摘要
【目的】探讨油柰(Prunus salicina lindley)WRKY33启动子在不同胁迫处理下的表达模式,为进一步深入研究油柰WRKY基因在油柰生长发育或抵御各种逆境胁迫中的作用机制等提供理论依据。【方法】利用MEGA6.06软件构建PsWRKY33的系统进化树。通过染色体步移技术克隆获得该基因启动子序列,利用PlantCARE数据库分析预测PsWRKY335′端上游启动子区域的顺式作用元件。利用拟南芥浸花法获得转基因植株。通过对不同胁迫处理下的各片段转基因幼苗进行组织化学染色和GUS酶活性测定。【结果】系统进化树分析表明PsWRKY蛋白与拟南芥WRKY33亲缘关系最近,故将该基因命名为PsWRKY33。获得PsWRKY33基因5′端上游启动子序列长度为1872 bp,经预测分析发现该启动子区域含有ABRE、ARE、LTR、MYB和W-box等响应不同植物激素等顺式作用元件,基于此构建3个不同长度的缺失片段。对转基因植株不同组织GUS染色发现PsWRKY33启动子主要表达在叶、花瓣和花序梗上,且随着片段的缺失其表达越低。在不同胁迫处理下,其GUS活性表达程度不同。在低温胁迫条件下,不同长度的PsWRKY33启动子片段均受到不同程度的诱导上调表达;而在SA胁迫下,各片段呈现出不同程度的诱导下调表达。【结论】PsWRKY33基因启动子可能参与调节油柰应答低温胁迫及外源激素SA的响应。
【Objective】Expression of PsWRKY33 promoter of Nane(Prunus salicina lindley)under stress was determined for a study on the mechanism of WRKY gene associated with the growth and stress-resistance of the plant.【Method】Unrooted phylogenetic tree representing relationships among protein sequences of PsWRKY33 was constructed using Nane and other plants through by MEGA 6.06 software.The promoter sequence was cloned by chromosome stepping technique.The cis-acting regulatory elements in the upstream promoter region at the 5′end of PsWRKY33 were predicted according to the PlantCARE database.Transgenic plants were obtained by using the Arabidopsis immersion method.Histochemical staining and GUS enzyme activity were performed on the transgenic seedlings under different stresses.【Result】The phylogenetic tree analysis showed that the PsWRKY protein was most closely related to Arabidopsis WRKY33,thus the gene was named PsWRKY33.The1872 bp length of 5′end upstream in PsWRKY33 was obtained,from Nane via genome walking technology.The promoter region contained motifs of hormones(ABRE and ARE)and biotic stresses(LTR,MYB,W-box,and other cis-acting elements).Then,the 35′-deletion fragments were constructed.The histochemical staining and GUS activity measurement showed that the full PsWRKY33 promoter fragment mainly expressed in leaves,petals,and peduncles,and its expression decreased with the loss of fragments.Under low temperature,the various fragments of PsWRKY33 promoter were upregulated,but downregulated under SA stress.【Conclusion】PsWRKY33 might response to low temperature and exogenous hormone SA stress in Nane.
作者
陈永萍
何水林
刘志钦
陈桂信
蒋际谋
CHEN Yongping;HE Shuilin;LIU Zhiqin;CHEN Guixin;JIANG Jimou(Fruit Research Institute,Fujian Academy of Agricultural Sciences/Fujian Breeding Engineering Technology Research Center for Longan and Loquat,Fuzhou,Fujian 350013,China;College of Agriculture,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2022年第2期170-177,共8页
Fujian Journal of Agricultural Sciences
基金
福建省农业高质量发展超越“5511”协同创新工程项目(KXXYJBG2021006)。
