砂糖橘精氨酸脱羧酶CrADC基因的克隆及表达分析  

Cloning and expression analysis of arginine decarboxylase gene(CrADC)from Citrus reticulata‘Shatangju’

作　　者：吴秀兰 任诗欣 李桂花[3] 唐文武[2] WU Xiulan;REN Shixin;LI Guihua;TANG Wenwu(College of Food and Pharmaceutical Engineering,Zhaoqing University,Zhaoqing 526061,Guangdong,China;College of Life Sciences,Zhaoqing University,Zhaoqing 526061,Guangdong,China;Vegetable Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,Guangdong,China)

机构地区：[1]肇庆学院食品与制药工程学院,广东肇庆526061 [2]肇庆学院生命科学学院,广东肇庆526061 [3]广东省农业科学院蔬菜研究所,广州510640

出　　处：《果树学报》2023年第7期1318-1329,共12页Journal of Fruit Science

基　　金：国家外专局项目(QN2022030024L);广东省大学生创新项目(S202010580073);肇庆市科技计划项目(2015B010201001);肇庆学院博士启动基金项目(611-612281)。

摘　　要：【目的】克隆砂糖橘精氨酸脱羧酶基因(CrADC),分析其在干旱胁迫下的表达模式,为探究CrADC基因调控多胺合成的抗旱分子机制提供理论参考。【方法】利用RT-PCR技术克隆砂糖橘CrADC基因,采用生物信息学进行蛋白序列及进化分析,利用qPCR检测不同组织和干旱胁迫下的基因相对表达量,并进行植物表达载体构建与烟草遗传转化验证。【结果】砂糖橘CrADC基因全长2262 bp,编码753个氨基酸,含有一个吡哆醛结合域。序列及进化分析显示果树ADC蛋白序列较保守且分为3类,起源于温带的苹果、李、枣、葡萄等8种落叶果树为一个进化分支,起源于热带或亚热带的柑橘、杧果、番木瓜等6种果树属于另一分支。qPCR实验表明,CrADC基因在砂糖橘叶、花、果肉和果皮组织均能表达,但不同时期叶片和果实不同部位的表达量差异显著,干旱胁迫24 h内的基因表达量会逐步上升。转基因实验表明,CrADC基因在烟草根、茎、叶组织中也能稳定表达,转基因系比对照烟草的电导率和丙二醛含量更低,过氧化氢酶和超氧化物歧化酶活性更高,表现出更好的抗旱生理特征。【结论】砂糖橘CrADC序列较保守,起源于亚热带或热带果树的进化分支。CrADC基因具有组织表达特异性,在干旱胁迫后24 h内该基因表达量上升,使转基因系比对照烟草具有更好的抗旱生理特性。【Objective】Shatangju(Citrus reticulata Blanco)is one of the most widely cultivated citrus in southern China and often encounters drought stress during cultivation.Polyamines can reduce drought damage by regulating stomatal closure and promoting root development.The arginine decarboxylase as a rate-limiting enzyme in polyamine synthesis,catalyzes conversion of arginine to putrescines,which is further converted into other polyamines.Therefore,in this study,arginine decarboxylase gene(CrADC)was cloned from Shatangju and its expression pattern was examined under drought stress,in order to provide understanding of the molecular mechanism regulating polyamines synthesis in drought resistance.【Methods】The cDNA sequence of CrADC was obtained by reverse transcription PCR(RTPCR).The coding sequences of CrADC was amplified from cDNA,then cloned into the vector pMD19-T and transformed into DH5αby heat shock.The DH5αwas cultured overnight at 37℃,then DNA from the plasmid was extracted and sequenced after PCR identification.Bioinformatics tools were used to analyze the characteristics and evolutionary relationship of the CrADC protein.The quantitative real-time PCR(qPCR)was used to detect the expression level of the CrADC gene in different tissues(young leaves,old leaves,flowers,30d fruit flesh,and 30d fruit peel)and at different times(0,3,6,9,12,24,36 h)after exposure to 10%PEG-6000 solution.Transgenic tobaccos were obtained by leaf disk transformation using Agrobacterium tumefaciens,and the expression level of the CrADC in the transgenic tobacco plants was detected by qPCR.Related physiological parameters,such as water loss(FL),electrolyte leakage(EL),malondialdehyde(MDA),and activities of catalase(CAT)and superoxide dismutase(SOD)were compared between transgenic lines(TL)and non-transgenic lines(CK)after drought stress.【Results】The cDNA sequence of the CrADC had 3076 bp including a 2262 bp open reading frame(ORF)encoding a protein with 753 amino acids.Bioinformatics analysis indicated the relative molecular

关 键 词：砂糖橘 CrADC基因克隆 干旱胁迫 表达分析 

分 类 号：S666.2[农业科学—果树学]