芜菁UF3GT基因的克隆及表达特性

目的:克隆‘津田’芜菁和‘赤丸’芜菁的UDP-葡萄糖∶类黄酮3-O-葡萄糖基转移酶(UF3GT)基因并研究其表达特性。方法:利用RT-PCR方法克隆‘津田’芜菁BrUF3GT1基因和‘赤丸’芜菁BrUF3GT2基因,并进行生物信息学分析;通过Northern杂交检测BrUF3GT1和BrUF3GT2基因的UV-A诱导表达特性;对BrUF3GT1和BrUF3GT2基因进行原核诱导表达。结果:BrUF3GT1和BrUF3GT2的开放读框为1407 bp,编码468个氨基酸残基;氨基酸序列分析显示,BrUF3GT1和BrUF3GT2与拟南芥UF3GT的同源性为87%,从第16~453位氨基酸残基的肽段具有糖基转移酶家族成员的结构域;BrUF3GT1和BrUF3GT2基因具有高度同源性,核苷酸序列在7个位点存在差异,推导的氨基酸序列在1个位点存在差异;Northern杂交结果显示,UV-A可以诱导BrUF3GT1和BrUF3GT2基因表达,基因的表达量与处理时间相关;原核诱导表达及纯化后可以获得相对分子质量分别约为51.88×103和51.89×103的BrUF3GT1和BrUF3GT2蛋白。结论:克隆了‘津田’芜菁和‘赤丸’芜菁的UF3GT基因,为初步阐明2种芜菁的花青素生物合成机理奠定了实验基础。

Cloning and Expression of UDP-Glucose： Flavonoid 3-O-GIucosyl- transferase Gene（UF3GT） in Turnip

Objective： To clone the UDPglucose：flavonoid 3Oglueosyhransferase（UF3GT） genes of ＇Tsuda＇ turnip and ＇Yurugi Akamaru＇ turnip, and to study the expression trait of the genes. Methods： BrUF3GT1 gene of ＇Tsuda＇ turnip and BrUF3GT2 gene of ＇Yurngi Akamarn＇ turnip were cloned by RTPCR method and analyzed by bioinformatics method. The expression characteristics of the genes induced by UVA were detected by Northern blot. Prokaryotic induced expression of BrUF3GT1 and BrUF3GT2 genes were conducted. Results： The open read ing frame of BrUF3GT1 and BrUF3GT2 genes contained 1407 bp encoding proteins of 468 amino acids. Amino ac id sequence analysis showed that BrUF3GT1 and BrUF3GT2 were 87% identitied to UF3GT of Arabidopsis thali aria, and the glycosyhransferase protein family domain was in the sequence from 16 to 453. BrUF3GT1 and BrUF3GT2 genes had high identity. The nucleotide sequence of BrUF3GT1 and BrUF3GT2 genes had 7 differenc es, as well as 1 in deduced amino acid sequence. The Northern blotting results showed that the expression of BrUF3GT1 and BrUF3GT2 genes could be induced by irradiation of UVA, and the expression of the genes was correlated with lightexposure time. The 51.88 kD and 51.89 kD proteins of BrUF3GT1 and BrUF3GT2 were suc cessfully purified after prokaryotic induced expression. Conclusion： The UF3GT genes were cloned from ＇Tsuda＇ turnip and ＇Yurugi Akamarn＇ turnip successfully, which established the experiment foundation of preliminarily clar ifying the mechanism of anthocyanin biosynthesis of these two turnips.