Molecular Cloning And Sequence Analysis Of Novel Retinoblastoma-related Gene From Barley

The plant RBR (retinoblastoma-related) protein is the homologue of animal pRB (retinoblastoma protein). They are widely expressed within various tissues, and actively interacted with other proteins, regulated the expression of the cell cycle related genes, and greatly affected the progress of cell proliferation, differentiation and endocycle. Identify the taxonomic status of RBR and genetic relationship with other plants though molecular characteristic analysis and sequence analysis. It is of great significance to the study of cell proliferation and differentiation mechanism in plant growth and development process. The aim of present study is to isolate RBR gene from GSHO1854, a shrunken endosperm mutational material of barley(Hordeum vulgare), and then, to perform multiple alignment analysis and construct phylogenetic tree. The main results were described as following:1. Various specific primers were designed for PCR amplification of the RBR gene from H. vulgare, on the basis of the conservative sequences of the reported plant RBR genes. The RBR gene sequences were obtained from both genomic DNA and total RNA of seedling. Five cDNA fragments (683bp,929bp,679bp,1213bp and 573bp respectively) and five DNA fragments (1237bp,1810bp,895bp,1828bp and 1214bp respectively) were cloned and sequenced. Then,3179bp of cDNA sequence and 5547bp of DNA sequence were obtained through fragment-splicing. The accession number of the two sequences in GenBank was GU121480 and GU121481, respectively. The cDNA sequence of HvRBR contains a complete ORF, consisting of 2825bp nucleotide acid, which could encode a protein of 974 amino acids with a molecular weight of 107.9 kDa and a pI of 7.5. By performing BLASTn program on NCBI website, these fragments had a highest sequence homology with reported RBR genes. Therefor, these sequences could be the fragments of RBR gene from barley.2. Through sequence alignment of DNA and cDNA sequence, HvRBR was more likely to contain 17 exons in the approximately 6000bp long DNA fragments. The highly conserved coding region of the A pocket domain was located on the longest exonlO of HvRBR, and the exonl2-15 encoded the conserved pocket B. The HvRBR amino acid sequence reveals 15 potential CDK phosphorylation sites and 19 positively charged residues in the pocket B domain. HvRBR protein contained a spacer region between conserved A and B domains. Although lower identity was found in this region, a conservative Cys653 was also found in all the plant RBR protein family members with similar position. It was suggested that the intra-or inter-molecular disulfide bond probably play an important role in affecting the structure and function of RBR protein.3. By performing multiple alignment analysis of amino acid sequences of HvRBR and other reported RB/RBR protein family members, HvRBR amino acid sequence shared the highest level of identity,84.3% with rice, but only about 50% amino acid identity with dicot RBR proteins (e.g. M. sativa or A. thaliana). Sequence analysis suggested that HvRBR was a novel member of subgroup C of plant RBR family.