百子莲脱水素基因ApY_(2)SK_(2)逆境应答模式及启动子调控功能研究  

Stress Response Pattern of Agapanthus praecox Dehydrin Gene ApY_(2)SK_(2) and Its Promoter Functional Analysis

作　　者：陈晓童 吕可 刘涛 张荻 CHEN Xiaotong;Lü Ke;LIU Tao;ZHANG Di(School of Design, Shanghai Jiao Tong University, Shanghai 200240, China;Hangzhou Qiandao Lake Global Tourism Co., Ltd, Hangzhou 311700, China)

机构地区：[1]上海交通大学设计学院风景园林系,上海200240 [2]杭州千岛湖全域旅游有限公司,杭州311700

出　　处：《西北植物学报》2021年第8期1267-1278,共12页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31670693,31971705);上海市自然科学基金(21ZR1434200)。

摘　　要：在百子莲胚性细胞中筛选到对超低温保存复合逆境具有积极响应的保护类蛋白脱水素(ApY_(2)SK_(2)),为探明ApY_(2)SK_(2)基因在复合逆境中的应答模式,该研究采用染色体步移技术克隆并分析了ApY_(2)SK_(2)编码基因上游1200 bp的启动子序列。结果表明:(1)序列分析显示,该启动子含有多个与逆境和激素诱导相关的顺式调控元件;实时荧光定量PCR结果表明,ApY_(2)SK_(2)基因的表达具有组织特异性,在百子莲的叶和果中表达量较高,且在多种胁迫处理与ABA激素诱导下,其表达量显著升高。(2)成功构建了5个ApY_(2)SK_(2)启动子不同缺失片段驱动GUS基因的融合表达载体,经农杆菌转化、抗性筛选和PCR检测鉴定,获得T3代纯和转基因拟南芥株系。(3)GUS组织化学染色结果显示,GUS基因在拟南芥幼苗全株、成年苗的叶、花和成熟果实中表达活性较强,但在未成熟果实中无明显表达;烟草瞬时表达结果显示,与对照组相比,在脱水胁迫和ABA处理下的ApY_(2)SK_(2)启动子不同缺失片段驱动GUS基因表达具有显著差异。(4)转基因拟南芥GUS活性测定结果显示,ApY_(2)SK_(2)启动子MBS元件和ABRE元件可响应干旱与渗透胁迫信号;ApY_(2)SK_(2)启动子LTR元件参与低温响应;ApY_(2)SK_(2)启动子-1199~-262 bp区域包含多个串联的ABRE顺式调控元件(-373~-211 bp)对响应ABA信号具有主要调控作用。该研究结果揭示了ApY_(2)SK_(2)启动子的组织特异性,且启动子上的关键顺式调控元件对不同的胁迫和激素信号响应具有决定性调控作用。ApY_(2)SK_(2) a dehydrin gene from Agapanthus praecox was identified to have the positive protective effect on embryonic cells during cryopreservation.In order to reveal the regulatory mechanism of ApY_(2)SK_(2) response to complex stresses of cryopreservation,a 1200 bp promoter sequence of ApY_(2)SK_(2) was cloned and analyzed by using Chromosome Walking technique.The results showed that:(1)ApY_(2)SK_(2) promoter contained multiple stress-,hormones response-and plant embryo development-related cis-acting elements.Quantitative Real-time PCR assay suggested that the ApY_(2)SK_(2) promoter had tissue-specific expression pattern,and ApY_(2)SK_(2) gene had the highest expression level in the leaves and fruits of A.praecox.This gene was significantly up-regulated response to various stress and ABA treatments.(2)Five different deletion fragments of ApY_(2)SK_(2) promoter were constructed into pBI121 expression vectors,and using Agrobacterium mediated transformation and PCR identification to obtain the A.thaliana T3 generation transgenic lines.(3)GUS histochemical staining results showed that GUS staining signal were mainly distribute at the whole seedling,and the leaves,flowers and mature fruits of adult transgenic plants.Furthermore,gene transient transformation in leaf epidermis of tobacco test showed that the driver activity of 5 different deletion promoter fragments were significantly different under PEG and ABA treatments.(4)Quantitative determination of GUS enzyme activity suggested that MBS element and ABRE element of the ApY_(2)SK_(2) promoter involved in response to drought and osmotic signals,respectively.The LTR element participates in the low temperature response.Additionally,-1199 to-262 bp of ApY_(2)SK_(2) promoter contained multiple tandem ABRE cis-acting regulators(locate at-373 to-211 bp)that play an important role in response to ABA signaling.This study finally confirmed that ApY_(2)SK_(2) promoter has tissue specific regulatory functions,and some core cis-acting elements play a decisive role in regul

关 键 词：百子莲 脱水素 逆境胁迫 启动子 

分 类 号：Q785[生物学—分子生物学] Q786