CLONING AND SEQUENCING OF B -CAROTENE HYDROXYLASE GENE FROM HAEMATOCOCCUS PLUVIALIS

Astaxanthin (3,3.-dihydroxy-B, B-carotene-4, 4.-dione), belonging to carotenoids, has been shown a series of biologiacal functions including an efficient antioxidant, enhancer of immune responses, an anti cancer agent, and so on. B-carotene hydroxylase (CRTR-B) is one of thekey enzymes involving in the natural astaxantin biosynthesis in H. pluvialis. crtR-B encoding B-carotene hydroxylase which is able to catalyse not only B-carotene to zeaxanthin but also canthaxanthin to astaxanthin. In this paper, crtR-B from H. pluvialis 34-1n was isolated and differences of crtR-B were observed between H. pluvialis 34-1n and H. pluvialis Flotow NIES-144. A pair of primers were designed according to B-carotene hydroxylase(GI: 5852869) cDNA sequence in Genbank. Total RNA of H. pluvialis 34-1n was extracted and was transcribed. A cDNA fragment of B-carotene hydroxylasegenewas amplified by using cDNA as templet and was further inserted into pMD 18-T simple Vector. The pMD 18-T simple Vector which contained the cDNA fragment was transformated into E. coli JM109 for sequencing. The sequence analysis revealed that the cDNA fragment contained an opening frameof 879bp encoding 292 amino acids. An amino acid absencewas found at the region of 33 compared with the published B-carotene hydroxylasegene and 4 amino acids differences were observed between the cloned gene and the sequence of H. pluvialis Flotow NIES-144. Multiple sequence alignment analysis indicated that the percentage identity of amino acid sequences ranged from 35. 3% to 9816%. There were only limited predicted amino acids sequence homologies to B-carotene hydroxylase in Alcaligenes sp and Erwinia uredovora.A higher amino acids homologywas shown to the higher plants (4016%) 4412%). The most significant homology was found between H. pluvialis 34-1n and C. reinhardtii (4910%). The identity of crtR-B from H. pluvialis 34-1n to H. pluvialis Flotow NIES-144 was 9816%.