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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1782bp,编码区长1134bp,编码377个氨基酸残基。Blastn分析表明,BnACTIN1基因与桑树(Morusalba: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18SrRNA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97cm时最高,约为株高11、150和220cm时表达量的230倍以上,是株高47cm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。
The endogenous actin gene fragment of ramie was obtained by PCR screening of cDNA library. The ramie Actin1 gene encoding gene (BnACTIN1) was obtained from Ramie No.1 in Boehmeria nivea (L.) Gaud. The full-length cDNA sequence (GenBank accession number: DQ665832, published on August 1, 2009) contains 1782 bp of the full-length cDNA sequence and 1134 bp in length encoding a protein of 377 amino acid residues. Blastn analysis showed that the BnACTIN1 gene was highly homologous to the actin gene sequences of plants such as Morusalba: DQ785808, Ricinus communis: AY360221 and Gossypium hirsutum: AY305723-305736. The deduced amino acid sequence of the molecular evolution analysis showed that the gene and the cotton Actin protein family AAP73451.1, AAP73457.1 clustered together, the closest relationship between the three. The fluorescence quantitative PCR system was established. The expression levels of 18SrRNA and Histone3 internal reference genes were normalized, and the expression level of BnACTIN1 gene in the ramie cell elongation was analyzed. The results showed that when the plant height was 97cm, the expression was about 230 times higher than that at the plant height of 11,150 and 220cm, which was 20 times higher than that of the 47cm plant height. This study revealed that the sequence of Actin1 obtained from BnACTIN1 was the gene encoding ramie and the highest expression period was at the period of rapid fiber elongation but earlier at the peak of fiber development, Myosin cytoskeletal formation related.