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.

作　　者：赵丽晶 刘英 习亚君 王小飞 高丽美[1] ZHAO Lijing;LIU Ying;XI Yajun;WANG Xiaofei;GAO Limei(College of Life Sciences, Shanxi Normal University, Taiyuan 030000, China)

机构地区：[1]山西师范大学生命科学学院,太原030000

出　　处：《西北植物学报》2022年第1期29-37,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：山西省青年科技基金(201801D221260)。

摘　　要：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

关 键 词：紫花苜蓿 MsUVR8 RACE技术 UV-B 功能分析 

分 类 号：Q785[生物学—分子生物学] Q786