Cloning, Sequencing And Evolution Of CAL Homologs In Brassica Species

Primers were designed according to the sequences of AtCAL, BoCAL, BobCAL and BoiCAL gene, and PCR amplifications were performed with the genomic DNA of Brussel Sprouts, Kale and Kohlrabi respectively, PCR fragments of about 1.6kb were obtained and designated correspondingly as BogCAL5', BoaCAL5' and BocCAL5'. Reclamation, purification and linkage of them respectively, then sequencing and analyses of according gene structure were made, results show that the complete sequence length of corresponding PCR product from Brussel Sprouts, Kohlrabi and Kale are 1665bp, 1650bp and 1650bp, containing the first two exons and introns and 22bp of the third exon. The whole-length of according cDNA sequence of the three species above are equal, 286bp in length, and encode 95 amino acids in all, containing the MADS domain and I region in accordingly encoding product.Extensive alignment between corresponding sequences and structures were made, and the results are as follows:1.Alignment between genomic DNA of three isolated fragments show great differences in the first intron of them. There exist successive deletions in the sequence of the genomic DNA of Kale and Kohlrabi, compared with that of Brussel Sprouts. However, the discrepancy has no remarkable affect on the relationship of them.2.Sequence alignments between the three fragments of according cDNA to find that BoaCAL and BocCAL share 97.9% identical percentage while the identity percentage between BogCAL and BoaCAL,BocCAL are 98.9%,98.2%, respectively. At the same time, there exists not some base deletion but only base substitution among them. There are 6 base transitions between the sequence of BoaCAL5'and BocCAL5', and 2 transitions and 1 base transversions between BoaCAL5' and BogCAL5', 3 transversions and 2 transitions between BocCAL5' and BogCAL5'.3.Alignment of according sequences of putative amino acid of CAL homologs from 8 plants reveals high conservation according to the MADS domains of them. Except for the amino acid sequence of Arabidopsis, the identical percentage of the other seven proteins is nearly up to 100%. Only one amino acid differs with the othersequences of amino acid. While great differences exist between the according amino acid sequence of Arabidopsis and the sequences of other seven one's, which is, 4 amino acids differs.4.Alignment was made between 8 second-structure of proteins referring to the difference of 8 cDNA sequences and corresponding amino acid sequences of them and the phylogenetic tree drawn among them and five pieces of conclusions obtained are as follows:There exists great difference in the four structures as " a turn and coil" of the second structure of proteins between BoaCAL5' and BocCAL5'; Difference also exists in the alignment of the four structures as above between BoaCAL5' and BocCAL5';(3)Only the structure of "turn" changes in the comparison of BogCAL5' with BoaCAL5' the same level of comparison as above;One amino acid differs between the protein sequence of BocCAL5' and BoCAL5',and accordingly," " structure exhibits difference between the above two;Both the amino acid sequence and the second-structure of proteins of BogCAL5' and BcpCAL5'.In addition, homologous alignments and analyses of identical percentage were made between corresponding cDNA sequences to CAL homologs of 8 plants in the same family, Brassicaceae. According to the information of above phylogenetic tree was drawn. Results show no differences between the phylogenetic tree and according botanical categories. At the same time, brussel sprouts and cauliflower are placed into the same category, while cabbage and broccoli are put into another one, which differs greatly with the results of classification by morphology and edible organ. Conclusively, morphological similarity and distance in phylogenetic tree are far from the same thing, and no inevitable relationship exhibits between them.