桑树类黄酮3–O–葡萄糖基转移酶基因的鉴定及主效基因功能分析

Identification of MaUFGTs from Mulberry and Function Analysis of the Major Gene

根据同源比对,从川桑(Morus notabilis)基因组数据库(morus.swu.edu.cn/morusdb/)中鉴定类黄酮3–O–葡萄糖基转移酶基因(MaUFGT)家族成员。采用实时荧光定量PCR(q RT-PCR)分析MaUFGT在桑树各组织和发育期的表达水平。随着果实发育成熟,MaUFGT1、MaUFGT2和MaUFGT3的表达模式相似,均呈上升趋势;在从顶芽依次向下不同叶位中,3个UFGT基因表达呈现先降低后升高,再降低又升高的趋势;在幼茎皮层和托叶中大量表达,幼茎表皮和木质部、叶柄中表达量较低,有的甚至不表达。MaUFGT2的表达量在3个基因中都是最高的,推测其是该基因家族的主效基因。从桑树品种‘嘉陵40’成熟果实中克隆了UFGT2基因,命名为MaUFGT2,登录号KP455729.1。其c DNA全长为1 386 bp,编码461个氨基酸,推测蛋白质分子量约为51 k D。序列比对结果发现所编码蛋白在不同物种间保守性不高。将MaUFGT2克隆到p ET-28a(+)原核表达载体后在大肠杆菌BL21(DE3)中进行诱导表达,表达产物经SDS-PAGE分析显示,融合蛋白大小约为55 k D,主要以包涵体形式表达,在上清液中也有少量表达。MaUFGT2蛋白经纯化和复性后,最后通过高效液相色谱(HPLC)分析该蛋白的酶活性,结果表明,体外酶活性鉴定MaUFGT2能够催化UDP葡萄糖转移至槲皮素形成槲皮素3–β–D–葡萄糖苷,验证了其具有糖基转移酶的功能。

The members of the MaUFGT（flavonoid 3-O-glucosyltransferase gene）family were identified from Morus notabilis genome database（http：//morus.swu.edu.cn/morusdb/）by means of homology alignment,and their expression in different tissues and at different growth stages of mulberry was analyzed with q RT-PCR（quantitative real-time fluorescent PCR）. With the development and maturation of the fruit,the expression of MaUFGT1,MaUFGT2 and MaUFGT3 showed an upward trend. Their expression generally dropped first and then increased,and was followed by another drop and another rise at different leaf positions. They were expressed abundantly in the cortex and stipule,but scantily in the epidermis,xylem and petiole. Of the three members of the MaUFGT family,MaUFGT2 always had the highest expression,therefore,is speculated to be the major gene. A full-length 1 386 bp c DNA of the MaUFGT2 gene,which encodes 461 amino acids,was cloned from the ripe fruit of mulberry‘Jialing 40＇. Its calculated molecular mass was about 51 k D. Sequence alignment showed that the encoded proteins were not highly conservative between different species. MaUFGT2 thus cloned was inserted into the vector p ET-28a（＋） and expressed in E. coli BL21（DE3）. SDS-PAGE results showed that the obtained MaUFGT2 protein was approximately 55 k D in size and was abundantly expressed in inclusion bodies and scarcely as a soluble protein. High performance liquid chromatography（HPLC）of the enzymatic activity of the purified and renaturated MaUFGT protein showed that the recombinant protein could catalyze UDP glucose to quercetin to form the quercetin 3-β-D-glucoside,thus confirming that MaUFGT2 possessed a glycosyltransferase function.