Prokaryotic Expression and Tissuespecific Expression of SmDL7H in Swertia mussotii Franch
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    Abstract:

    In this research, according to the SmDL7H gene sequence of transcriptome of Swertia mussotii, we obtained the cDNA complete sequences by using method of homologous cloning, bioinformatics analysis, constructed prokaryotic expression vector, transformed into Escherichia coli and tissuespecific expression analysis, in order to identify the function of the 7deoxyloganic acid7hydroxylase(SmDL7H)which is a key enzyme of the secoiridoid pathway in the S. mussotii. This work will also provide a foundation to study the secoiridoid pathway in S. mussotii. The results showed that: (1) SmDL7H gene from S. mussotii (GeneBank accesion number: MH243070) was cloned; SmDL7H gene open reading frame(ORF)of SmDL7H was 1 554 bp in length, which encoded 517 amino acids, with the isoelectric point of 9.02 and molecular mass of 59.5 kD; bioinformatics forecasted the SmDL7H protein with no signal peptide. (2) Phylogenetic analysis showed that SmDL7H protein shares high identity with Gentiana rigescens, Catharathus roseus, Cinchona calisaya and etc plants. (3) SmDL7H gene connected with expression vector pET28a and then transformed into E.coil Rosetta(DE3)for heterologous expression. The recombinant protein was induced by 0.1 mol/L IPTG at 25 ℃ for 12 h. Prokaryotic expression analysis showed that the expressed protein was 59.5 kD and accorded with our forecast. (4) qRTPCR analysis indicated that SmDL7H was expressed in leaves, stems, flowers, roots and callus, and the highest in leaves, the lowest in roots.

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LI Xiaoxue, WANG Yong, SUN Jiqi, ZHU Yerong, MA Lin, XIANG Beibei. Prokaryotic Expression and Tissuespecific Expression of SmDL7H in Swertia mussotii Franch[J]. Acta Botanica Boreali-Occidentalia Sinica,2018,38(8):1375-1381

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  • Online: September 20,2018
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