胡萝卜DcPSY2基因克隆及其在非生物胁迫响应中的表达分析  

Cloning and Expression Analysis of DcPSY2 Gene Under Abiotic Stress in Carrot(Daucus carota)

作　　者：丁旭 王雅慧 李彤 张榕蓉 徐志胜[1] 熊爱生[1] DING Xu;WANG Ya-Hui;LI Tong;ZHANG Rong-Rong;XU Zhi-Sheng;XIONG Ai-Sheng(College of Horticulture,Nanjing Agricultural University/State Key Laboratory of Crop Genetics and Germplasm Enhancement/Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China,Nanjing 210095,China)

机构地区：[1]南京农业大学园艺学院/作物遗传与种质创新国家重点实验室/农业农村部华东地区园艺作物生物学与种质创制重点实验室

出　　处：《农业生物技术学报》2019年第12期2110-2119,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31872098);江苏省杰出青年基金(No.BK20130027);江苏高校优势学科建设项目(PAPD)

摘　　要：类胡萝卜素(carotenoids)作为一种重要的营养物质,广泛存在于胡萝卜(Daucus carota)中。番茄红素(lycopene)是一种主要的类胡萝卜素,对植物生长发育和逆境响应有重要作用。八氢番茄红素合成酶(phytoene synthase,PSY)是番茄红素合成的关键酶之一。利用RT-PCR(reverse transcription PCR)技术从胡萝卜’君川红’中克隆获得ORF长为1317 bp、编码438个氨基酸的DcPSY2基因(GenBank No.NM001329167.1)。将DcPSY2与其他17种植物中PSY蛋白的氨基酸序列进行多重比对,结果显示其总体相似性为82.22%,可见PSY蛋白是高度保守的蛋白质。进化关系表明,胡萝卜DcPSY2蛋白与红木(Bixa orellana)PSY蛋白进化关系最近。胡萝卜DcPSY2蛋白是亲水性、非分泌蛋白,且无信号肽,属于类异戊二烯合成C1超级家族(isoprenoid biosyn C1 superfamily)成员,在各细胞器中均有分布。二级结构预测表明,胡萝卜DcPSY2由53.65%的α-螺旋、5.48%的β-折叠、12.79%的延伸主链和28.08%的随机卷曲组成。qRT-PCR结果显示,DcPSY2基因在高温(38℃)、低温(4℃)、干旱(200 g/L PEG6000)和高盐(200 mmol/L NaCl)4种非生物胁迫下均有明显响应。在高温和高盐胁迫下,DcPSY2基因在处理2 h后表达量达到峰值;在低温和干旱胁迫下,DcPSY2基因的表达量在处理后1 h时显著上调(P<0.05)。本研究结果为DcPSY2基因在胡萝卜生长发育中的功能研究及其在抗逆育种中的应用研究提供了基础资料。Carotenoids,as an important nutrient,are very common in carrot(Daucus carota).Lycopene,a kind of carotenoids,plays important roles in growth,development,and response to abiotic stress in higher plant.Phytoene synthase(PSY)is one of the key enzymes of lycopene biosynthesis.In this study,DcPSY2(GenBank No.NM001329167.1)was cloned from carrot cv.’Junchuanhong’by RT-PCR(reverse transcription PCR).It contained an ORF with the length of 1317 bp and encoded 438 amino acids.The amino acid sequences of DcPSY2 and the PSY proteins of 17 other plants were compared,the results showed that the overall similarity of PSY proteins was 82.22%,which indicated that PSY proteins were conserved.Evolutionary relationship exhibited that DcPSY2 protein had the closest evolutionary relationship with the PSY protein in mahogany(Bixa orellana).DcPSY2 protein was a hydrophilic and non-secretory protein without signal peptide.It belonged to isoprenoid biosyn C1 superfamily and was distributed in various organelles.Secondary structure prediction demonstrated that DcPSY2 contained 53.65%α-helix,5.48% β-fold,12.79% extended main chain and 28.08%random curl.The results of qRT-PCR showed that DcPSY2 gene expression changed significantly in response to 4 kinds of abiotic stresses,including high temperature(38℃),low temperature(4℃),drought(200 g/L PEG6000)and high salt(200 mmol/L NaCl).Under high temperature or high salt treatments,the relative expression level of DcPSY2 reached the peak after 2 h.Whereas under low temperature or drought treatments,the expression level of DcPSY2 increased significantly at 1 h(P<0.05).The results provide a basic material for the functional study of DcPSY2 gene in carrot growth and its application study in stress resistance breeding.

关 键 词：胡萝卜 八氢番茄红素合成酶(PSY) 基因克隆 表达分析 非生物胁迫 

分 类 号：S631.2[农业科学—蔬菜学]