摘要
芽茎是药用植物老鸦瓣重要繁殖器官,前期老鸦瓣转录组数据显示MYB是芽茎发育过程中表达最活跃转录因子家族之一。为研究MYB转录因子在芽茎发育阶段可能发挥的作用,该研究基于老鸦瓣MYB基因家族表达谱数据,筛选并克隆得到1个高效响应芽茎发育的MYB转录因子,命名为AeMYB4。该基因开放阅读框为756 bp,编码251个氨基酸,预测的氨基酸序列中包含2个高度保守的SANT结构域和冷胁迫因子CBF结合位点,系统进化分析结果显示AeMYB4和拟南芥AtMYB15聚为一类,属于S2亚家族R2R3-MYB,该亚族转录因子多数和低温胁迫应答相关。构建GFP-AeMYB4融合表达载体转入根癌农杆菌后瞬时侵染本氏烟草叶片后显微观察,发现AeMYB4蛋白定位于细胞核;构建pGBKT7-AeMYB4融合表达载体转入酵母菌中进行转录活性检测,结果表明AeMYB4为转录激活因子。基因表达分析显示AeMYB4表达具有组织特异性,主要在芽茎发育阶段表达,且在芽茎发育中期表达最活跃,推测AeMYB4可能在芽茎发育过程中发挥重要作用。该研究为进一步探讨AeMYB4转录因子在芽茎中的功能提供了研究基础。
Stolon is an important organ for reproduction and regeneration of Amana edulis.Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A.edulis stolon.In order to study the possible role of MYB transcription factors in stolon development,the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors.In the present study,sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids,and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF.By multiple sequence alignment and phylogenetic analysis,it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana,belonging to subgroup S2 of R2 R3-MYB.And most of the transcription factors in this subfamily are related to low temperature stress.The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana,and the results showed the protein was located in the nucleus.To investigate the transcriptional activation,the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells,which proved that AeMYB4 was a transcription activator with strong transcriptional activity.Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves,flowers,and bulbs of A.edulis,which indicated that AeMYB4 transcription factor was tissue-specific in expression,mainly in the stolon development stage,and that the expression was the most active in the middle stage of stolon development,suggesting that AeMYB4 gene may play an important role in stolon development.This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A.edulis.
作者
徐碧霞
朱再标
郭巧生
张军霞
XU Bi-xia;ZHU Zai-biao;GUO Qiao-sheng;ZHANG Jun-xia(Institute of Chinese Medicinal Materialsy Nanjing Agricultural University,Nanjing 210095,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2021年第17期4395-4402,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81773834)。
关键词
老鸦瓣
MYB转录因子
克隆
特性分析
Amana edulis
MYB transcription factor
cloning
characteristic analysis
