Cloning And Expression Vector Construction Of Phytochrome Genes PHYB From A. Edgeworthii Benth

A . edgeworthii Benth is a kind of annual winding herb. The ground seed of A . edgeworthii Benth is oblate, the color of seed coat that with gray and white spot on it is dark brown. There is smooth, lustrous and hard wax on the surface which is leather qualitative. the rate of hard real is 98.3% under natural conditions.PHYB genes are the most important one gene sequence in the plant photomorpho-genesis. Phytochrome (PHY) involve in many physiological processes such as plant seed germination, stemelongation, cotyledon extention,seedling growth and flowering control. There are five family members in PHYs, which are PHYA, PHYB, PHYC, PHYD, PHYE respectively. Phytochrome has two types of Pâ… and Pâ…¡, P I mainly in the yellow tissues, Pâ…¡mainly in green tissues. Pâ… phytochrome is a PHYA, phytochrome is Pâ…¡has PHYB, PHYC, PHYD, PHYE. Each phytochrome can be interchangeable in two configurations:red absorption (Pr) and far-red light absorption (Pfr). Pr absorbs red light energy into Pfr, Pfr absorbs far red light into Pr. Pfr is the active form of phytochrome.In order to reveal the construction features of wild A.edgeworthii Benth. PHYB genes in the molecular and provide a theoretical basis for the expression and regulation mechanism. Sequence analysis showed that the DNA contains 4 introns and 5 exons sequences, length of 6560 bp, exon and intron sequences between the cut line with GT/AG rule. On the basis of comparing the sequence to Glycine max and Medicago sativa, the homologies of nucleotide were 98% and 94%.PHYB gene whose sequence is 3472 bp in length was clones by RT-PCR with all open reading flame (ORF) of 3317 bp and encoded 1105 amino acid residues with a predicted molecular weight of 123.78 kDa and an isoelectric point of 5.99. The protein sequence comparison showed that the predicted protein had higher identifies to those from and Glycine max PHYB-4 Medicago sativa and Lotus japonicus (98%, 94% and 84%), named AephyB, genbank accession number:JF412664.The intermediate vector Psk-intPHYB is constructed with inverted repeats, which could be applied to other RNA interference. Firstly, sequences of PHYB genes submitted on NCBI were analysised, and 480 bp fragment of the conserved domain were selected as RNAi target sequence. Two pairs of primer with restriction sites were used to amplify the sense and anti-sense fragments, with double enzyme digestion, the sense and anti-sense fragments were linked into intermediate vectors. In order to produce a intron-hairpin structure, there is a maize intron between sense and anti-sense fragment.According constructed with inverted repeats of intermediate vector Psk-intPHYB, became applicable to build one plant RNAi expression vector by Agrobacterium-mediated. It is pJIM19PHYB. Using Spel and Xhol restriction enzyme cut inverted repeat sequence, connected to the plant expression vector pJIM19, construction RNAi expression vector.