The Gene Cloning And Sequence Analysis Of The Pnae In Rauvolfia Yunnanensis

As a medicinal plant, Rauvolfia contains about200indole alkaloids Among them one of the most intriguing alkaloids was ajmaline, comprising a highly complicated hexacyclic ring system bearing nine chiral carbon centers, ajmaline’s anabolism included more than10different steps,and the PNAE enzyme is just in the center position of those steps.It is the key enzyme which catalyze the polyneuridine aldehyde(PNA) into the epi-vellosimine,this reaction’s rate decides the productivity of ajmaline.At present there is no report about the cloning of PNAE in Rauvolfia yunnanensis Tsiang,so the clone of the PNAE will have a profound effect on the endeavor to increasing the productivity of ajmaline in Rauvolfia yunnanensis Tsiang.Following is the result of this experiment:1)After searched the related protein and cDNA sequences at the NCBI website,we compared them by the blast software to get the roughly conserved domains in its cDNA sequence. According to some identified mRNA sequence of the polyneuridine aldehyde esterase (PNAE) on the NCBI website, a number of pairs of primers were designed. the high-quality total RNA was obtained from Rauvolfia yunnanensis’ fresh leaves which was cultured in our lab’s greenhouse,then the partial inter-sequence of the PNAE cDNA was obtained by RT-PCR technique,finally through the technique called rapid-amplification of cDNA ends(RACE),the sequence in both two flanks of the known sequence was acquired. The full length sequence (1004bp) of the cDNA of PNAE in Rauvolfia yunnanensis has been got and pieceed together.2)The full length sequence (1004bp) of the cDNA of PNAE included an open-reading-frame (ORF) which encoded264amino acids. Sequence analysis showed that the homologous rate of the PNAE gene’s amino acid sequence among each species was ranging from40%to60%and PNAE gene is not highly conservative.3)The further analysis of all the known PNAE in plant species found that there are two conservative domains in the amino acid sequence, which revealing that PNAE gene has two highly conserved domains among different plant species.One of the them is similar to a four-hydrophobic-amino-acid common conservative domains in hydrolytic enzyme (a glycine around a histidine).Another domain has a same amino acid sequence with the A/b hydrolase super family’s conservative domains,that shows the PNAE enzyme is a new member of A/b hydrolase super family.4)In general,the further cloning of the pnae in other species could make use of the two highly common conservative domains as a reference...