何首乌中UDP-鼠李糖合成酶基因FmRHM1/2的克隆与鉴定

Cloning and characterization of UDP-L-rhamnose synthase 1/2 from Fallopia multiflora

UDP-鼠李糖是一种由UDP-鼠李糖合酶(RHM)催化合成的鼠李糖供体,而鼠李糖是鼠李糖苷化合物的重要组成部分,植物中只有少数基因编码的酶参与UDP-鼠李糖生物合成。本研究基于何首乌(Fallopia multiflora(Thunb.) Harald.)转录组数据,首次克隆得到2个RHM基因(FmRHM1和FmRHM2),并进行生物学信息分析、体外功能鉴定及组织特异性分析。结果显示FmRHM1/2基因的开放阅读框均为2 013 bp,均编码670个氨基酸,推测蛋白质分子质量均为75.6 kDa,理论等电点分别为6.20和7.19,具有RHM酶家族的特征信号序列(GxxGxxG/A和YxxxK);多序列比对与系统进化树显示, FmRHM与其他物种的RHM具有同源性。体外酶促反应结果显示,重组蛋白FmRHM1和FmRHM2均具有催化活性,可将UDP-葡萄糖转化为UDP-鼠李糖。组织特异性表达显示, FmRHM1和FmRHM2基因在根中的表达量最低,并与茎和叶相比均存在显著性差异。本研究首次报道了何首乌RHM,并验证了其催化功能,为进一步研究微生物合成UDP-鼠李糖奠定基础。

UDP-rhamnose is a rhamnose donor in a reaction catalyzed by UDP-rhamnose synthase(RHM),and plays an important role in the biosynthesis of rhamnoside compounds. The current literature suggests that there are only a few genes can encode the corresponding enzymes to participate in UDP-rhamnose biosynthesis in plants. In this study, two RHM genes(FmRHM1 & 2) were first cloned by using the transcriptomic data of Fallopia multiflora(Thunb) Harald and the multidimensional analysis, including bioinformatics, functional identification in vitro and tissue-specific expression analysis. The results showed that the open reading frame(ORF) of FmRHM1 & 2 genes both were 2 013 bp, encode proteins consisting of 670 amino acids with a calculated molecular mass of 75.6 kDa,and the theoretical isoelectric points of 6.20 and 7.19, respectively. Bioinformatic analysis also indicated that FmRHM1 & 2 contained 2 special sequences of GxxGxxG/A and YxxxK. The phylogenetic analysis showed that the FmRHM gene has a high homology with RHM of other species. The results of enzyme activity in vitro revealed that both recombinant FmRHM1 and FmRHM2 have catalytic activities for converting UDP-glucose into UDPrhamnose. Measurements of tissue-specific expressions showed that the expression levels of FmRHM1 and FmRHM2 were lower in roots. On the contrary, the 2 genes showed significantly high expression in the stems and leaves. In conclusion, we have cloned and characterized the RHM gene function for the first time in F. multiflora.Here we have provided the preliminary data suggesting the need for further research on UDP-rhamnose biosynthesis by microorganisms.