Beet M14 Strain M14 Of Beta Corolliflora Expression Gene-100, M14 Of Beta Corolliflora-265, M14 Of Beta Corolliflora-g12, G Full-length Cdna Cloning And Sequence Analysis

Apomictic monosomic addition line M14 of Beta corolliflora Zoss,was the progeny from the hybridization of Beta vulgaris L. and Beta corolliflora Zoss. in sugar beet. Constituted of the normal 18 Beta vulgaris L. chromosomes with the No.9 chromosome of Beta corolliflora Zoss, M14 was found having a chromosome transmission frequency 96.5%. The reason of apomixis in M14 was interpreted as possible existence of apomixis gene(s) in the additional chromosome, thus M14 is a precious material for research of apomixis. Meanwhile M14 was cold and disease resistant. Study of the specifically expressed genes in M14 will provide direct molecular evidence to reveal the special reproductive process of M14, and will help us to discover and use excellent genes of M14.In order to isolate and identify the apomixis gene(s) of M14, we have obtained 298 differential expression ESTs by the method of SSH and DDRT-PCR, three of which were selected as candidate ESTs in this study. SMART RACE was played to obtained full-length cDNA sequences of gene M14-100, M14-265 and M14-G12, which carry the three ESTs respectively. The sequences were then identified by RT-PCR, and bioinformatic analysis was also carried out.The results showed that M14-100 (GenBank accession No. EU529829) was 1 707bp in length, and shared 60~70% similarity with cyclin related genes from Arabidopsis thaliana by the method of bioinformatic analysis. But the function of cyclin was not clear. The longest ORF of M14-100 contained 324 amino acids. Molecular weight of its putative protein was 34.4 kD, and theoretical isoelectric point was 5.11. It has strong hydrophilicity, and is non-secretion protein. Secondary structure prediction of M14-100 putative protein indicated that it contained 6α-helices and 3β-strands.M14-265(GenBank accession No. EU529830) was 1 006bp in length. By the method of bioinformatic analysis we didn't find any genes which share similarity with it, thus we deduced that M14-265 was a new gene. There is no significant ORF in M14-265, and the longest ORF contained 81 amino acids. M14-265 maybe encode a noncoding RNA or short-peptide. Molecular weight of its putative protein was 9.3 kD, and theoretical isoelectric point was 4.69. It is non-secretion protein. Secondary structure prediction of M14-100 putative protein indicated that it contained 1α-helices and 2β-strands.M14-G12 (GenBank accession No. EU529828) was 873bp in length, and shared 99% similarity with calmodulin gene from Arabidopsis thaliana. It was suggested that M14-G12 was calmodulin gene of M14, and its ORF contained 149 amino acids. Molecular weight of its coding protein was 16.8 kD. Theoretical isoelectric point was 4.11. Its total average hydrophilicity was -0.620. Secondary structure prediction of M14-G12 coding protein indicated that it was anα-helix rich protein. Three-dimensional structure prediction of M14-G12 coding protein showed that it contained four helix-spring-helix structures, which kept accordance with the function of calmodulin.