火龙果DREB1D基因的克隆及在转基因拟南芥中的功能分析  

Cloning of DREB1D gene from pitaya and its functional analysis in transgenic Arabidopsis thaliana

作　　者：张璐芳 侯黔东 蔡晓薇 杨鵾[1] 文晓鹏[1] ZHANG Lufang;HOU Qiandong;CAI Xiaowei;YANG Kun;WEN Xiaopeng(College of Life Sciences/Institute of Agro-bioengineering,Guizhou University/Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guiyang 550025,Guizhou,China)

机构地区：[1]贵州大学生命科学学院·农业生物工程研究院·山地植物资源保护与种质创新教育部重点实验室,贵阳550025

出　　处：《果树学报》2023年第7期1330-1341,共12页Journal of Fruit Science

基　　金：国家自然科学基金项目(32060663)。

摘　　要：【目的】DREB(dehydration responsive element binding)是一类脱水响应元件结合蛋白,在植物响应高温、干旱、高盐和低温等多种非生物胁迫过程中发挥关键作用。以紫红龙火龙果(Hylocereus monacanthus)为材料,克隆得到DREB转录因子,并命名为HmDREB1D(HU02G01866.1),探究其生物学功能。【方法】构建HmDREB1D基因植物过表达载体,通过亚细胞定位分析HmDREB1D基因在细胞中的位置。异源转化拟南芥,对T3代纯合系转基因拟南芥(OE3、OE4、OE5)进行生物学功能验证。【结果】火龙果HmDREB1D基因的开放阅读框全长723 bp,产生的蛋白定位于细胞核内,属DREB1s亚家族,具有典型的AP2结构域。将HmDREB1D基因转化至拟南芥获得超表达转基因株系,与野生型相比,转基因株系表现出较高的抗逆性。在干旱胁迫下,转基因植株T3代纯合系种子的萌发率高于野生型。转基因植株的叶片在逆境胁迫下表现出更低的电导率及更高的保护性酶活性。实时荧光定量PCR分析显示,RD20、HSP70和COR15A等逆境胁迫响应基因在HmDREB1D基因超表达植株中具有更高的表达量。【结论】过表达HmDREB1D基因通过调控抗逆相关基因表达,加速清除植株内的活性氧,增强植株的抗逆性。【Objective】DREB(dehydration responsive element binding)proteins are widely present in plants and are primarily involved in the abiotic stress response of plants.The two primary DREB transcription factors are DREB1 and DREB2,with DREB1 being mostly associated to low temperature and drought stress and DREB2 being primarily related to drought,salt,and high temperature stress.Pitaya(Hylocereus monacanthus)belongs to the cactus plants,because of its high nutritional value and strong resistance stress,it is popular with customers in karst regions like Guizhou and Guangxi.DREBs were found responsive to drought stress in pitaya,leaving the underlying mechanism unrevealed.This study intends to clone HmDREB1D(HU02G01866.1)gene and verify its biological function.【Methods】The pCambia35s-HmDREB1D-GFP plant overexpression vector was constructed by seamless cloning technology and transformed into Tobacco.The fluorescence signal of HmDREB1D was observed under a laser confocal microscope to determine the subcellular location.The expression vector of pCambia35sHmDREB1D was constructed.The HmDREB1D gene was transformed into A.thaliana,a total of 6 transgenic A.thaliana plants were obtained,and 3 overexpressed transgenic Arabidopsis(OE3,OE4 and OE5)plants were chosen for further biological verification.After surface sterilization,HmDREB1D transgenic Arabidopsis(OE3,OE4 and OE5)and the wild type Arabidopsis seeds were sown in 250 mmol·L^(-1)mannitol 1/2 MS medium for drought stress treatment(16 h/8 h day/night cycle,24℃).The germination rate was counted after 7 days.The seedlings grew in 1/2 MS medium for 7 days,and then were transplanted into 1/2 MS medium containing 250 mmol·L^(-1)mannitol for drought stress treatment(16 h/8 h day/night cycle,24℃).The root length and fresh weight were measured 7 days after the treatment.Transgenic Arabidopsis(OE3,OE4 and OE5)and the wild type Arabidopsis seeds were surface sterilized,sown in 1/2 MS medium and cultured for 7 days,and then transplanted in pots filled with nutrient soil

关 键 词：火龙果 HmDREB1D 功能分析 非生物胁迫 亚细胞定位 

分 类 号：S667.9[农业科学—果树学]