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

doi:10.7668/hbnxb.20195096

Abstract

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

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.

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Figures/Tables 9

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

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

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

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

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.

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.

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

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.

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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