摘要
【目的】克隆栽培烟草K326着丝粒特异组蛋白(Centromeric histone H3,CENH3)基因,并对其进行生物信息及表达特性分析,为探明NtCENH3基因的单倍体诱导功能及烟草品种快速改良提供依据。【方法】以栽培烟草K326为材料,利用同源克隆技术获取NtCENH3-1和NtCENH3-2基因的cDNA全长序列,运用生物信息学软件分析蛋白的序列特征;通过亚细胞定位技术验证基因的表达部位;利用荧光定量PCR检测NtCENH3-1和NtCENH3-2基因在烟草不同组织部位的表达情况。【结果】NtCENH3-1和NtCENH3-2包含7个外显子和6个内含子,编码区(CDS)长度分别为468 bp和471 bp,分别编码156个和157个氨基酸,蛋白的分子量分别为17.64 kDa和17.75 kDa。进化树分析表明,栽培烟草CENH3-1和CENH3-2基因分别来源于祖先种绒毛烟草和林烟草,2个基因均定位于细胞核中。RT-PCR分析显示,NtCENH3-1和NtCENH3-2在不同组织中呈差异性表达,且在雌蕊及幼嫩的果实中表达量较高。【结论】在烟草中成功克隆着丝粒特异组蛋白NtCENH3-1和NtCENH3-2基因,烟草着丝粒特异组蛋白基因参与烟草各组织的生长发育过程。
【Objective】NtCENH3-1 and NtCENH3-2 genes from Nicotiana tabacum K326 were cloned and their bioinformation and expression profile were analyzed,which provided the basis for exploring the haploid induction function of NtCENH3 gene and the rapid improvement of tobacco variety.【Method】Using the cultivated tobacco K326 as the research material,the full-length cDNA sequences of NtCENH3-1 and NtCENH3-2 genes were cloned by homologous cloning technology and the sequence characteristics of the protein were analyzed by bioinformatics.Subcellular localization was conducted to verify the expression tissue of the protein;Finally,the expression profile of NtCENH3-1 and NtCENH3-2 genes in different tobacco tissues was detected by fluorescence quantitative PCR.【Result】NtCENH3-1 and NtCENH3-2 contained 7 exons and 6 introns.The coding regions(CDS)were 468 bp and 471 bp in length,encoding 156 and 157 amino acids,respectively.The molecular weights of the proteins were 17.64 kDa and 17.75 kDa.The phylogenetic tree showed that NtCENH3-1 and NtCENH3-2 were originated from the ancestral species of Nicotiana tomentosiformis and Nicotiana sylvestris,respectively,and both genes were located in nucleus.RT-PCR analysis indicated that NtCENH3-1 and NtCENH3-2 were differentially expressed in different tobacco tissues,and highly expressed in female flower and young fruit.【Conclusion】NtCENH3-1 and NtCENH3-2 genes were successfully cloned from K326 and were involved in the growth and development stages of different tobacco tissues.
作者
曹领改
张莉莉
张盼
张吉顺
张孝廉
CAO Linggai;ZHANG Lili;ZHANG Pan;ZHANG Jishun;ZHANG Xiaolian(Guizhou Academy of Tobacco Sciences,Guiyang,Guizhou 550081;College of Agriculture,China Agriculture University,Beijing 100094,China)
出处
《贵州农业科学》
CAS
2023年第11期1-8,共8页
Guizhou Agricultural Sciences
基金
贵州省烟草科学研究院科技项目“着丝粒组蛋白CENH3在烟草单倍体诱导中的功能研究”(GZYKY2021-03)
贵州省科技厅基金项目“烟草单倍体诱导相关NtPLA基因的克隆及功能鉴定”[黔科合基础(2020)1Y067]
中国烟草总公司贵州省公司科技项目“烟草单碱基编辑系统的建立及应用”(2022XM04)
贵州省烟草科学研究院科技项目“NtBGAL在烟草半乳糖基化改造中的功能研究及应用”(GZYKY2021-06)。
关键词
烟草
CENH3
基因克隆
生物信息
表达特性
生长发育
tobacco
CENH3
gene cloning
bioinformation
expression characteristics
growth and development
