普通烟草β-半乳糖苷酶基因(NtBGAL)的生信分析及其在不同组织及原核诱导的表达  

Bioinformatics analysis and expression in different tissues and prokaryotic induction of NtBGAL gene from Nicotiana tabacum

作　　者：曹领改 刘杰 张洁 张盼 余世洲 CAO Ling-gai;LIU Jie;ZHANG Jie;ZHANG Pan;YU Shi-zhou(Guizhou Academy of Tobacco Science,Guiyang 550081,China;Beijing Life Science Academy,Beijing 100101,China)

机构地区：[1]贵州省烟草科学研究院,贵阳550081 [2]北京生命科技研究院,北京100101

出　　处：《西南农业学报》2024年第6期1220-1227,共8页Southwest China Journal of Agricultural Sciences

基　　金：贵州省烟草科学研究院科技项目(GZYKY2021-06);中国烟草总公司重大科技项目(110202101032(JY-09));北京生命科技研究院科技项目(2023000CC0080)。

摘　　要：【目的】探明普通烟草β-半乳糖苷酶基因NtBGAL的表达模式与蛋白特征,为后期创制烟草多用途利用的底盘材料提供依据。【方法】通过比对普通烟草K326基因序列与本氏烟草NbBGAL1基因的同源性,以K326的cDNA为模板经PCR技术克隆获得NtBGAL基因,随后利用生信工具软件分析其基因结构及蛋白理化性质,并采用qRT-PCR检测该基因在烟草不同组织部位(根、茎、叶和花)的表达模式,最后开展原核诱导表达、蛋白纯化及蛋白表征研究。【结果】克隆获得烟草NtBGAL基因,其与本氏烟草NbBGAL1基因同源性最高,二者具有相同的结构域(Motif);该基因定位于9号染色体上,含有18个内含子,mRNA长度为6649 bp,CDS长度为2541 bp,编码846个氨基酸,翻译的β-半乳糖苷酶属于GH35家族,NtBGAL蛋白的分子量为92.44 kDa,理论等电点(pI)为6.8,GRAVY为-0.222,定位于细胞壁。该基因在普通烟草不同组织部位的表达量为花>叶>茎>根,差异显著。在37℃下进行原核诱导,NtBGAL蛋白有明显表达;采用包涵体纯化方法获得较高纯度的NtBGAL蛋白。NtBGAL蛋白酶促反应最适温度为30~40℃,最适pH为4.0~6.5,Mn^(2+)浓度为0.05~0.15 mmol/L时对NtBGAL酶活性有增强作用。【结论】研究明确NtBGAL基因的组织表达模式及蛋白表征,为进一步通过改造NtBGAL基因,利用普通烟草生产人源化糖蛋白奠定基础。【Objective】The expression pattern and protein characteristics of theβ-galactosidase gene NtBGAL from Nicotiana tabacum were explored to provide a basis for the future development of tobacco as a versatile platform material.【Method】Through comparing the homologous between the gene sequences of N.tabacum K326 and NbBGAL1 gene of N.benthamiana,NtBGAL gene was cloned through PCR by taking cDNA of K326 as the template.Subsequently,bioinformatics tools were used to analyze its gene structure and protein physicochemical properties.The gene expression pattern in different tobacco tissues(roots,stems,leaves and flowers)was examined using qRT-PCR.Finally,the research on prokaryotic expression,protein purification and protein characterization of the gene was carried out.【Result】The cloned tobacco NtBGAL gene obtained in the study exhibited the highest homology with the NbBGAL1 gene in N.benthamiana,and both possessed identical structural domains(Motif).The gene was located on chromosome 9,and it contained 18 introns.The mRNA length of the gene was 6649 bp.The CDS was 2541 bp encoding 846 amino acids.The enzyme belonged to the GH35 family.The NtBGAL protein had a molecular weight of 92.44 kDa,a theoretical isoelectric point(pI)of 6.8,GRAVY score of-0.222,and was localized on the cell wall.There was a significant difference in gene expression levels of NtBGAL in different tissues of N.tabacum,followed by flower>leaf>stem>root.The prokaryotic expression was performed at 37℃,NtBGAL protein had obvious expression,and the higher purified NtBGAL protein was obtained by purification method of inclusion body.The optimal temperature for the enzymatic reaction of NtBGAL protein was 30-40℃and the optimal pH was 4.0-6.5.NtBGAL enzyme activity was enhanced at Mn^(2+)concentrations ranging from 0.05 to 0.15 mmol/L.【Conclusion】The study clarifies the tissue expression pattern and protein characterization of the NtBGAL gene,which lays the foundation for further modification of the NtBGAL gene to produce humanized glycop

关 键 词：烟草 NtBGAL基因 Β-半乳糖苷酶 生信分析 原核诱导 蛋白表征 

分 类 号：S572[农业科学—烟草工业]