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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

Target sequences of HMG gene for RNA interference and plasmid vector for cotton genetic transformation. (A) Two sequences of HMG gene were used for RNAi target sequences in this experiment. The HMGi 1 sequence was indicated in red font and the HMGi 2 sequence was indicated in blue font. (B) Schematic representation of T-DNA region of the expression cassette of HMGR RNA interference vector.
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Figure 1: Target sequences of HMG gene for RNA interference and plasmid vector for cotton genetic transformation. (A) Two sequences of HMG gene were used for RNAi target sequences in this experiment. The HMGi 1 sequence was indicated in red font and the HMGi 2 sequence was indicated in blue font. (B) Schematic representation of T-DNA region of the expression cassette of HMGR RNA interference vector.

Mentions: Two target fragments (namely HMGi1 and HMGi2) in the coding sequence of HMGR gene (GenBank accession no. GU584103) were chosen for RNAi targets including a 455bp and a 874bp fragments, as shown in Figure 1A respectively. Two pairs of primers with attB1 and attB2 adaptors were designed according to target fragments. All the sequences of primers for HMGi1 and HMGi2 were displayed in Table 1. The purified PCR products were incorporated into vector pHellsgate4 according to the manufacturer's recommendations 31, which harboring a NPT II gene as the select marker. The T-DNA region of the plastid was shown in Figure 1B.Then the constructed vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation, whose positivity was verified by PCR. The Agrobacterium tumefaciens-mediated transformation of cotton was applied according to our previous reports 29, 32-34.


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)

Target sequences of HMG gene for RNA interference and plasmid vector for cotton genetic transformation. (A) Two sequences of HMG gene were used for RNAi target sequences in this experiment. The HMGi 1 sequence was indicated in red font and the HMGi 2 sequence was indicated in blue font. (B) Schematic representation of T-DNA region of the expression cassette of HMGR RNA interference vector.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Target sequences of HMG gene for RNA interference and plasmid vector for cotton genetic transformation. (A) Two sequences of HMG gene were used for RNAi target sequences in this experiment. The HMGi 1 sequence was indicated in red font and the HMGi 2 sequence was indicated in blue font. (B) Schematic representation of T-DNA region of the expression cassette of HMGR RNA interference vector.
Mentions: Two target fragments (namely HMGi1 and HMGi2) in the coding sequence of HMGR gene (GenBank accession no. GU584103) were chosen for RNAi targets including a 455bp and a 874bp fragments, as shown in Figure 1A respectively. Two pairs of primers with attB1 and attB2 adaptors were designed according to target fragments. All the sequences of primers for HMGi1 and HMGi2 were displayed in Table 1. The purified PCR products were incorporated into vector pHellsgate4 according to the manufacturer's recommendations 31, which harboring a NPT II gene as the select marker. The T-DNA region of the plastid was shown in Figure 1B.Then the constructed vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation, whose positivity was verified by PCR. The Agrobacterium tumefaciens-mediated transformation of cotton was applied according to our previous reports 29, 32-34.

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