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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

The body size, net weight gain and abnormal pupation of cotton bollworm larvae fed on leaves of negative control and positive transgenic cotton leaves. (A) and (B) The larvae feeding on leaves of negative(on the left) and positive(on the right) transgenic plants for 144 h show difference in body size. The growth of cotton bollworms was conspicuously impaired by the ingestion of transgenic leaves expressing dsRNAs. (C) The net weight gain of cotton bollworm larvae fed with transgenic plants expressing ds-HMGRs was impaired. The net weight gain of transgenic and control group did not show significant difference before 96 hours, but became distinct at 120 hours after feeding. The test was repetitiously performed for three times. The student's t-test was used to perform the statistical analyses of the data. **statistically significant at 0.01. (D) The phenotype of pupae pupated from larvae reared on positive and negative transgenic leaves. Larvae fed on leaves expressing dsRNA of HMGR pupated on time but the appearance of their pupae were distinctive from control. Their exocuticle were significantly thinner and softer than control, whose internal structure can be easily observed by naked eye.
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Figure 7: The body size, net weight gain and abnormal pupation of cotton bollworm larvae fed on leaves of negative control and positive transgenic cotton leaves. (A) and (B) The larvae feeding on leaves of negative(on the left) and positive(on the right) transgenic plants for 144 h show difference in body size. The growth of cotton bollworms was conspicuously impaired by the ingestion of transgenic leaves expressing dsRNAs. (C) The net weight gain of cotton bollworm larvae fed with transgenic plants expressing ds-HMGRs was impaired. The net weight gain of transgenic and control group did not show significant difference before 96 hours, but became distinct at 120 hours after feeding. The test was repetitiously performed for three times. The student's t-test was used to perform the statistical analyses of the data. **statistically significant at 0.01. (D) The phenotype of pupae pupated from larvae reared on positive and negative transgenic leaves. Larvae fed on leaves expressing dsRNA of HMGR pupated on time but the appearance of their pupae were distinctive from control. Their exocuticle were significantly thinner and softer than control, whose internal structure can be easily observed by naked eye.

Mentions: As described previously, newly hatched larvae were very sensitive to plant-derived dsRNA and as much as 80% of tested larvae died 72 h after feeding on the transgenic leaves. So, we also evaluated the effect of transgenic plant derived dsRNAs on the development of relatively older insects (two-instar larvae). Net weight gains and phenotype of two-instar larvae reared on negative control and transgenic leaves were measured to evaluate the suppression efficiency of dsRNAs. The difference on net weight gain between transgenic and control groups was not conspicuous within 96 hours of bioassay, but turned out to be obviously significant (P < 0.01) after 120 hours (Fig. 7C). As is illustrated, the difference was culminated when larvae were fed with negative control and transgenic leaves for 144 hours then decreased a bit 24 hours later, which suggested that the transgenic plant-derived dsRNAs not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. In the terms of morphological structures, accordingly, the larvae reared on positive transgenic leaves presented smaller body size than those on negative control leaves (Fig. 7A for CK and 7B for HMGi), but any other morphological malformation has not been observed in this experiment. Whether the transgenic plants expressing dsHMGR affect the percentage and time of pupation is not clear based on our available data because most of the tested larvae died before pupation or the larvae number was not even in dsRNAs treated and control group. However, lower frequency of abnormal pupation phenotype was observed and shown in Figure 7D. The larvae reared on transgenic leaves expressing dsHMGRs could pupate on time but their cuticulae were extremely thin and soft (Fig. 7D, on the right), which made the internal structure of pupae can be clearly observed by naked eye. This change may be due to the inhibition effect of plant-delivered dsRNAs on the cuticulae development and the ecdysis of tested larvae.


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)

The body size, net weight gain and abnormal pupation of cotton bollworm larvae fed on leaves of negative control and positive transgenic cotton leaves. (A) and (B) The larvae feeding on leaves of negative(on the left) and positive(on the right) transgenic plants for 144 h show difference in body size. The growth of cotton bollworms was conspicuously impaired by the ingestion of transgenic leaves expressing dsRNAs. (C) The net weight gain of cotton bollworm larvae fed with transgenic plants expressing ds-HMGRs was impaired. The net weight gain of transgenic and control group did not show significant difference before 96 hours, but became distinct at 120 hours after feeding. The test was repetitiously performed for three times. The student's t-test was used to perform the statistical analyses of the data. **statistically significant at 0.01. (D) The phenotype of pupae pupated from larvae reared on positive and negative transgenic leaves. Larvae fed on leaves expressing dsRNA of HMGR pupated on time but the appearance of their pupae were distinctive from control. Their exocuticle were significantly thinner and softer than control, whose internal structure can be easily observed by naked eye.
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Related In: Results  -  Collection

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Figure 7: The body size, net weight gain and abnormal pupation of cotton bollworm larvae fed on leaves of negative control and positive transgenic cotton leaves. (A) and (B) The larvae feeding on leaves of negative(on the left) and positive(on the right) transgenic plants for 144 h show difference in body size. The growth of cotton bollworms was conspicuously impaired by the ingestion of transgenic leaves expressing dsRNAs. (C) The net weight gain of cotton bollworm larvae fed with transgenic plants expressing ds-HMGRs was impaired. The net weight gain of transgenic and control group did not show significant difference before 96 hours, but became distinct at 120 hours after feeding. The test was repetitiously performed for three times. The student's t-test was used to perform the statistical analyses of the data. **statistically significant at 0.01. (D) The phenotype of pupae pupated from larvae reared on positive and negative transgenic leaves. Larvae fed on leaves expressing dsRNA of HMGR pupated on time but the appearance of their pupae were distinctive from control. Their exocuticle were significantly thinner and softer than control, whose internal structure can be easily observed by naked eye.
Mentions: As described previously, newly hatched larvae were very sensitive to plant-derived dsRNA and as much as 80% of tested larvae died 72 h after feeding on the transgenic leaves. So, we also evaluated the effect of transgenic plant derived dsRNAs on the development of relatively older insects (two-instar larvae). Net weight gains and phenotype of two-instar larvae reared on negative control and transgenic leaves were measured to evaluate the suppression efficiency of dsRNAs. The difference on net weight gain between transgenic and control groups was not conspicuous within 96 hours of bioassay, but turned out to be obviously significant (P < 0.01) after 120 hours (Fig. 7C). As is illustrated, the difference was culminated when larvae were fed with negative control and transgenic leaves for 144 hours then decreased a bit 24 hours later, which suggested that the transgenic plant-derived dsRNAs not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. In the terms of morphological structures, accordingly, the larvae reared on positive transgenic leaves presented smaller body size than those on negative control leaves (Fig. 7A for CK and 7B for HMGi), but any other morphological malformation has not been observed in this experiment. Whether the transgenic plants expressing dsHMGR affect the percentage and time of pupation is not clear based on our available data because most of the tested larvae died before pupation or the larvae number was not even in dsRNAs treated and control group. However, lower frequency of abnormal pupation phenotype was observed and shown in Figure 7D. The larvae reared on transgenic leaves expressing dsHMGRs could pupate on time but their cuticulae were extremely thin and soft (Fig. 7D, on the right), which made the internal structure of pupae can be clearly observed by naked eye. This change may be due to the inhibition effect of plant-delivered dsRNAs on the cuticulae development and the ecdysis of tested larvae.

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