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

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.

An integration of phenotypic and transcriptomic data analysis reveals yield-related hub genes in Jatropha curcas inflorescence

“…The RNA interference or the RNAi silencing mechanism has emerged as a powerful tool for in planta gene expression manipulation and have been actively adopted in J . curcas [ 71 , 72 , 73 , 74 ]. The mechanism silences the expression of a target gene, introduced through its silencing machinery [ 75 ].…”

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing

“…Our studies on C1 transcripts in developing endosperm and curcin proteins in mature endosperm demonstrated that the expression of the C1 gene was efficiently suppressed or completely silenced by the C1 promoter-driven RNAi-mediated gene silencing. Patade et al, (2014) made a 35S promoter-driven RNAi cassette for curcin genes and used Agrobacterium -mediated in planta transformation to produce transformed jatropha plants [ 27 ]. The authors reported that the transcripts of curcin precursor gene were reduced by more than 98 % to undetectable level [ 27 ].…”

“…Patade et al, (2014) made a 35S promoter-driven RNAi cassette for curcin genes and used Agrobacterium -mediated in planta transformation to produce transformed jatropha plants [ 27 ]. The authors reported that the transcripts of curcin precursor gene were reduced by more than 98 % to undetectable level [ 27 ]. However, the research paper did not provide any data on molecular analysis of stable insertion of T-DNA in jatropha genome, biochemical analysis on curcin proteins in the leaves and seeds of transformed plants.…”

Fueling the future; plant genetic engineering for sustainable biodiesel production

“…Some research works have successfully used the RNAi technology to reduce curcins in Jatropha. Suppression of this gene could reduce the curcin up to 98% (Patade et al, 2014;Gu et al, 2015). The cDNA of the gene encoding curcin, i.e., curcin2 was obtained from J. curcas leaves (Tong et al, 2007).…”