向日葵ACC氧化酶基因(HaACO1)的克隆及表达分析  

Cloning and Expression Analysis of ACC Oxidase Gene(HaACO1) from Sunflower( Helianthus annuus L.)

作　　者：孙瑞芬[1] 张艳芳[1] 郭树春[1] 于海峰[1] 李素萍[1] 乔慧蕾[1] 聂惠[1] 安玉麟[1] 

机构地区：[1]内蒙古农牧业科学院,呼和浩特010031

出　　处：《中国生物工程杂志》2015年第9期21-27,共7页China Biotechnology

基　　金：内蒙古自治区自然科学基金(2012MS0315);内蒙古农牧业创新基金(2013CXJJN14)资助项目

摘　　要：目的：克隆向日葵中的ACC氧化酶基因（HaACO1）,并对其进行生物信息学分析及盐胁迫表达分析,为理解向日葵ACC氧化酶生理功能并加强对ACO基因的利用奠定基础。方法：以前期从盐胁迫的内葵杂4号根中获得的ACC氧化酶基因片段4-4-7TDF（KM823963）为基础,通过RT-PCR和5＇/3＇RACE技术克隆ACO基因的全长cDNA序列,利用生物信息学软件对获得的cDNA序列及编码的蛋白质序列进行分析。同时采用PCR方法克隆基因组DNA（genemic DNA,gDNA）序列,并对其进行结构分析。利用实时荧光定量PCR分析Na Cl胁迫下向日葵根、下胚轴、叶中HaACO1的表达量和不同NaCl浓度及不同胁迫时间下根中Ha ACO1的表达量。结果：Ha ACO1的cDNA序列全长为1 135bp,其开放阅读框为942bp,编码313个氨基酸。预测其分子质量和等电点分别为35.84k Da和5.13,基因登录号为KP966508。HaACO1与已报道的多种植物的ACO基因核苷酸序列及其推导的氨基酸序列有较高的相似性,分别为76%~83%和77%~88%。gDNA起始密码子至终止密码子序列长1 018bp,包含2个外显子和1个内含子,基因登录号为KP988289。实时荧光定量PCR分析表明向日葵HaACO1在不同器官及不同NaCl浓度、不同时间诱导下存在特异性表达差异。结论：获得的向日葵HaACO1是植物ACO家族成员之一,该基因应答盐胁迫具有独特的表达模式。Objective：In an effort to understand the physiological functions of 1-aminocyclopropane-1-carboxylic acid（ACC） oxidase and exploit ACO gene more profitably,an ACC oxidase gene（HaACOl） was cloned from sunflower and analyzed by bioinformatics method,and expression pattern of HaACOl gene in sunflower was revealed under salt stress.Method：The full length cDNA sequence of ACO gene in sunflower was obtained by RT-PCR and 5＇/3＇RACE methods based on the sequence of 4-4-7TDF（accession No.KM823963）,a gene fragment of ACC oxidase from Inner Mongolia Sunflower Hybrid No.4.The cDNA sequence and its encoded protein was analyzed by bioinformatics method.The genemic DNA（gDNA） of ACO sequence was cloned by PCR.The expression level of HaACOl gene in roots,hypocotyl and leaves under NaCl stress was tested by quantitative Real-time PCR（qRT-PCR）.Results：The full length sequence of HaACOl gene in sunflower was1 135 bp,and its open reading frame（ORF） was 942 bp,then the protein sequence analysis results showed that the HaACOl gene encoded 313 amino acids with molecular weight 35.84 kDa and pI 5.13.The sequence accession No.in GenBank was KP966508.The nucleotide sequence and the amino acid sequence of HaACOl was highly homologous to other species（75%~83%,77%~88%respectively）.The full length of the coding region of gDNA was 1 018 bp,comprised of two exons separated by one intron.The sequence has been submitted to GenBank（Accession No.KP988289）.The results of qRT-PCR showed that the expression level of HaACOl had specific expression differences in different organs.Different concentration of NaCl treatmet or different induction time with NaCl also lead to the specific expression of HaACOl in sunflower.Conclusion：HaACOl belongs to ACO gene family in plants,and this gene has distinctive expression pattern when plants response to salt stress.

关 键 词：向日葵 ACC氧化酶(ACO) 盐胁迫 基因表达 

分 类 号：S565.5[农业科学—作物学]