拟南芥<i>NBS1</i>可变剪切体响应DNA损伤修复的功能研究

doi:10.7668/hbnxb.20195096

摘要/Abstract

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。

关键词: 拟南芥, NBS1, 可变剪切, DNA损伤修复, 功能分析

Abstract:

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.

Key words: Arabidopsis, NBS1, Alternative splicing, DNA damage repair, Functional analysis

浦霞, 吕春桃, 张宇, 徐慧妮, 余迪求, 孙旭东. 拟南芥NBS1可变剪切体响应DNA损伤修复的功能研究[J]. 华北农学报, 2025, 40(1): 84-92. doi: 10.7668/hbnxb.20195096.

PU Xia, LYU Chuntao, ZHANG Yu, XU Huini, YU Diqiu, SUN Xudong. Functional Research of Arabidopsis NBS1 Alternative Splicing in Response to DNA Damage Repair[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 84-92. doi: 10.7668/hbnxb.20195096.

http://www.hbnxb.net/CN/Y2025/V40/I1/84

图/表 9

表1 试验所用引物序列

Tab.1 Sequence of primers used in the experiment

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	引物类型 The type of primers
GFP-NBS1-SalⅠ-F	GCAGCGGCCGTCGACATGGTTTGGGGTCTCTTTC	基因克隆
GFP-NBS1-EcoRⅠ-R	GTTGATTCAGAATTCTCAACTTCCAGAGAGAAAC
GFP-NBS1-3-SalⅠ-F	GCAGCGGCCGTCGACATGGTTTGGGGTCTCTTTCC
GFP-NBS1-3-EcoRⅠ-R	GTTGATTCAGAATTCTCAACTTCCAGAGAGAAAC
Seq-pRI101-GFP-F	CGACAACCACTACCTGAGCA	转基因植株鉴定
GFP-NBS1-EcoRⅠ-R	GTTGATTCAGAATTCTCAACTTCCAGAGAGAAAC
Seq-pRI101-GFP-F	CGACAACCACTACCTGAGCAC
GFP-NBS1-3-EcoRⅠ-R	GTTGATTCAGAATTCTCAACTTCCAGAGAGAAACC
LP	GAATGCAGCGAAGAAGATGAG	突变体鉴定
RP	AAACCCAAGAAGAAATGTGGG
BP	ATTTTGCCGATTTCGGAAC
RP	AAACCCAAGAAGAAATGTGGG
qPCR-UBQ10-F	AACTTTGGTGGTTTGTGTTTTGG	qRT-PCR检测NBS1基因相对
qPCR-UBQ10-R	TCGACTTGTCATTAGAAAGAAAGAGATAA	表达量
qPCR-AtNBS1-F	CAGGAGGGGAAGGAGTTGTG
qPCR-AtNBS1-R	CGTTCCACGCTTTCTAGCCT

图1 NBS1与NBS1-3的序列差异分析

Fig.1 Sequence difference analysis of NBS1 and NBS1-3

图2 NBS1及可变剪接体NBS1-3蛋白结构分析 A.NBS1蛋白结构预测;B.可变剪切体NBS1-3蛋白结构预测。

Fig.2 Structure analysis of NBS1 and spliceosome NBS1-3 protein A.NBS1 protein structure prediction;B.Structure prediction of spliceosome NBS1-3 protein.

图3 烟草中AtNBS1与AtNBS1-3的亚细胞定位分析

Fig.3 Subcellular localization analysis of AtNBS1 and AtNBS1-3 in tobacco Bar=20 μm。

图4 转基因及突变体拟南芥鉴定 A.AtNBS1过表达拟南芥株系鉴定;B.AtNBS1-3过表达拟南芥株系的鉴定;C.拟南芥突变体nbs1的鉴定;M.5 000 bp Marker;P.pRI101-NBS1为阳性对照;WT和—.阴性对照;a.LP+RP引物对;b.BP+RP引物对。

Fig.4 Identification of transgenic and mutant Arabidopsis A.Identification of AtNBS1 overexpressed Arabidopsis; B.Identification of AtNBS1-3 overexpressed Arabidopsis;C.Identification of Arabidopsis mutant nbs1; M.5 000 bp Marker;P.pRI101-NBS1 is positive control;WT and—.Negative control;a.LP+RP primer pair;b.BP+RP primer pair.

图5 不同浓度DNA损伤剂MMS处理后的种子萌发率分析 A.MMS处理不同时间后的种子萌发率统计;B.MMS处理14 d后种子的萌发率统计;C.MMS处理14 d后种子的生长情况。不同小写字母表示差异达到显著水平(P<0.05)。图6—8同。

Fig.5 Analysis of seed germination rates after treatment with the DNA damaging agent MMS A.Statistics of seed germination after MMS treatment;B.Statistics of seed germination after 14 days of MMS treatment;C.Growth situation of seeds after 14 days of MMS treatment.Different lowercase letters indicate a significant difference at P<0.05.The same as Fig.6—8.

图6 MMS处理后植物表型分析

Fig.6 Phenotype analysis of plants after MMS treatment Bar=1 cm

图7 NBS1表达水平检测 A.不同浓度MMS处理下拟南芥NBS1基因的表达水平分析;B.WT、NBS1过表达株系中NBS1基因的表达水平分析;C.WT、NBS1-3过表达株系中NBS1基因的表达水平分析。

Fig.7 qRT-PCR analysis of NBS1 A.Analysis of the expression level of NBS1 gene under different concentrations of MMS;B.Analysis of the expression level of NBS1 gene in WT and overexpressed lines;C.Analysis of the expression level of NBS1 gene in WT and NBS1-3 overexpressed lines.

图8 MMS处理后拟南芥根尖DNA损伤程度分析 A.WT、突变体nbs1及NBS1-OE-1根尖PI染色结果;B.NBS1-OE-3、NBS1-3-OE-1及NBS1-3-OE-3根尖PI染色结果;C.WT、nbs1、NBS1-OE-1、NBS1-OE-3、NBS1-3-OE-1及NBS1-3-OE-3拟南芥幼苗根尖细胞DNA损伤面积统计。

Fig.8 Analysis of the extent of DNA damage in Arabidopsis root tips after MMS treatment A.Results of PI staining WT,mutant nbs1 and NBS1-OE-1 root tips;B.Results of PI staining of NBS1-OE-3,NBS1-3-OE-1 and NBS1-3-OE-3 root tips; C.Statistical of DNA damage area in root tip cells of WT,nbs1,NBS1-OE-1,NBS1-OE-3,NBS1-3-OE-1 and NBS1-3-OE-3 Arabidopsis seedlings.

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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

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