紫花苜蓿UV-B光受体基因MsUVR8的克隆及功能研究

Cloning and Functional Study of UV-B Photoreceptor Gene MsUVR8 in Medicago sativa L.

该研究采用RACE扩增技术克隆了一个紫花苜蓿UV-B光受体基因(MsUVR8),在生物信息学分析基础上,采用农杆菌介导法获得了该基因过表达愈伤组织,并对UV-B辐射处理后MsUVR8过表达愈伤组织及其野生型中的类黄酮、黄酮醇、花青素、过氧化氢(H_(2)O_(2))、超氧阴离子(O_(2)^(-·))含量以及UV-B信号通路相关基因的表达进行检测分析,以探讨MsUVR8基因的生物学功能,为揭示植物响应UV-B胁迫的分子机制奠定理论基础。结果表明:(1)成功克隆获得紫花苜蓿MsUVR8基因CDS序列834 bp,且MsUVR8与蒺藜苜蓿MtUVR8基因序列相似度高达95%以上;MsUVR8蛋白形成了不完整的β-折叠结构,系统发育分析显示其与鹰嘴豆属于同一分支。(2)对MsUVR8过表达系检测发现,紫花苜蓿MsUVR8过表达愈伤组织(UVR8-OE)中类黄酮含量较野生型愈伤组织(WT)明显升高,而且经UV-B辐射后的UVR8-OE类黄酮物质含量较WT进一步显著升高。(3)DPBA荧光标记实验发现,UV-B辐射大大促进了细胞中黄酮醇的合成,且UV-B辐射后的UVR8-OE中黄酮醇含量最高。(4)DAB和NBT染色显示,UV-B处理后WT中活性氧(H_(2)O_(2)和O_(2)^(-·))的积累增加,而在UV-B辐射处理与未处理的UVR8-OE中H_(2)O_(2)和O_(2)^(-·)的积累无明显差异,表明MsUVR8可增强植物组织细胞的抗氧化性能,并可降低UV-B胁迫引起的氧化损伤。(5)UV-B辐照后,WT中PAL、CHS和FLS表达被激活而显著提高,UVR8-OE中的4种基因表达均达到最大,且较其他3个处理组均显著增强。研究认为,紫花苜蓿MsUVR8被UV-B激活后,促进了类黄酮合成相关基因的表达,并激活了类黄酮合成关键酶的活性,从而提高了类黄酮物质的合成效率,增强了UV-B胁迫条件下植物愈伤组织的抗氧化能力。

In this study,a UV-B photoreceptor gene(MsUVR8)of alfalfa was cloned by RACE amplification.On the basis of bioinformatics analysis,Agrobacterium mediated method was used to obtain the overexpressed callus of MsUVR8.The contents of flavonoids,flavonols,anthocyanins,hydrogen peroxide(H_(2)O_(2)),superoxide anion(O_(2)^(-·))and UV-B signaling pathway related genes in MsUVR8 overexpressed callus and wild type after UV-B radiation treatment were detected and analyzed.The purpose of this study is to supply a theoretical foundation for revealing the molecular mechanism of plant response to UV-B stress.The results showed that:(1)the CDS sequence of MsUVR8 in alfalfa was 834 bp,and the similarity between MsUVR8 and MtUVR8 gene of Medicago truncatula was more than 95%.The MsUVR8 protein formed an incompleteβ-folded structure,and phylogenetic analysis showed that it belongs to the same branch as chickpea.(2)It was found that the content of flavonoids in MsUVR8 overexpressed callus(UVR8-OE)of alfalfa was significantly higher than that of wild-type callus(WT),and the content of flavonoids in UVR8-OE after UV-B radiation was further significantly higher than that of WT.(3)DPBA fluorescence labeling showed that UV-B radiation greatly promoted the synthesis of flavonols,and UVR8-OE had the highest flavonols content after UV-B radiation.(4)DAB and NBT staining showed that the accumulation of reactive oxygen species(H_(2)O_(2) and O_(2)^(-·))in WT increased after UV-B treatment,but there was no significant difference between UV-B-treated and untreated UVR8-OE,indicating that MsUVR8 could enhance the antioxidant capacity of plant tissues and cells and reduce the oxidative damage caused by UV-B stress.(5)After UV-B irradiation,the expression of PAL,CHS and FLS in WT was activated and significantly increased,and the expression of four genes in UVR8-OE reached the maximum,which was significantly increased compared with the other three treatment groups.The results showed that alfalfa MsUVR8 activated by UV-B promoted the expression of genes related to flavonoid synthesis and activated the activities of key enzymes in flavonoid synthesis,thus improving the efficiency of flavonoid synthesis and enhancing the antioxidant capacity of plant callus under UV-B stress.