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

Gu K, Tian D, Mao H, Wu L, Yin Z - BMC Plant Biol. (2015)

Bottom Line: The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette.The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable.Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Republic of Singapore. keyu@tll.org.sg.

ABSTRACT

Background: Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves.

Results: A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette.

Conclusion: The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

No MeSH data available.


Related in: MedlinePlus

PCR analysis of T0-1 and T0-35 and their T1 progeny. The DNA sequences of primers are listed in Table 1. pCMFC1, control plasmid; MD44, wild-type control. T0-1/T1-1 and T0-1/T1-2, T1 plants derived from T0 plant T0-1; T0-35/T1-1 and T0-35/T1-2, T1 plants derived from T0 plant T0-35
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Fig3: PCR analysis of T0-1 and T0-35 and their T1 progeny. The DNA sequences of primers are listed in Table 1. pCMFC1, control plasmid; MD44, wild-type control. T0-1/T1-1 and T0-1/T1-2, T1 plants derived from T0 plant T0-1; T0-35/T1-1 and T0-35/T1-2, T1 plants derived from T0 plant T0-35

Mentions: In total, twelve transgenic T0 plants were obtained after Agrobacterium-mediated transformation of jatropha cotyledon discs [25]. Initial PCR analysis indicated that all of the twelve T0 plants carried C1 promoter-driven RNAi cassette for the C1 gene, showing the amplification of Gus linker (Table 2). However, six of the twelve T0 plants gave amplification of the F1-R2 fragment, indicating that they carried marker-free T-DNA (Table 2). The transgenic T0 plants grew and developed normally compared to wild-type MD44 in the same growth condition. T1 seeds from the T0 plants were collected and used for further molecular analysis. Embryos of the T1 seeds were dissected and germinated on seed germination medium, while the endosperm from the same set of T1 seeds was analyzed individually for C1 proteins by western blot analysis. T1 plants were transplanted to soil and used for molecular characterization of the transgenes. Initial screening identified five T0 plants, T0-1, T0-29, T0-35, T0-40A and T0-48. They produced T1 seeds that had lower C1 content than non-transgenic MD44 seeds (Fig. 2a). Among the five transgenic lines, T1 seeds derived from T0-1 and T0-35 had the lowest level of C1 content (Fig. 2a, lanes 2 and 4). Both T0-1 and T0-35 carried marker-free T-DNA, showing the amplification of F1-R2 fragment (Table 2; Fig. 3). However, PCR analysis indicated that they also carried the Hpt gene (Table 2; Fig. 3). The results suggested that the two T0 plants carried both marker-free and non-marker-free T-DNAs. T1 plants T0-1/T1-1, T0-1/T1-2, T0-35/T1-1 and T0-35/T1-2 inherited the marker-free T-DNAs from the respective T0 plants, showing the amplification of the Gus linker and F1-R2 fragments (Fig. 3). However, they also showed the amplification of F2-R2 fragment (Fig. 3). In addition, T0-1/T1-1 and T0-1/T1-2 still contained the Hpt gene (Fig. 3). The results suggested that the T1 plants carried either non-marker-free T-DNA or truncated T-DNA.Table 2


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

Gu K, Tian D, Mao H, Wu L, Yin Z - BMC Plant Biol. (2015)

PCR analysis of T0-1 and T0-35 and their T1 progeny. The DNA sequences of primers are listed in Table 1. pCMFC1, control plasmid; MD44, wild-type control. T0-1/T1-1 and T0-1/T1-2, T1 plants derived from T0 plant T0-1; T0-35/T1-1 and T0-35/T1-2, T1 plants derived from T0 plant T0-35
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4599812&req=5

Fig3: PCR analysis of T0-1 and T0-35 and their T1 progeny. The DNA sequences of primers are listed in Table 1. pCMFC1, control plasmid; MD44, wild-type control. T0-1/T1-1 and T0-1/T1-2, T1 plants derived from T0 plant T0-1; T0-35/T1-1 and T0-35/T1-2, T1 plants derived from T0 plant T0-35
Mentions: In total, twelve transgenic T0 plants were obtained after Agrobacterium-mediated transformation of jatropha cotyledon discs [25]. Initial PCR analysis indicated that all of the twelve T0 plants carried C1 promoter-driven RNAi cassette for the C1 gene, showing the amplification of Gus linker (Table 2). However, six of the twelve T0 plants gave amplification of the F1-R2 fragment, indicating that they carried marker-free T-DNA (Table 2). The transgenic T0 plants grew and developed normally compared to wild-type MD44 in the same growth condition. T1 seeds from the T0 plants were collected and used for further molecular analysis. Embryos of the T1 seeds were dissected and germinated on seed germination medium, while the endosperm from the same set of T1 seeds was analyzed individually for C1 proteins by western blot analysis. T1 plants were transplanted to soil and used for molecular characterization of the transgenes. Initial screening identified five T0 plants, T0-1, T0-29, T0-35, T0-40A and T0-48. They produced T1 seeds that had lower C1 content than non-transgenic MD44 seeds (Fig. 2a). Among the five transgenic lines, T1 seeds derived from T0-1 and T0-35 had the lowest level of C1 content (Fig. 2a, lanes 2 and 4). Both T0-1 and T0-35 carried marker-free T-DNA, showing the amplification of F1-R2 fragment (Table 2; Fig. 3). However, PCR analysis indicated that they also carried the Hpt gene (Table 2; Fig. 3). The results suggested that the two T0 plants carried both marker-free and non-marker-free T-DNAs. T1 plants T0-1/T1-1, T0-1/T1-2, T0-35/T1-1 and T0-35/T1-2 inherited the marker-free T-DNAs from the respective T0 plants, showing the amplification of the Gus linker and F1-R2 fragments (Fig. 3). However, they also showed the amplification of F2-R2 fragment (Fig. 3). In addition, T0-1/T1-1 and T0-1/T1-2 still contained the Hpt gene (Fig. 3). The results suggested that the T1 plants carried either non-marker-free T-DNA or truncated T-DNA.Table 2

Bottom Line: The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette.The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable.Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Republic of Singapore. keyu@tll.org.sg.

ABSTRACT

Background: Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves.

Results: A marker-free RNAi construct carrying a β-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette.

Conclusion: The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

No MeSH data available.


Related in: MedlinePlus