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。
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 Actinl gene from ramie cultivar Zhongzhu 1 was cloned （GenBank accession number： DQ665832） by using degenerate 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 motifs of Actinl gene contained six ATP binding sites, six profilin binding sites and nine gelsolin binding sites. The cDNA sequence of Actinl 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 clone 18S rRNA and Histone3 genes and establish the fluorescence quantitative PCR system. The system was used to study the expression of Actinl gene in different fiber development phases by using 18S rRNA and Histone3 genes as inner references. The results showed that Actinl 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 BnACT1N1 gene expression increased slowly in 11 to 47 cm of plant height,
出处
《作物学报》
CAS
CSCD
北大核心
2010年第1期101-108,共8页
Acta Agronomica Sinica
基金
国家“十一五”科技支撑计划项目(2006BAD06B03)
国家公益性行业(农业)科研专项经费项目(nyhyzx07-018)资助
