Limits...
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

RT-PCR and qRT-PCR analysis of T1 transgenic cotton plants. (A) RT-PCR analysis of HMGi1 transgenic lines. Different transgenic lines expressed dsHMGR while the expression of dsRNA was absent in negative control; CK:  control; numbers marked above the gel indicating corresponding lines. (B) RT-PCR analysis of HMGi2 transgenic lines. Different transgenic lines expressed dsRNA while the expression of double-strand HMGR was absent in negative control. The relative expression of dsHMGRs in the HMGi1 (C) and HMGi2 (D) transgenic lines were verified by qRT-PCR. The lines, with intense signals in electrophoretogram, were verified to show higher expression levels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4582153&req=5

Figure 4: RT-PCR and qRT-PCR analysis of T1 transgenic cotton plants. (A) RT-PCR analysis of HMGi1 transgenic lines. Different transgenic lines expressed dsHMGR while the expression of dsRNA was absent in negative control; CK: control; numbers marked above the gel indicating corresponding lines. (B) RT-PCR analysis of HMGi2 transgenic lines. Different transgenic lines expressed dsRNA while the expression of double-strand HMGR was absent in negative control. The relative expression of dsHMGRs in the HMGi1 (C) and HMGi2 (D) transgenic lines were verified by qRT-PCR. The lines, with intense signals in electrophoretogram, were verified to show higher expression levels.

Mentions: RT-PCR and qRT-PCR were performed to detect the expression level of dsRNA-HMGR in T1 lines from each positive T0 transgenic plant. The result of RT-PCR showed that dsHMGR was expressed in all 11 positive lines (5 lines from HMGi1 and 6 from HMGi2, Fig. 4A and B), while the transcription levels of dsRNAs in these lines appeared diversity, which were quantified by qRT-PCR. All the semi-quantitative results from RT-PCR were confirmed by qRT-PCR suggesting that the dramatic variation of dsRNAs expression existed in different transgenic lines (Fig. 4C and D). For example, the dsHMGR expression level in line L13 of HMGi1 was 9.13 times higher than in the line L58-4.Three lines with relatively higher expression level of dsRNAs were selected for further insect feeding bioassay to analyze the mortality of larvae and the efficiency of RNA interference.


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)

RT-PCR and qRT-PCR analysis of T1 transgenic cotton plants. (A) RT-PCR analysis of HMGi1 transgenic lines. Different transgenic lines expressed dsHMGR while the expression of dsRNA was absent in negative control; CK:  control; numbers marked above the gel indicating corresponding lines. (B) RT-PCR analysis of HMGi2 transgenic lines. Different transgenic lines expressed dsRNA while the expression of double-strand HMGR was absent in negative control. The relative expression of dsHMGRs in the HMGi1 (C) and HMGi2 (D) transgenic lines were verified by qRT-PCR. The lines, with intense signals in electrophoretogram, were verified to show higher expression levels.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: RT-PCR and qRT-PCR analysis of T1 transgenic cotton plants. (A) RT-PCR analysis of HMGi1 transgenic lines. Different transgenic lines expressed dsHMGR while the expression of dsRNA was absent in negative control; CK: control; numbers marked above the gel indicating corresponding lines. (B) RT-PCR analysis of HMGi2 transgenic lines. Different transgenic lines expressed dsRNA while the expression of double-strand HMGR was absent in negative control. The relative expression of dsHMGRs in the HMGi1 (C) and HMGi2 (D) transgenic lines were verified by qRT-PCR. The lines, with intense signals in electrophoretogram, were verified to show higher expression levels.
Mentions: RT-PCR and qRT-PCR were performed to detect the expression level of dsRNA-HMGR in T1 lines from each positive T0 transgenic plant. The result of RT-PCR showed that dsHMGR was expressed in all 11 positive lines (5 lines from HMGi1 and 6 from HMGi2, Fig. 4A and B), while the transcription levels of dsRNAs in these lines appeared diversity, which were quantified by qRT-PCR. All the semi-quantitative results from RT-PCR were confirmed by qRT-PCR suggesting that the dramatic variation of dsRNAs expression existed in different transgenic lines (Fig. 4C and D). For example, the dsHMGR expression level in line L13 of HMGi1 was 9.13 times higher than in the line L58-4.Three lines with relatively higher expression level of dsRNAs were selected for further insect feeding bioassay to analyze the mortality of larvae and the efficiency of RNA interference.

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