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