[关键词]
[摘要]
目的 克隆细胞色素P450s(CYP450s)家族成员的2个基因,探究红花Carthamus tinctorius幼叶在逆境胁迫下CYP450s基因的表达与红花总黄酮含量变化关系。方法 以吉红1号为材料,对CYP450s家族的2个基因CYP81E8、CYP71A1进行基因克隆表达及分析。研究了茉莉酸甲酯(methyl jasmonate,MeJA)、干旱(PEG-6000)、光及暗处理4种逆境胁迫对红花总黄酮含量的影响,分析了CtCYP81E8和CtCYP71A1基因的表达与叶片总黄酮含量之间的相关性。结果 CtCYP81E8编码区序列为1014 bp,编码337个氨基酸;CtCYP71A1编码区序列为1287 bp,编码428个氨基酸。2个基因在不同胁迫条件下的表达量及总黄酮含量的变化表明:MeJA处理后,与野生型相比,叶片中的总黄酮含量均有不同程度的增加。CtCYP81E8、CtCYP71A1的表达量与总黄酮的积累量呈正相关;当采用PEG-6000处理后,CtCYP81E8处于正调控,而CtCYP71A1处于负调控状态,红花的叶片中总黄酮含量均显著增加;光胁迫下,在一定时间内总黄酮含量与CtCYP71A1基因表达量呈正相关,与CtCYP81E8基因表达量呈负相关,暗胁迫下,总黄酮含量与CtCYP81E8及CtCYP71A1基因表达趋势一致,都在一定时间内呈正相关。结论 成功克隆了红花CtCYP81E8及CtCYP71A1基因。qRT-PCR分析表明,CtCYP81E8、CtCYP71A1基因可能在红花幼叶期对逆境胁迫的适应中发挥重要作用,为红花CYP450s家族基因在红花黄酮代谢合成的功能研究提供理论依据。
[Key word]
[Abstract]
ObjectiveTwo genes of members of the CYP450s family were cloned to explore the relationship between the expression of CYP450s genes and the changes in total flavonoids in young leaves of Honghua Carthamus tinctorius under stress. MethodsUsing Jihong No. 1 as the material, the two genes CYP81E8 and CYP71A1 of the CYP450s family were cloned, expressed and analyzed. The effects of methyl jasmonate, drought (PEG-6000), light and dark treatments on the content of total flavonoids in C. tinctorius were studied, and the correlation between the expression of CtCYP81E8 and CtCYP71A1 genes and the content of total flavonoids in leaves was analyzed. ResultsThe CtCYP81E8 coding region sequence was 1014 bp, encoding 337 amino acids; the CtCYP71A1 coding region sequence was 1287 bp, encoding 428 amino acids. The expression of the two genes under different stress conditions and the changes in total flavonoid content indicated that the total flavonoid content in the leaves increased to varying degrees after treatment with methyl jasmonate compared with the wild type. The expression of CtCYP81E8 and CtCYP71A1 was positively correlated with the accumulation of total flavonoids; when treated with PEG-6000, CtCYP81E8 was under positive regulation, while CtCYP71A1 was under negative regulation, and the total flavonoid content in the leaves of C. tinctorius increased significantly; Under light stress, the total flavonoid content was positively correlated with CtCYP71A1 gene expression in a certain period of time, and negatively correlated with CtCYP81E8 gene expression. Under dark stress, the total flavonoid content was consistent with CtCYP81E8 and CtCYP71A1 gene expression trends, and both showed a positive correlation within a certain period of time. ConclusionThe CtCYP81E8 and CtCYP71A1 genes of C. tinctorius were successfully cloned. qRT-PCR analysis showed that CtCYP81E8 and CtCYP71A1 genes may play an important role in the adaptation of C. tinctorius young leaves to adversity stress. This study provides a theoretical basis for the study of CYP450s family genes in flavonoid metabolism and synthesis of C. tinctorius.
[中图分类号]
R282.12
[基金项目]
:国家自然科学基金资助项目(31771868);国家自然科学基金资助项目(81760679);吉林省科技厅项目(20190201172JC);吉林省科技厅项目(20190201175JC);吉林农业大学大学生创新创业训练计划项目;贵州省中医药管理局项目