摘要
【目的】克隆红花油体蛋白基因CtOleosin,研究其表达特性,为探索其功能奠定基础。【方法】以红花种子为研究对象,于开花后4,8,12,16,20,24,28,32d,提取红花种子总RNA,利用RT-PCR技术克隆红花CtOleosin全长cDNA片段,生物信息学方法分析其特性及结构功能;利用Protparam在线工具分析CtOleosin蛋白的理化性质,用Blastp比对该蛋白与其他物种相关蛋白的同源性,使用SMART软件进行功能区分析;利用荧光定量方法检测该基因在种子不同发育时期的表达量。【结果】克隆了红花CtOleosin基因,其全长639bp,编码213个氨基酸,蛋白理论分子质量约为21.48ku,理论等电点9.39,带正电荷残基18个,负电荷残基14个;红花CtOleosin基因编码产物与向日葵Oleosin的同源性高达59.11%,属于油体蛋白家族成员,此蛋白不存在信号肽,无糖基化位点,存在2个跨膜区;以EF1α作为内参基因分析发现,CtOleosin基因表达量在红花种子发育初期逐渐升高,28d时达到最高,之后又有所下降。【结论】成功克隆了红花CtOleosin基因,明了了其特性、功能、编码产物的基本理化性质,以及其在红花种子不同发育时期的表达量变化情况。
【Objective】CtOleosin was cloned from Carthamus tinctorius L.and its expression characteristics were explored to lay foundation for its function exploration.【Method】The total RNA was extracted from safflower seeds 4,8,12,16,20,24,28 and 32 d after flowering and the cDNA of CtOleosin was cloned by RT-PCR.Its characteristics and structure function were studied by bioinformatics method,its physicochemical properties were analyzed by Protparam tool,and the homology was compared with other species using Blastp.The SMART software was used for functional area analysis,and the expression in different development periods was detected by qRT-PCR.【Result】CtOleosin was cloned and its cDNA was 639 bp with 213 amino acids.The molecular weight of the encoded protein was about 21.48 ku and theoretical isoelectric point was 9.39 with positively charged residues of 18 and negatively charged residues of 14.The CtOleosin encoding product had a closer genetic relationship with sunflower Oleosin with homology of 59.11%,belonging to the Oleosin family.The protein had no signal peptide and no glycosylation site,but had two transmembrane regions.EF1αwas selected as the appropriate reference gene and the expression level of CtOleosin was gradually increased in different development periods with the highest at 28 d,and then clecreased.【Conclusion】The CtOleosin gene was successfully cloned,the characteristics,functions and encoded products were cleared,and the expression in safflower seeds at different stages was analyzed.
作者
卢玉彬
迟孟涵
孙晓玉
温世雄
王清曼
陈希
强卫东
杨晶
LU Yubin;CHI Menghan;SUN Xiaoyu;WEN Shixiong;WANG Qingman;CHEN Xi;QIANG Weidong;YANG Jing(Ministry of Education Engineering Research Center of Bioreactor and Pharmaceutical Development,Jilin Agriculture University,Changchun,Jilin 130118,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2018年第11期121-128,共8页
Journal of Northwest A&F University(Natural Science Edition)
基金
2017年吉林省大学生创新创业训练计划项目(2017470)
关键词
红花
油体蛋白
克隆
生物信息学
safflower
CtOleosin
clone
biological information
