拟南芥NBS1可变剪切体响应DNA损伤修复的功能研究  

Functional Research of Arabidopsis NBS1 Alternative Splicing in Response to DNA Damage Repair

作　　者：浦霞 吕春桃 张宇[2] 徐慧妮[3] 余迪求 孙旭东[2] PU Xia;LYU Chuntao;ZHANG Yu;XU Huini;YU Diqiu;SUN Xudong(School of Ecology and Environmental Science,Yunnan University,Kunming 650091,China;Kunming Institute of Botany,Chinese Academy of Sciences,Kunming 650201,China;Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China)

机构地区：[1]云南大学生态与环境学院,云南昆明650091 [2]中国科学院昆明植物研究所,云南昆明650201 [3]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《华北农学报》2025年第1期84-92,共9页Acta Agriculturae Boreali-Sinica

基　　金：云南省研究专项-重大项目(202401BC070001);国家自然科学基金项目(32370405)。

摘　　要：DNA损伤会对植物的生长发育造成严重的影响,NBS1是参与损伤修复的重要基因,为研究NBS1与其可变剪切体NBS1-3之间的功能差异。根据NBS1和NBS1-3基因序列分别设计特异性引物,以野生型拟南芥叶片RNA反转录得到的cDNA第一链为模板,克隆NBS1和NBS1-3,对2个基因序列和蛋白质三维结构进行分析。同时,创制了NBS1、NBS1-3过表达株系,获得突变体nbs1纯合株系,并检测NBS1基因在NBS1及NBS1-3过表达植株的相对表达量。为进一步阐明NBS1与可变剪接体NBS1-3之间的功能差异,用0.6 mmol/L甲基黄酸甲酯(MMS)对野生型、nbs1突变体和过表达植株进行处理,观察损伤面积。定量检测结果显示,NBS1基因在NBS1及NBS1-3过表达植株的表达量均高于野生型。根尖PI染色结果表明,0.6 mmol/L MMS处理后,nbs1突变体植株相对损伤面积最大,而NBS1-3过表达植株相对损伤面积最小,其次依次为NBS1过表达植株和野生型。因此,在DNA损伤修复方面,NBS1-3可能比NBS1发挥更大的作用。DNA damage significantly hinders impact plant growth and development.NBS1 plays an important role in the cellular response to DNA damage,to analyze the functional difference between NBS1 and its alternative splicing isoform,NBS1-3.Specific primers were designed to clone NBS1 and NBS1-3 from the complementary DNA of Arabidopsis thaliana,and the sequences of the NBS1,NBS1-3 and the three-dimensional structure of the proteins were analyzed.Transgenic Arabidopsis thaliana lines overexpressing of NBS1 and NBS1-3 were generated respectively,and nbs1 homozygous mutant plants were identified,detecting the expression levels of NBS1 in overexpressed lines NBS1 and NBS1-3.To further elucidate the functional distinction between NBS1 and NBS1-3,wild type,mutant and overexpressed plants were treated with 0.6 mmol/L Methyl Methanesulfonate(MMS)and observed damage area.The results of quantitative showed that the expression levels of NBS1 in overexpressed lines NBS1 and NBS1-3 were higher than wild type.PI staining of root tips showed that after 0.6 mmol/L MMS treatment,the mutant showed the highest relative damage area,while the NBS1-3 overexpressed lines exhibited the least damage,followed by NBS1 overexpressed lines and wild type.These results suggest a potentially more important role of NBS1-3 in DNA damage repair compared to NBS1.

关 键 词：拟南芥 NBS1 可变剪切 DNA损伤修复 功能分析 

分 类 号：S567[农业科学—中草药栽培]