榅桲C4H基因的克隆、序列分析及表达  

Cloning,sequence analysis and expression of C4H gene in Cydonia oblonga

作　　者：车玉红[1] 杨波[2] 郭春苗[2] 刘晨曦 木巴热克·阿尤普 吴津蓉[1] 杜鹃[1] CHE Yuhong;YANG Bo;GUO Chunmiao;LIU Chenxi;Mubareke·Ayoupu;WU Jinrong;DU Juan(Xinjiang Agricultural Vocational Technical College,Changji 831100,Xinjiang,China;Institute of Horticulture,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,Xinjiang,China;Animal Biotechnological Research Center,Xinjiang Academy of Animal Science,Urumqi 830011,Xinjiang,China)

机构地区：[1]新疆农业职业技术学院,新疆昌吉831100 [2]新疆农业科学院园艺作物研究所,新疆乌鲁木齐830091 [3]新疆畜牧科学院生物技术研究所,新疆乌鲁木齐830011

出　　处：《经济林研究》2020年第2期1-8,共8页Non-wood Forest Research

基　　金：新疆维吾尔自治区自然科学基金项目(2018D01A45)。

摘　　要：【目的】肉桂酸-4-羟化酶(cinnamate 4-hydroxylase,C4H)是调节木质素合成的关键基因,榅桲是新疆特色经济果树之一。目前,有关榅桲C4H基因的序列信息尚不明确。为了获得该基因的序列信息及其在果实不同发育时期的差异性表达规律,为深入研究该基因在榅桲果实发育过程中的作用和功能奠定基础。【方法】以榅桲果实为研究材料,基于GenBank已登录近源物种的C4H基因的cDNA序列,提取其果实的总RNA并反转录为cDNA,利用同源克隆的方法获得榅桲C4H基因的cDNA序列,并对该序列进行生物信息学分析,同时检测在榅桲果实开花后不同时间点C4H基因表达量的差异。【结果】同源克隆结果表明:C4H基因编码区的序列全长为1 518 bp,编码505个氨基酸,其蛋白的分子质量为58.28 kD。生物信息学分析结果显示:榅桲C4H蛋白为亲水性不稳定蛋白质,其二级结构以α-螺旋和无规卷曲结构为主。系统进化分析结果显示:榅桲C4H蛋白与甜樱桃(Prunus avium,XP021806844.1)、山杏(Prunus armeniaca,VVA16404.1)的C4H蛋白同源性均较高,其相似性分别为92.87%和91.24%。实时荧光定量PCR的结果显示:随着榅桲果实的发育,在开花后10 d的果肉中C4H基因的表达量最高,随着果实的不断发育,果肉中C4H基因的表达量逐渐减少。【结论】成功获得了榅桲C4H基因全长编码区序列,并发现了C4H基因在果实发育初期的表达最高,这与榅桲果实的木质化程度密切相关。【Objective】Cinnamate 4-hydroxylase(C4H)gene is a key gene regulating synthesis of lignin.Cynthia oblonga is one of special non-wood fruit trees in Xinjiang.At present,sequence information of C4H gene in C.oblonga is undefined.To obtain sequence information and differential expression rule of C4H gene at different fruit development stages,and to lay a foundation for further studying function of this gene during fruit development in C.oblonga.【Method】C.oblonga fruits are used as research materials.Based on cDNA sequence of C4H gene in GenBank.Total RNA and cDNA were obtained from fruits,and cDNA sequence of C4H gene was cloned by using homologous cloning method.Bioinformatics analysis of sequence was carried out.Meanwhile,expression of C4H gene in fruits at different time points after flowering was detected.【Result】The results of homologous cloning indicate that total length of coding region in C4H gene is 1518 bp,which encodes 505 amino acids,and molecular mass of protein is 58.28 kD.The results of bioinformatics analysis show that C4H protein in C.oblonga is a kind of hydrophilic unstable protein,and its secondary structure is dominated byα-helix and random curl.The results of phylogenetic analysis indicate that C4H protein in C.oblonga has high homology with those from Prunus avium(XP_021806844.1)and Prunus armeniaca(VVA16404.1),and similarities are 92.87%and 91.24%,respectively.The results of real-time fluorescence quantitative PCR show that expression of C4H gene is the highest on the 10th day after flowering.With development of fruit,expression of C4H gene is decreased gradually.【Conclusion】Full-length coding region of C4H gene in C.oblonga was successfully obtained.Expression of C4H gene is highest at initial fruit development period and closely related to degree of fruit lignification.

关 键 词：榅桲 C4H基因 克隆 序列分析 基因表达 

分 类 号：S661.9[农业科学—果树学]