Cloning And Sequence Analysis Of PHYB,CRY1,CRY2B,PHYA Genes Of Medicago Sativa And Gene Prediction Of Six Photoreceptors Of Medicago Truncatula

Alfalfa(Medicago sativa) is an important forage legume, the characteristics of fall dormancy is related with cold hardiness and production. Fall dormancy of alfalfa is a photoperiod response. Phytochrome and cryptochrome are the receptors of photoperiod response. So, study the ralations between the photoreceptors and fall dormancy maybe a good way to reveal the the mechanism of alfalfa's fall dormancy.In this study, the gene of 6 photoreceptors (three kind photoreceptors) of barrel medic (Medicago truncatula) were predicted by the methods and protocols of Bioinformatics, there was Phytochrome A(PHYA), Phytochrome B(PHYB), Cryptochrome 1(CRY1), Cryptochrome 2A(CRY2A), Cryptochrome 2B(CRY2B) and Phototropin 1(PHOT1).Three predicted genes of barrel medic (PHYB, CRY1, CRY2B) were used as PCR primer design templates to cloning and sequencing the corresponding gene of alfalfa. The result was : 7150bp of genomic DNA and 3425bp(encode to 1141aa) of coding sequence of PHYB were got , and the length covered 99.0% of the full CDS of predicted PHYB in barrel medic. 4318bp of genomic DNA and 2030bp(encode to 676aa) of coding sequence were obtain, the number of CRY2B was 2251bp(genomic DNA) and 1453bp(coding sequence), and they coverd 99.5% and 80.3% of the full CDS of predicted CRY1 and CRY2B in barrel medic respectively. In addition, exon - intron boundaries and conserved region were analyzed of the three gene.This experiment used the method of Genome Walking(Chromosome Walking) to get the 5'-end sequence of PHYA and PHYB of alfalfa , the result was the length of 1309bp and 1752bp was obtained respectively, indicated Genome Walking was a good method to obtain the unknown sequence adjacent to known sequence.Used by a pair of PCR primers (length of production about 5kb), cloned and got the sequence of PHYB gene of two different types of fall dormancy of alfalfa .The analysis suggested that there had some difference between the two sequences. Not only the length of four bases in non-coding region, but also had 4 base and 26 base of nucleotides in coding region and non-coding region. And the differences in coding region lead to 4 amino acid differences.