梁平柚鼠李糖基转移酶基因(Cm1,2RhaT)的克隆与表达分析  

Cloning and Expression Analysis of a 1,2Rhamnosyl-transferase Gene(Cm1,2RhaT) from Citrus maxima cv.Liangping

作　　者：张军[1] 陈雪[1] 陶静静[1] 马婧[1] 眭顺照[1] 李名扬[1] 

机构地区：[1]西南大学园艺园林学院,南方山地园艺学教育部重点实验室,重庆市花卉工程技术研究中心,重庆400715

出　　处：《农业生物技术学报》2013年第5期511-521,共11页Journal of Agricultural Biotechnology

基　　金：教育部博士点基金(No.200806351015)

摘　　要：柚皮苷是一种双氢黄酮类化合物的苦味剂,鼠李糖基转移酶是柚皮苷的合成过程中的关键酶。本研究通过RT-PCR方法从梁平柚(Citrumaxima cv.Liangping)扩增到鼠李糖基转移酶(Cm1,2RhaT)基因,构建了该基因的原核表达载体pET28a-CmRhaT并转化到大肠杆菌(Escherichia coli)BL21(DE3)中,诱导表达并分离纯化得到融合蛋白。利用SYBR GreenⅠ实时荧光定量PCR检测Cm1,2RhaT基因在梁平柚等5个柑橘品种叶片、果实及不同发育时期的表达差异。研究结果获得Cm1,2RhaT的DNA和cDNA序列(GenBank登录号:JQ217381),cDNA长1436bp,推测的编码蛋白Cm1,2RhaT包含453个氨基酸,理论分子量为51.2kD。分离纯化得到了较高浓度(0.526mg/mL)和纯度(95%)的融合蛋白。定量分析表明,该基因在梁平柚的果实和叶片中均有表达。从开花后30～180d,该基因的表达量总体上随着果实的成熟而逐渐降低。其同源基因在其它品种中也呈现和梁平柚基本一致的波动性表达模式。本研究从梁平柚中扩增出一个Cm1,2RhaT基因,其表达的酶具有典型的糖基转移基团,是柑橘苦味物质之一黄烷酮合成中的关键酶,参与柑橘次生代谢过程,可能是无苦味柑橘品种改良的一个切入点。Naringin is a bittering agent of kinds of hydrotestosterone flavonoids and the rhamnosyltransferase is a key enzyme in the synthesis of naringin. In this study we cloned a 1,2rhamnosyltransferase gene（Cm1, 2RhaT） which wa from Citrus maxima（Burm.） Merr. cv. Liangpin Yu. The Cm1, 2RhaT gene expression vector pET28a-CmRhaT was constructed by the gene fragment cloned into the prokaryotic expression vector pET-28a （＋）, which was then transformed into Escherichia coil BL21（DE3）. The objective fusion protein was induced and expressed. Further fusion protein was induced to express and purified. The SYBR Green Real-time qRT- PCR was employed to analyze the expression differences of Cm1,2RhaT in the fruits and leaves＇s different developmental stages of Citrus maxima （Burm.） Merr. cv. Liangpin Yu results showed that Cm1,2RhaT was isolated and characterized from and other four citrus varieties.The Citrus maxima （Burro.） Merr. cv.Liangpin Yu. The accessed cDNA （GenBank accession No. JQ217381） was 1 436 bp in length encoding 453 deduced amino acids with a predicted molecular mass of 51.2 kD. We obtained the fusion protein after isolation and purification. Its concentration and purity were 0.526 mg/mL and 95%, respectively. Real-time fluorescence qPCR（RT-PCR）showed that the Cm1,2 RhaT expressed in the Liangpingyou＇s fruits and leaves. The expression level of Cm1,2 RhaT was reduced gradually with the fruit developing from 30-80 d after flowering. Its homologous genes in other species also showed the same as volatility expression patterns as in Liangpingyou. Cm1, 2RhaT which was amplified from Citrus maxima （Burm.） Merr. cv. Liangpin Yu expressed a kind of enzyme with typical glycosyl transfer group. It is a key enzyme in the synthesis of fiavanone which is one of Citrus＇ s bitter substances. The enzyme involves in secondary metabolism process of Citrus. To sum up, the research and results has certain significance to not-bitter citrus＇s varieties improvement.

关 键 词：鼠李糖基转移酶 CM1 2RhaT基因 原核表达 苦味 基因表达 

分 类 号：S666.301[农业科学—果树学]