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Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

Tian G, Cheng L, Qi X, Ge Z, Niu C, Zhang X, Jin S - Int. J. Biol. Sci. (2015)

Bottom Line: In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation.In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves.The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Science and Technology, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China.

ABSTRACT
RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control.

No MeSH data available.


Related in: MedlinePlus

Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.
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Figure 3: Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.

Mentions: In this experiment, YZ-1, an elite cotton cultivar was used for genetic transformation. Following callus induction (Fig.2A), somatic embryogenesis (Fig.2B) and plant regeneration were realized (Fig.2C-D). All regenerated plantlets were cultured in nutrients-containing water for acclimation (Fig.2E) and then transferred to pots (Fig.2F). Totally, 30 independent positive transgenic T0 plants (15 for each vector of HMGi1 and HMGi2 respectively) were verified by PCR analysis (Fig.3A). Most of these T0 plants were fertile and exhibited normal growth and phenotypes when compared to the wild type control plants containing no constructs. A Kanamycin resistant analysis 30 was performed to screen theT1 population from the positive transgenic T0 plants. As shown in Figure 2G, two types of root phenotypes were observed after the kanamycin test in that some plantlets showed a strong root system with a tap root and lateral roots, and other plantlets had a weak root phenotype without lateral roots (Fig. 2G). A subsequent PCR analysis revealed that only the plants with strong branch roots were PCR positive. The PCR data were in accordance with the root phenotype, suggesting that the antibiotics selection as described here is an effective method for the primary screening of transgenic progeny. Totally, 6 T0 transgenic lines contained a single copy T-DNA insertion and 5 lines had multiple T-DNA copies as verified by the Southern blot data (Fig. 3B). All these PCR and Southern blot positive T0 regeneration plants exhibited normal phenotype and were selected for further analysis.


Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

Tian G, Cheng L, Qi X, Ge Z, Niu C, Zhang X, Jin S - Int. J. Biol. Sci. (2015)

Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4582153&req=5

Figure 3: Molecular analysis for the putative transgenic cotton plants. (A) PCR analysis for HMGi1 and HMGi2 putative transgenic plants. M:Marker; N:Negative control; P: Positive control; Numbers marked above the gel indicating the corresponding T0 transgenic plants. (B) Southern blotting analysis of transgenic T0 plants. M: DNA molecular weight marker DIG-labeled (0.12-23.1 kb)(Roche, Germany); P: positive control; B: blank lane (no DNA loading); N: negative control plant DNA; Lane: 1-10 different HMGi transgenic lines.
Mentions: In this experiment, YZ-1, an elite cotton cultivar was used for genetic transformation. Following callus induction (Fig.2A), somatic embryogenesis (Fig.2B) and plant regeneration were realized (Fig.2C-D). All regenerated plantlets were cultured in nutrients-containing water for acclimation (Fig.2E) and then transferred to pots (Fig.2F). Totally, 30 independent positive transgenic T0 plants (15 for each vector of HMGi1 and HMGi2 respectively) were verified by PCR analysis (Fig.3A). Most of these T0 plants were fertile and exhibited normal growth and phenotypes when compared to the wild type control plants containing no constructs. A Kanamycin resistant analysis 30 was performed to screen theT1 population from the positive transgenic T0 plants. As shown in Figure 2G, two types of root phenotypes were observed after the kanamycin test in that some plantlets showed a strong root system with a tap root and lateral roots, and other plantlets had a weak root phenotype without lateral roots (Fig. 2G). A subsequent PCR analysis revealed that only the plants with strong branch roots were PCR positive. The PCR data were in accordance with the root phenotype, suggesting that the antibiotics selection as described here is an effective method for the primary screening of transgenic progeny. Totally, 6 T0 transgenic lines contained a single copy T-DNA insertion and 5 lines had multiple T-DNA copies as verified by the Southern blot data (Fig. 3B). All these PCR and Southern blot positive T0 regeneration plants exhibited normal phenotype and were selected for further analysis.

Bottom Line: In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation.In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves.The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae.

View Article: PubMed Central - PubMed

Affiliation: College of Plant Science and Technology, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China.

ABSTRACT
RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control.

No MeSH data available.


Related in: MedlinePlus