野大麦肌动蛋白基因片段的克隆及其表达特征分析  

Cloning and Expression of Actin Gene Fragment in Hordeum Brevisubulatum

作　　者：袁惠君[1] 关玉晨 王春梅[2] 冯欢 张欢欢 袁毅君[3] YUAN Huijun;GUAN Yuchen;WANG Chunmei;FENG Huan;ZHANG Huanhuan;YUAN Yijun(School of Life Science and Engineering,Lanzhou University of Technology,Lanzhou 730050,China;Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS,Lanzhou 730050,China;College of Bioengineering and Biotechnology,Tianshui Normal University,Tianshui 741000,China)

机构地区：[1]兰州理工大学生命科学与工程学院,兰州730050 [2]中国农业科学院兰州畜牧与兽药研究所,兰州730050 [3]天水师范学院生物工程与生物技术学院,天水741000

出　　处：《中国草食动物科学》2024年第4期30-38,共9页China Herbivore Science

基　　金：甘肃省自然科学基金项目(22JR5RA040);国家自然科学基金项目(32060390)。

摘　　要：野大麦(Hordeum brevisubulatum)是重要的野生禾本科牧草,具有多种抗逆性。为深入挖掘野大麦的抗逆基因,本研究以RT-PCR法克隆了野大麦肌动蛋白基因(Actin,ACT)的核心片段。采用BLAST序列比对,qRT-PCR实时定量方法对其序列特征和表达特性进行了分析。结果表明,野大麦ACT的基因片段长518 bp,编码171个氨基酸。BLAST分析表明,野大麦的肌动蛋白基因片段与大麦(Hordeum vulgare)ACT基因核苷酸序列的一致性达97%,与ACT蛋白氨基酸序列的同源性达91%,并将其命名为HbACT。与11种相关植物进行ACT氨基酸序列比对发现,HbACT含152个保守氨基酸和19个非保守氨基酸,说明HbACT蛋白具有高度保守性。qRT-PCR分析表明,在不同盐浓度处理条件下,不同处理时间野大麦各器官的HbACT的Ct平均值为19.30,地上部的变异系数为3.5,峰度系数为0.262;地下部的变异系数为5.3,峰度系数为0.409,均属于尖峰分布,Ct值的稳定系数均小于1.7。综上所述,HbACT基因表达稳定,可作为内参基因用于研究野大麦功能基因的表达模式分析。Wild barley(Hordeum brevisubulatum)is an important gramineous forage.It has a variety of stress resistance.To further explore and study the stress resistance genes of H.brevisubulatum,this study cloned the core fragment of the H.brevisubulatum actin gene(ACT)using RT-PCR method.Its sequence and expression characteristics were analyzed by BLAST sequence alignment and qRT-PCR real-time quantitative method.The results showed that the ACT gene fragment of H.brevisubulatum was 518 bp in length and encoded 171 amino acids.ACT gene fragment of H.brevisubulatum was 97%consistent with the nucleotide sequence of Hordeum vulgare ACT gene,and the amino acid sequence homology of ACT protein was 91%by through BLAST analysis.The actin gene fragment of H.brevisubulatum was identified and named HbACT.The amino acid sequences of HbACT were compared with those of other 11 plants,and 152 conserved amino acids and 19 non-conserved amino acids were found,which proved that HbACT protein was highly conserved.qRT-PCR analysis showed that the mean Ct value of HbACT in the organs of H.brevisubulatum treated with different salt concentrations and salt treatment time was 19.30,the coefficient of variation in the ground part was 3.5,and the coefficient of kurtosis was 0.262.The variation coefficient and kurtosis coefficient of the underground part were 5.3 and 0.409,both belonging to the peak distribution.The results of RefFinder comprehensive analysis showed that the stability coefficient of Ct value was less than 1.7.In conclusion,HbACT gene expression was stable.It could be used as an internal reference gene to study the expression pattern analysis of functional genes in H.brevisubulatum.

关 键 词：野大麦 肌动蛋白基因 克隆 表达模式分析 

分 类 号：S54[农业科学—作物学]