文献类型：期刊文献

中文题名：落叶松LkTCTP启动子的克隆及表达分析

英文题名：Isolation and Characterization of a Larix Promoter Derived from the Translationally Control Tumor Protein(TCTP)Gene

作者：黄雨芹[1] 张如鑫[2] 张馨元[3] 王俊臣[1] 齐力旺[1] 张立峰[1]

第一作者：黄雨芹

机构：[1]中国林业科学研究院林业研究所,林木遗传育种全国重点实验室,国家林业和草原局林木育种与培育重点实验室,北京100091;[2]河北农业大学生命科学学院,河北保定071001;[3]北京林业大学林学院,北京100083

年份：2025

卷号：38

期号：2

起止页码：48-57

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：;北大核心:【北大核心2023】；

基金：农业生物育种国家科技重大专项(2023ZD04058);国家自然基金项目(32171811);中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2019SY011)。

语种：中文

中文关键词：落叶松;TCTP;启动子克隆;体细胞胚;表达分析

外文关键词：Larix;TCTP;promoter cloning;somatic embryogenesis;expression analysis

分类号：S791.223

摘要：[目的]通过研究落叶松LkTCTP及其启动子的表达特性,揭示该基因在体细胞胚发育中的功能,评估它作为组成型启动子的可行性。[方法]利用染色体步移技术,获得LkTCTP启动子序列;采用qRT-PCR检测其在不同温度、激素处理下的表达模式;构建2.0 kb的LkTCTP启动子融合GUS载体,在模式植物及落叶松中研究其启动子活性及组织定位。[结果]获得了长度约为3.0 kb的LkTCTP启动子序列,生物信息学预测该区域含有多个与激素和抗逆相关的顺式作用元件,如AuxRR-core/TGA-element、TATC-box、TCA-element、LTR、MBS/TC-rich repeats等;高温(42、37℃)和激素(75μmol·L^(-1)ABA、50μmol·L^(-1)GA、50μmol·L^(-1)IAA、50μmol·L^(-1)SA)处理均可诱导LkTCTP上调;LkTCTP 2.0 kb启动子在拟南芥中驱动GUS呈组成型表达,也可用于目的蛋白的亚细胞定位研究;落叶松胚性细胞瞬时侵染6 d时表现出明显的GUS染色,引入P19蛋白可增加外源基因的表达强度;转基因体细胞胚苗的子叶、下胚轴以及根部均可观察到GUS染色,且子叶基部表达量明显高于叶尖,但在落叶松中的表达具有组织特异性。[结论]本文克隆了LkTCTP的启动子序列,发现其表达水平受温度、激素影响;在拟南芥幼苗及落叶松体细胞胚发育阶段均呈组成型表达,但在落叶松幼苗的表达具有组织特异性。上述结果表明LkTCTP参与落叶松体细胞胚发育,揭示了其启动子作为组成型启动子的应用潜力。本研究为开发适用于落叶松的基因工程元件提供理论依据。
[Objective]To reveal LkTCTP function during somatic embryogenesis and evaluate its feasibility as a constitutive promoter,based on its expression patterns and promoter activity analysis.[Method]The promoter region of LkTCTP was cloned using an improved genome walking technique.The expression patterns of LkTCTP under different temperature and hormone treatments were examined by qRTPCR.Promoter activity and tissue localization were analyzed in model plants and Larix by constructing an expression vector for the fusion of LkTCTP promoter and GUS.[Result]A~3.0 kb fragment of the LkTCTP promoter was obtained.In this region we identified many important potential cis-acting elements related to hormones and stress resistance predicted by PLANTCARE,such as AuxRR core/TGA element,TATC box,TCA element,LTR,MBS/TC rich repeats.The expression of LkTCTP could be induced and upregulated by high temperature(42℃,37℃)and hormone treatments(75μmol·L^(?1)ABA、50μmol·L^(?1)GA、50μmol·L^(?1)IAA、50μmol·L^(?1)SA).The 2.0 kb promoter of LkTCTP could drive constitutive expression of GUS in Arabidopsis and could also be used for subcellular localization of transient expressed in tobacco.Notably,significant GUS staining was observed in Larix embryonic cells following a brief 6-day infection,particularly when the P19 protein,which suppresses transcription silencing and enhances exogenous gene expression,was introduced.GUS staining was also evident in transgenic somatic embryo seedlings,with expression levels at the base of the cotyledons is significantly higher than at the leaf tips,indicating tissue-specific expression in Larix.[Conclusion]The prediction of cis-acting elements in the LkTCTP promoter and its response to temperature and hormones,suggests that it may be closely related to stress resistance responsiveness.The expression patterns in model plants and Larix indicate its feasibility as a constitutive promoter.However,further validation using promoters of different lengths is still needed to provide a theoretical basis for the development of genetic engineering elements suitable for Larix.