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作物学报 ›› 2010, Vol. 36 ›› Issue (1): 101-108.doi: 10.3724/SP.J.1006.2010.00101

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

苎麻Actinl基因克隆及其在韧皮部纤维不同发育阶段的表达

马雄风1,2,喻春明1,唐守伟1,朱爱国1,王延周1,朱四元1,刘建新1,熊和平1*   

  1. 1中国农业科学院麻类研究所/农业部麻类遗传改良与工程微生物重点开放实验室，湖南长沙410205；2中国农业科学院棉花所，河南安阳455000
  • 收稿日期:2009-03-23 修回日期:2009-07-24 出版日期:2010-01-12 网络出版日期:2009-10-13
  • 通讯作者: 熊和平,E-mail:ramiexhp@2118.cn;Tel:0731-8998500
  • 基金资助:

    本研究由国家“十一五”支撑计划项目（2006BAD06B03）和国家公益性行业（农业）科研专项经费项目（nyhyzx07-018）资助。

Cloning and Tissue Expression of Acting1 Gene in Different Fiber Development Phases of Ramie [Boehmeria nivea (Linn.) Gaud]

MA Xiong-Feng1,2,YU Chun-Ming1,TANG Shou-Wei1,ZHU Ai-Guo1,WANG Yan-Zhou1,ZHU Si-Yuan1,LIU Jian-Xin1,XIONG He-Ping1*   

  1. 1Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences/Key Laboratory of Genetic Improvement & Engineering Microbiology for Bast Fiber Crops,Changsha 420105,China;2Institute of Cotton Research,Chinese Academy of Agricultural Sciences,Anyang 455000,China
  • Received:2009-03-23 Revised:2009-07-24 Published:2010-01-12 Published online:2009-10-13
  • Contact: XIONG He-Ping,E-mail:ramiexhp@2118.cn;Tel:0731-8998500

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被引次数

11

摘要：

通过cDNA文库的PCR筛选法获得苎麻内源肌动蛋白基因片段，采用苎麻[Boehmeria nivea (L.) Gaud]中苎1号为材料，结合RACE技术获得了苎麻的Actin1蛋白编码基因(BnACTIN1)的cDNA全长序列(GenBank登录号为DQ665832，2009年8月1日公布), 该基因全长cDNA序列1 782 bp，编码区长1 134 bp，编码377个氨基酸残基。Blastn分析表明，BnACTIN1基因与桑树(Morus alba: DQ785808)、蓖麻子(Ricinus communis: AY360221)、棉花(Gossypium hirsutum: AY305723-305736)等植物的肌动蛋白基因序列有高度的同源性。对推导的氨基酸序列进行分子进化分析表明，该基因与棉花Actin蛋白家族AAP73451.1、AAP73457.1聚为一类，三者之间的亲源关系最近。建立荧光定量PCR体系，用18S rRNA和Histone3内参基因对表达水平均一化，分析BnACTIN1基因在苎麻纤维细胞伸长过程中的表达水平变化。结果表明，在株高97 cm时最高，约为株高11、150和220 cm时表达量的230倍以上，是株高47 cm时期的20倍。本研究揭示的所获得BnACTIN1序列为苎麻的Actin1蛋白编码基因，其最高表达时期开始于纤维快速伸长时期，但稍早于纤维发育的高峰期，推测该基因可能与韧皮部纤维伸长过程中的肌细胞骨架形成有关。

关键词: 苎麻, 肌动蛋白, 基因克隆, 实时定量PCR

Abstract:

Ramie [Boehmeria nivea (L.) Gaud] is an important natural fiber crop, and its quality improvement is a challenge for ramie breeders in breeding program. Plant fiber development is a complex process, which involves many genes and enzymes. Actin protein cytoskeleton plays a significant role in cytomorphology including cell elongation of ramie fiber. It is promising for ramie quality improvement by purposely regulating Actin gene expression with gene engineering technique. In the present study, the full-length cDNA sequence of Actin1 gene from ramie cultivar Zhongzhu 1 was cloned (GenBank accession number: DQ665832) by usingdegenerate primer RT-PCR method, RACE technology and screening with a full-length cDNA library. The full-length cDNA was composed of 1 782 bp sequences including an open reading frame (ORF) of 1 134 bp region which encoded 377 amino acids. Bioinformatic analysis showed that the conserved motifsof Actin1gene contained six ATP binding sites, six profilin binding sites and nine gelsolin binding sites. The cDNA sequence of Actin1 shared high sequence homology with that from other crops previously reported, which was close to that from Morus alba (GenBank accession number: DQ785808), Ricinus communis (AY360221) and Gossypium hirsutum (AY305723-305736). Gene sequence analysis showed that the putative amino acid sequence and Gossypium hirsutum Actin (AAP73451.1, AAP73457.1) were gathered to a same group. Degenerate primer RT-PCR method was used to clone18S rRNA and Histone3 genes and establish the fluorescence quantitative PCR system. The system was used to study the expression of Actin1 gene in different fiber development phases by using 18S rRNA and Histone3 genes as inner references. The results showed that Actin1 could express in all kinds of ramie fiber development phases, and the mRNA was 230 times higher in 97 cm of plant height than 11, 150 and 220 cm, 20 times higher than in 47 cm. The BnACTIN1 gene expression increased slowly in 11 to 47 cm of plant height, the peak in the plant height from 47 to 97 cm, in then rapidly declined in 150 cm of plant height, and kept the lowest level till the plant height was up to 220 cm. The high expression of BnACTIN1 gene presented at the beginning of the rapid elongation of the fiber cell, but slightly earlier than the peak period of fiber development. We speculated that ramie actin cytoskeleton plays a important role in the process of phloem fiber elongation.

Key words: Ramie, Actin protein, Gene cloning, Real-time quantitative PCR

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