油茶ABI5基因的克隆及其表达分析

Cloning and expression analysis of ABI5 from Camellia oleifera

以油茶(Camellia oleifera)主栽品种‘华硕’为试验材料,通过RT-RCR的方法克隆出油茶脱落酸不敏感蛋白5(abscisic acid-insensitive 5,ABI5)全长cDNA序列,命名为CoABI5,利用不同的在线网站对CoABI5基因进行生物信息学分析,通过原核表达、亚细胞定位和实时荧光定量PCR分别对该基因的表达情况、表达部位和表达模式进行研究分析。结果表明:该序列CDS长度为795 bp,编码264个氨基酸,其中与茶树(Camellia sinensis)的ABI5相似性最高,达到97.73%。原核表达实验发现融合蛋白pCold-CoABI5-TF在37℃诱导4 h后,在85 kDa处出现一条蛋白条带,与理论融合蛋白的分子量一致。亚细胞定位显示该基因定位于细胞核。实时荧光定量PCR结果表明,CoABI5基因在油茶花粉中的表达量最高,自交24 h花柱中的表达量大于‘华硕’ב华鑫’异交,异交48 h子房中的表达量大于自交。本研究对进一步挖掘该基因在油茶后期自交不亲和过程中发挥的作用提供研究基础,对油茶自交不亲和分子机制解析具有重要的意义。

In this study,the Camellia oleifera cultivar‘Huashuo’was used as the material to clone the abscisic acid-insensitive 5 of Camellia oleifera by RT-PCR method,which was named CoABI5.The CoABI5 gene was analyzed by bioinformatics using different online sites,and the expression,expression site and expression pattern of CoABI5 gene were analyzed by using prokaryotic expression,subcellular localization and real-time quantitative RT-PCR.The results showed that the sequence CDS length of CoABI5 was 795 bp and encoded 344~363 amino acids,CoABI5 had the highest similarity(97.3%)with the ABI5 of Camellia sinensis.Prokaryotic expression showed that fusion protein pCold-CoABI5-TF was induced for 4 h at 37℃,it had a protein band at 85 kDa,which was consistent with the molecular weight of the theoretical fusion protein.Subcellular localization showed that CoABI5 gene was located in the nucleus.Real-time quantitative RT-PCR results showed that CoABI5 gene was highly expressed in pollens,and the expression of CoABI5 gene in style of selfs 24 h was higher than that of‘Huashuo’and‘Huaxin’outcrosses,the expression of CoABI5 gene in ovary of outcrosses 48 h was higher than that of selfs.This study provides a research basis for the further exploring the role of CoABI5 gene in late self-incompatibility of Camellia oleifera and it is great significance for the analysis of the molecular mechanism of self-incompatibility of Camellia oleifera.