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Bulletin of Botanical Research ›› 2015, Vol. 35 ›› Issue (3): 355-362.doi: 10.7525/j.issn.1673-5102.2015.03.006

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Construction of RNA interference vector carrying two genes of DS and CAS of Betula platyphylla Suk(Birch.)

LIANG Tian1;MA Hong-Si1;YIN Jing1*;ZHAN Ya-Guang1,2;ZHANG Meng-Yan1   

  1. 1.College of Life Sciences,Northeast Forestry University,Harbin 150040;
    2.State Key Laboratory of Tree Genetics Breeding,Northeast Forestry University,Harbin 150040
  • Online:2015-05-20 Published:2015-06-24
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Abstract: Cycloartenol synthase(CAS) and dammarenediol synthase(DS) are the key enzymes of branch pathway of birch in birch triterpenoid synthesis process. Silencing of DS gene and CAS gene help triterpenoid synthesis precursor-2,3-oxidesqualene to synthesize betulin and oleanolic acid. PCR method of homologous sequences and a new vector pRNAi-GG were used to construct RNAi expression vectors of CAS gene and DS gene. The cloning part cDNA sequence of DS and CAS gene, respectively, CAS gene fragment length was 300bp, DS gene fragment length was 509bp, and their similarity reached 100% and 92%, respectively, compared with BPX1 and OSCBPD of Betula platyphylla var. japonica. With the principle of primer design and pRNAi-GG instruction, we connected the fragments on forward and contrary direction to the both sides of PDK intron of pRNAi-GG. The recombinant vectors were transformed into competent E.coli cells Trans1-T1. By PCR and sequence detection, the interference vector of DS gene and interference vector of DS+CAS two genes were constructed successfully. Our study will lay the foundation for the genetic modification of blocking-up triterpenoid branch pathways and promote the precursor to synthetize betulin and oleanolic acid in the triterpenoids metabolism pathway of white birch.

Key words: Betula platyphylla Suk(Birch.), triterpenoid, RNA interference, cycloartenol synthase(CAS), dammarenediol synthase(DS)

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