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      RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

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          Abstract

          Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

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          Author and article information

          Journal
          Mol. Biol. Rep.
          Molecular biology reports
          1573-4978
          0301-4851
          Jul 2014
          : 41
          : 7
          Affiliations
          [1 ] Defence Institute of Bio-Energy Research, Haldwani-263 139, Nainital, Uttarakhand, India, vikaspatade1981@gmail.com.
          Article
          10.1007/s11033-014-3301-8
          24574003
          6181285c-307b-4c9e-80eb-e5b60e05e212
          History

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