雨生红球藻β-胡萝卜素酮化酶基因克隆、分析及叶绿体表达载体构建

Cloning and Characterization of β-Carotene Ketolase Gene from Haematococcus pluvialis and Construction of its Chloroplast Transformation Expression Vector

β-胡萝卜素酮化酶(BKT)是雨生红球藻虾青素合成途径中的关键酶。根据GenBank中所收录的雨生红球藻BKT的cDNA序列设计特异性引物,以高光照处理的雨生红球藻细胞的总RNA为模板,采用RT-PCR的方法克隆出了β-胡萝卜素酮化酶基因(bkt)。序列测定结果表明,bkt全长960bp编码320个氨基酸。克隆的bkt序列与GenBank收录的BKT的cDNA(AY603347)序列只相差一个碱基。并对其编码的氨基酸进行了二级结构的预测和疏水性分析。同时将所克隆的bkt构建入衣藻叶绿体表达载体p64Dbkt,为基因的进一步表达研究及探索其作用机制奠定了基础。

β-carotene ketolase （BKT） is the key enzyme for the biosynthesis of astaxanthin. The specific primer was designed based on the sequence of BKT cDNA in the GenBank, and the total RNA of Haematococcus pluvialis was isolated from the high light treatment cells. The β-carotene ketolase gene fragment was amplified by RT-PCR. DNA sequencing results showed that bkt was composed of 960 bp and encoded 320 amino acid residues. Sequence alignments with BLAST revealed that there was only one base pair different from BKT cDNA （AY603347）. Further, the secondary structure and hydrophobicity of BKT were analyzed, and the bkt chloroplast expression vector p64Dbkt was constructed. The achievement of this study has laid a foundation for the further research on expressing bkt.