Molecular Cloning Of Auxin-related Genes From Moso Bamboo Seedlings And The Effects Of Radiation Treatments On Gene Expression

Moso Bamboo（Phyllostachys edulis） is one of a typical clonal plant, which is native to subtropical China. It has long been the most important economic bamboo in China, for its high production, fast growth, giant size, good quality for timber use and wide distribution. However, little is known about sexual propagation of Moso bamboo and developmental mechanism of seedlings. To further explore the molecular mechanism of the growth and develepment of Phyllostachys edulis, Auxin-related genes:auxin binding protein gene （PheABP1） and auxin response factor gene （PheARF1） were researched in this study.（1） A full length of PheABP1 gene was cloned from young blades of Phyllostachys edulis seedlings by RT-PCR and RACE. Sequence analysis indicated that:the cloned PheABP1 gene consisted of 902 nucleotides, encoding a 2.2×104 daltons peptide containing 205 amino acids with pI of 6.32. The homology of the amino acid of PheABPl and Oryza sativa, Avena sativa, Zea Mays, Sorghum bicolor and etc, was all over 76%. This gene encoded a protein sequence containing the typical auxin-binding protein domain, which belonged to the Cupin₂ protein superfamily; the phylogenic tree determined that the ABP1 of Avena sativa was most closely related to the Phyllostachys edulis. The N-terminal 34 amino acid residues of PheABP1 was presumed to be signal peptide region, with a positive charge and strong hydrophobicity, the mature protein had 172 amino acids; PheABPl protein sequence contained an N-glycosylation site, a casein kinase II phosphorylation site, two N-acylation sites, one amidation site, a endoplasmic reticulum binding site （KDEL） and other motifs; PSORT predicted that PheABP1 protein located on the cytoplasmic membrane.PheABPl gene expressions in different tissues and different treatments of radiation dose between one-year-old and three-year-old seedlings were measured using the real time quantitative PCR method, the reference gene to target genes was Actin gene. The results showed that:PheABPl gene relative expression in tissues of germination of seedlings is much higher than three-year-old seedlings. During the period of seeds germination, the relative expression of radicle was the highest; for three-year-old seedlings, the relative expression of leaves and shoots were higher, followed by the rhizome, the roots and stems were the lowest. When treating with 25Gy radiation, there was the highest overall expression level for PheABPl gene.（2） PheARFl gene was cloned from young blades of phyllostachys edulis seedlings by RT-PCR and RACE. The full-length cDNA sequence of PheARFl gene contained 2870bp, its coding sequence was 2487bp and by which 829 amino acids with B3、Auxin_resp and AUX-IAA sequences were encoded. The homology of the the amino acid sequence of PheARF1 gene, and Oryza sativa, Avena sativa, Zea Mays and Sorghum bicolor was more than 90%. Cluster analysis showed that the ARF1 protein of Oryza sativa was most related to PheARF1 protein.Using the real time quantitative PCR method, we concluded that:the relative expression level of PheARF1 gene in the germination of seedlings was higher than three-year-old seedlings. During seed germination, the relative expression of embryo was the highest; for three-year-old seedlings, PheARF1 gene expression was very low in different organizations except for the bamboo shoots. Among the radiation treatments, there was the highest overall expression level with 25Gy for PheARF1 gene.