向日葵ACC氧化酶基因(<em>HaACO1</em>)的克隆及表达分析

doi:10.13523/j.cb.20150904

中国生物工程杂志

2015, Vol. 35

Issue (9): 21-27 DOI: 10.13523/j.cb.20150904

研究报告

向日葵ACC氧化酶基因(HaACO1)的克隆及表达分析

孙瑞芬, 张艳芳, 郭树春, 于海峰, 李素萍, 乔慧蕾, 聂惠, 安玉麟

内蒙古农牧业科学院呼和浩特 010031

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

SUN Rui-fen, ZHANG Yan-fang, GUO Shu-chun, YU Hai-feng, LI Su-ping, QIAO Hui-lei, NIE Hui, AN Yu-lin

Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences, Huhhot 010031, China

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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NaCl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 HaACO1的表达量.结果:HaACO1 的cDNA序列全长为1135bp,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35.84kDa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1018bp,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.

关键词： 向日葵; ACC氧化酶(ACO); 盐胁迫; 基因表达

Abstract:

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 (HaACO1) was cloned from sunflower and analyzed by bioinformatics method, and expression pattern of HaACO1 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 HaACO1 gene in roots, hypocotyl and leaves under NaCl stress was tested by quantitative Real-time PCR (qRT-PCR). Results: The full length sequence of HaACO1 gene in sunflower was 1 135bp, and its open reading frame (ORF) was 942bp, then the protein sequence analysis results showed that the HaACO1 gene encoded 313 amino acids with molecular weight 35.84kDa and pI 5.13. The sequence accession No. in GenBank was KP966508. The nucleotide sequence and the amino acid sequence of HaACO1 was highly homologous to other species (75%~83%, 77%~88% respectively). The full length of the coding region of gDNA was 1 018bp, 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 HaACO1 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 HaACO1 in sunflower. Conclusion: HaACO1 belongs to ACO gene family in plants, and this gene has distinctive expression pattern when plants response to salt stress.

Key words: Sunflower ACC oxidase(ACO) Salt stress Gene expression

收稿日期: 2015-04-02 出版日期: 2015-09-25

ZTFLH:

S565.5

基金资助:

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

通讯作者: 安玉麟 E-mail: nkyanyulin@163.com

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引用本文:

孙瑞芬, 张艳芳, 郭树春, 于海峰, 李素萍, 乔慧蕾, 聂惠, 安玉麟. 向日葵ACC氧化酶基因(HaACO1)的克隆及表达分析[J]. 中国生物工程杂志, 2015, 35(9): 21-27.

SUN Rui-fen, ZHANG Yan-fang, GUO Shu-chun, YU Hai-feng, LI Su-ping, QIAO Hui-lei, NIE Hui, AN Yu-lin. Cloning and Expression Analysis of ACC Oxidase Gene ( HaACO1) from Sunflower (Helianthus annuus L.). China Biotechnology, 2015, 35(9): 21-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150904 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I9/21

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