海南杜鹃热诱导基因RhRCA1启动子的克隆与功能分析

为了阐述RhRCA1基因的调控机制,以海南杜鹃(Rhododendron hainanense)3年生扦插苗为材料,分析RhRCA1高温响应和组织表达特性;并克隆获得RhRCA1的启动子序列,将该启动子分别驱动荧光素酶报告基因在烟草中瞬时表达、GUS报告基因在拟南芥中稳定表达,分析该启动子活性、组织特异性和热诱导性。结果显示:(1)25℃条件下,RhRCA1表达量极低,37℃处理后其表达量显著升高,呈现典型的热诱导特性,并且RhRCA1在绿色组织中的表达量显著高于非绿色组织,呈现组织特异性;(2)获得的启动子长1 624 bp,其含有多个非生物胁迫响应、光响应、组织特异性等相关元件;(3)构建RhRCA1启动子和萤光素酶融合的植物表达载体,在烟草叶片中瞬时表达,荧光成像结果表明RhRCA1启动子能强烈响应高温胁迫;(4)构建RhRCA1启动子和GUS融合的植物表达载体,转化拟南芥植株筛选至T3代,GUS染色结果显示,高温能显著诱导RhRCA1启动子在拟南芥子叶、成熟叶、茎、萼片、果荚等绿色组织中的表达。研究结果表明RhRCA1启动子是1个兼具高温诱导型和组织特异性的启动子,可应用于植物...

Isolation and Function Analysis of the Promoter of a Thermal Inducible Gene RCA1 in Rhododendron hainanense

In order to analysis the regulatory mechanism and expression profile of RhRCA1,three-year-old cutting seedlings of Rhododendron hainanense were used as the experimental material to isolate the promoter of RhRCA1.This promoter was then used to drive the Luciferase and GUS reporters in tobacco and Arabidopsis thaliana respectively,to figure out its activity,tissue specificity and thermal inducibility.The results showed that:(1)Remarkable high expression of RhRCA1 was observed in the quantitative experiment after high temperature treatment,which appeared typical thermal inducibility.(2)The promoter of RhRCA1 with a length of 1624 bp was cloned and the promoter contained multiple ciselements related to abiotic stress response,light signals and tissue specificity.(3)A plant expression vector was constructed using the RhRCA1 promoter to drive Luciferase report gene,and was transformed into tabacco to analyse transient expression.The results showed that RhRCA1 promoter could strongly respond to heat stress.(4)The promoter and GUS fusion expression vector were constructed and transformed into Arabidopsis.Analysis of T3 generation transgenic Arabidopsis tissues with histochemical staining showed that heat stress had a significant impact on the induction of RhRCA1 promoter expression in green tissues,including cotyledon,mature leaf,young stem,sepal and seedpods.These results indicated that the promoter of RhRCA1 is a heat-inducible and tissue-specific promoter.This promoter is ideal to drive other genes to improve the thermostability of plants under high temperature by genetic engineering,which has promising applications under the tendency of global warming.