川黔紫薇LeAGL11基因克隆表达分析及转录自激活检测  

Cloning,expression analysis and autonomous transcriptional activation testing of transcription factor LeAGL11 from Lagerstroemia excelsa (Dode) Chun

作　　者：李雪露 陆柳淑 邓涪元 李露 雷宇行 彭继庆[1] 何钢[1] 乔中全[2] LI Xuelu;LU Liushu;DENG Fuyuan;LI Lu;LEI Yuxing;PENG Jiqing;HE Gang;QIAO Zhongquan(College of Life and Environmental Sciences,Central South University of Forestry&Technology,Changsha 410004,Hunan,China;Hunan Provincial Key Laboratory of Tree Clone Breeding,Hunan Academy of Forestry,Changsha 410004,Hunan,China)

机构地区：[1]中南林业科技大学生命与环境科学学院,湖南长沙410004 [2]湖南省林业科学院林草培育研究所,湖南长沙410004

出　　处：《中南林业科技大学学报》2024年第6期197-206,共10页Journal of Central South University of Forestry & Technology

基　　金：湖南省自然科学基金项目(2023JJ30338);湖南省科技创新计划项目(2022RC1012);长沙市杰出创新青年培养计划项目(kq2106090)。

摘　　要：【目的】探究转录因子AGL11在紫薇属紫薇自交及川黔紫薇与紫薇杂交过程中不同时间的差异性表达及其在酵母中的转录自激活特性,为研究紫薇远缘杂交结实率低的分子机制奠定基础。【方法】以‘紫韵’紫薇自交授粉和 ‘紫韵’紫薇ב川黔1号’川黔紫薇杂交授粉后 24、48 和72 h的雌蕊为材料,克隆LeAGL11基因,通过生物信息学分析其理化性质等,采用实时荧光定量方法分析该基因在不同授粉方式和不同阶段的相对表达量,通过DNA重组技术构建LeAGL11的酵母表达载体,转化至Y2HGold感受态细胞内进行转录自激活检测。【结果】从川黔紫薇中克隆得到LeAGL11基因,该基因的编码序列长666 bp,编码221个氨基酸,LeAGL11蛋白无信号肽和跨膜结构域,不属于膜蛋白,属于亲水性蛋白。氨基酸序列比对和进化树分析显示其与紫薇、石榴和葡萄的AGL11蛋白都有较高的相似度;蛋白质结构预测和序列分析表明其具有MADS-box基因家族中典型的MADS-box和K-box保守结构域。实时荧光定量试验分析不同阶段的相对表达量表明LeAGL11基因在杂交后呈现出上调的趋势,在自交后呈现出先下调后上调的趋势,在自交授粉24 h时相对表达量最高,自激活检测发现pGBKT7-LeAGL11重组质粒在缺色氨酸的培养基中生长,在SD/-Trp+AbA+X-α-gal培养基中没有发生颜色变化。【结论】LeAGL11基因在紫薇自交过程及川黔紫薇和紫薇杂交过程中的相对表达量具有一定的差异性,且LeAGL11无转录自激活活性,可用于后续试验。【Objective】To investigate the differential expression of AGL11 in self-and cross pollinations between Lagerstroemia indica and Lagerstroemia excelsa (Dode) Chun,and self-activation characteristics of the transcription factor AGL11,it can lay a foundation for the study of the biological function of LeAGL11 in the distant cross incompatibility between L.excelsa and L.indica.【Method】Wecloned the LeAGL11 and analyzed its physicochemical property through bioinformatics,RT-qPCR was used to assess and compare the expression of LeAGL11 in pistils of different developmental stages (24 h,48 h and 72 h after ‘Ziyun’בZiyun’ and ‘Ziyun’בL.excelsa No.1’),a pGBKT7-AGL11 yeast expression vector was constructed by DNA recombination technology and transformed into the Y2HGold yeast strain.【Result】The coding sequence of LeAGL11 was 666 bp,translating 221 amino acids,it had no signal peptide and transmembrane domain,it was hydrophilic protein but not membrane protein,the sequence alignments of amino acids and phylogenetic analysis showed that it had a high degree of similarity with the AGL11 protein of L.indica,Punica granatum and Vitis vinifera.The protein structure prediction and sequence analysis indicated that it had a typical MADS-box and K-box conserved domains of the MADS-box gene family.The relative expression of this gene was highest expressed at 24 h after self-pollination,showing an upward trend in selfing,and firstly decreased and then increased up in hybridization.Self-activation showed pGBKT7-LeAGL11 plasmid did not turn blue in SD/-Trp + AbA + X-α-gal medium.【Conclusion】The relative expression of the LeAGL11 gene during selfing in L.indica and hybridization between L.indica and L.excelsa is characterized by a certain degree of variability,the LeAGL11 gene has no self-activation activity and could be used for subsequent yeast two hybrid experiments.

关 键 词：紫薇属 远缘杂交 AGL11 基因克隆 基因表达 

分 类 号：S718.46[农业科学—林学]