摘要
为了解启动子在龙眼AGO1基因(DlAGO1)表达调控中的作用,根据龙眼基因组数据,以龙眼胚性愈伤组织的DNA为模板克隆DlAGO1基因的启动子序列,利用Methprimer、BPDG和PlantCARE在线软件对该序列进行生物信息学分析,将该启动子序列定向替换pCAMBIA1300载体的CaMV35S启动子,构建DlAGO1基因启动子与GUS基因的融合表达载体,瞬时转化本氏烟烟草叶片,进行GUS组织化学染色分析,并用不同浓度的赤霉素(GA_(3))、茉莉酸甲酯(MeJA)、水杨酸(SA)和脱落酸(ABA)喷洒烟草叶片,用实时荧光定量仪(real-time quantitative PCR,RT-qPCR)分析不同浓度激素处理下GUS基因的相对表达情况.结果显示:分离克隆得到1437 bp的DlAGO1基因启动子序列,该序列含有2个CpG岛分布区,2段核心启动子区域,还含有多种顺式元件,包括大量的TATA-box和CAAT-box核心元件以及GA_(3)、MeJA、ABA、光、逆境、胚乳表达等响应元件;转基因烟草叶片被中度染色,ABA处理能够诱导DlAGO1启动子的活性.综上所述,龙眼DlAGO1基因启动子可以驱动下游GUS基因的表达,DlAGO1基因参与ABA等激素的响应,可能参与抗逆应答机制,结果可为进一步分析该基因的激素应答和抗逆机制奠定基础.(图7表1参31)
In this study,we aimed to understand how DlAGO1 expression is regulated in longans.Based on longan genome data,the promoter sequence of DlAGO1 was cloned using EC DNA as the template.Bioinformatics analysis was conducted using Methprimer,BPDG,and PlantCARE online software.The DlAGO1 promoter sequence of DlAGO1 was inserted into the pCAMBIA1300 vector and a fusion expression vector was constructed.Tobacco leaves were transformed and GUS histochemical staining was performed.Furthermore,tobacco leaves were sprayed with different concentrations of gibberellic acid(GA_(3)),methyl jasmonate(MeJA),salicylic acid(SA),and abscisic acid(ABA),and real-time quantitative PCR(qRT-PCR)was used to analyze the relative expression of the GUS gene during exposure to the different concentrations of hormones.An isolated 1437 bp promoter sequence of DlAGO1 contained two CpG island distribution regions,two core promoter regions,and mutiple cis-elements which contained TATA-box,CAAT-box,GA_(3),MeJA,ABA,light,stress,and endosperm expression response elements.All transgenic tobacco leaves were stained,but the staining was shallow.ABA treatment induced DlAGO1 promoter activity.These findings suggest that the DlAGO1 promoter could drive the expression activity of GUS,participate in responses to hormones like ABA,and possibly participate in an antiviral mechanism.The findings lay a foundation for further analysis of ABA and response to it.
作者
陈荣珠
王小平
申序
林争春
赖钟雄
CHEN Rongzhu;WANG Xiaoping;SHEN Xu;LIN Zhengchun;LAI Zhongxiong(Department of Pharmacy,Zhangzhou Health Vocational College,Zhangzhou 363000,China;Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2022年第2期485-490,共6页
Chinese Journal of Applied and Environmental Biology
基金
漳州市科技局重大专项(ZZ2018ZD2019)
漳州卫生职业学院科技创新团队培育项目(Kjcx-3)
福建省高原学科建设经费(102/71201801101)
福建农林大学科技创新专项基金(KFA19037A)
国家自然科学基金项目(31572088)资助。
