关键词: 桑树, 果实, 类黄酮3&ndash, O&ndash, 葡萄糖基转移酶, UFGT基因, 表达, 酶活性分析

Abstract:

The members of the MaUFGT（flavonoid 3-O-glucosyltransferase gene）family wereidentified 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 qRT-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 cDNA 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 kD. Sequence alignment showed that the encoded proteins were not highly conservative between different species. MaUFGT2 thus cloned was inserted into the vector pET-28a(+) and expressed in E. coli BL21(DE3). SDS-PAGE results showed that the obtained MaUFGT2 protein was approximately 55 kD 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.

Key words: mulberry, fruit, flavonoid 3-O-glucosyltransferase, UFGT gene, expression, enzymatic activity analysis