金柑磷酸蔗糖磷酸酶的基因克隆、表达与蛋白结构分析  

Cloning,Expression and Structural Analysis of Sucrose Phosphate Phosphatase From Fortunella crassifiolia Swingle

作　　者：龙凌云[1] 黄秋岚 黄秋伟[1] 檀小辉[1] 李慧敏[1] 刘功德[1] 单彬[1] 毛立彦[1] LONG Lingyun;HUANG Qiulan;HUANG Qiuwei;TAN Xiaohui;LI Huimin;LIU Gongde;SHAN Bin;MAO Liyan(Guangxi Subtropical Crops Research Institute/Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable,Ministry of Agriculture and Rural Affairs/Guangxi Key Laboratory of Quality and Safty Control for Subtropical Fruits,Nanning,Guangxi 530001)

机构地区：[1]广西壮族自治区亚热带作物研究所/农业农村部亚热带果品蔬菜质量安全控制重点实验室/广西亚热带特色水果质量安全控制重点实验室,广西南宁530001

出　　处：《核农学报》2023年第12期2349-2360,共12页Journal of Nuclear Agricultural Sciences

基　　金：广西壮族自治区农业科学院基本科研业务专项项目(桂农科2022YM16);国家现代农业产业技术体系广西创新团队建设专项(nycytxgxcxtd-2021-05-03);广西重点研发计划项目(桂科AB19245035)。

摘　　要：磷酸蔗糖磷酸酶(SPP)是植物蔗糖生物合成最后一步催化反应的关键酶,对调控植物生长发育不同时期的蔗糖合成过程有重要作用。为探索金柑(Fortunella crassifiolia Swingle)SPP基因及其编码蛋白的序列特征及其在果实发育过程的表达特性,本研究以四倍体品种脆蜜金柑(F.crassifiolia Swingle cv.Cuimi)为试验材料,采用反转录PCR和cDNA末端快速克隆(RACE)方法从金柑果肉中克隆SPP基因,对其进行生物信息学和原核表达分析,并检测果实发育不同时期该基因在果肉中的表达量。结果表明,FcSPP基因的cDNA序列全长1638 bp,最长开放阅读框(ORF)为1191 bp,编码396个氨基酸,理论等电点为6.57,为稳定的亲水性蛋白,二级结构主要由α-螺旋和无规则卷曲组成。FcSPP蛋白含有S6PP和S6PP_C保守结构域,属于植物磷酸蔗糖磷酸酶家族成员。系统进化分析结果显示,FcSPP蛋白与甜橙SPP同源性最高,在进化树上与拟南芥等双子叶植物SPP划归为一类。此外,本研究构建了FcSPP基因的原核表达载体,诱导表达获得纯化的FcSPP蛋白,分子量鉴定为45.96 kDa。定量分析结果表明,脆蜜金柑果实发育4个时期FcSPP基因相对表达量呈逐步上升趋势,果实膨大期、成熟期的果肉组织中该基因表达量显著高于初果期和生长期(P<0.05)。本研究为解析金柑果实蔗糖生物合成的分子机制及调控蔗糖生物合成相关基因的研究提供了理论基础。Sucrose phosphate phosphatase(SPP)is a key enzyme that catalyzes the final step of sucrose biosynthesis in plants,and plays an important role in the regulation of sucrose synthesis at different stages of plant growth and development.In order to investigate the sequence characteristics of SSP gene and SSP proteins in Fortunella crassifiolia Swingle and its expression characteristics during fruit development,the FcSPP gene was cloned from F.crassifiolia Swingle cv.Cuimi(tetraploid)flesh by reverse transcription PCR and RACE methods,its bioinformatics and prokaryotic expression analysis also were performed.In addition,the expression levels of FcSPP in the flesh at different stages of fruit development were detected.The results showed that the full length of cDNA sequence of FcSPP gene was 1638 bp,with 1191 bp open reading frame(ORF),encodes 396 amino acids and the theoretical isoelectric point was 6.57.Meanwhile,FcSPP was a stable hydrophilic protein with a secondary structure mainly composed ofα-helical and random coil.FcSPP protein contains conserved S6PP and S6PP_C domains which were belonged to the plant sucrose phosphate phosphatase family.Phylogenetic analysis showed that FcSPP shared the highest similarity with SPP of Citrus sinensis,and was classified with dicotyledonous plants such as Arabidopsis SPP in the phylogenetic tree.In addition,the prokaryotic expression vector for the FcSPP gene was constructed,and the purified FcSPP protein was obtained by induced expression with a molecular weight of 45.96 kDa.Quantitative real time PCR analysis indicated that the relative expression level of FcSPP gene showed gradual rising trend in the four stages of fruit development.And the expression level of FcSPP gene in the flesh tissue expansion and maturity stages were significantly higher(P<0.05)than that in initial fruit and growth stages.This study provides a theoretical basis for understanding the molecular mechanism of sucrose biosynthesis and exploring the genes regulating sucrose biosynthesis in citrus fruits.

关 键 词：金柑 磷酸蔗糖磷酸酶 基因克隆 实时荧光定量PCR 

分 类 号：S666[农业科学—果树学]