bl2（DE3），并通过不同浓度的IPTG诱导表达，SDS-PAGE检测表达产物。并构建表达载体pC5GT并转化农杆菌GV3101.用菌液浸泡花序法对拟南芥进行遗传转化。结果显示：克隆获得的Vam5GT cDNA全长1 497 bp，开放阅读框1 347 bp，编码448个氨基酸，该基因表达产物相对分子质量为49.84 ku，等电点为5.23，是不稳定蛋白。该基因属于UDPGT超基因家族，不包含信号肽。Vam5GT在山葡萄花后4周的果皮中表达丰度最高，其他时期表达逐渐下降；该基因原核表达产物与预期大小一致，表明原核表达成功，在含50 mg/L Kanomycin的培养基上对拟南芥T₀代种子进行筛选，先后得到2个阳性幼苗，转化率为0.05%。对移栽成活的2株抗性植株进行PCR检测为阳性。

Abstract:

In order to explore the expression and function of 5GT gene in the synthesis of anthocyanins in Vitis amurensis,the full-length cDNA sequence of V.amurensis 5GT gene was cloned by RT-PCR with V.amurensis (GenBank accession number GU237133).The protein was bioinformatically analyzed to predict its function.The expression of Vam5GT gene in V.amurensis was detected by qPCR.The complete ORF of the gene was ligated into the prokaryotic expression vector pET28a.Escherichia coli Bl2 (DE3) was induced and its expression was induced by different concentrations of IPTG.The expression product was detected by SDS-PAGE.The expression vector pC5GT was constructed and transformed into Agrobacterium GV3101.The genetic transformation of Arabidopsis thaliana was carried out by soaking inflorescence with bacterial solution.The results showed that the cloned Vam5GT cDNA was 1 497 bp in length and 1 347 bp of ORF,encoding 448 amino acids.This protien's relative molecular mass was 49.84 ku and isoelectric point was 5.23 indicating it was unstable protein.This gene belongs to the UDPGT supergene family and does not contain a signal peptide.The expression abundance of Vam5GT was highest in the pericarp of V.amurensis at 4 weeks after flowering,and the expression was decreased in other periods.The prokaryotic expression product of this gene was consistent with the expected size,indicating that the prokaryotic expression was successful,and it was on the medium containing 50 mg/L kanomycin.T₀ generation seeds were screened.Two positive seedlings were obtained successively,and the transformation rate was 0.05%.The two resistant plants transplanted and survived were positive for PCR detection.