Gene Cloning and Bioinformatics Analysis of Limonene-3-hydroxylase from Schizonepeta tenuifolia / 中国实验方剂学杂志

ABSTRACT

Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.