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. 2015 Sep 15;11(11):1296-305.
doi: 10.7150/ijbs.12463. eCollection 2015.

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

Geng Tian  1 , Linlin Cheng  1 , Xuewei Qi  1 , Zonghe Ge  1 , Changying Niu  1 , Xianlong Zhang  1 , Shuangxia Jin  1
Affiliations

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

Geng Tian et al. Int J Biol Sci. .

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.

Keywords: 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR); RNA interference; cotton bollworm; double-stranded RNAs; pest control.; transgenic cotton.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
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.
Figure 2
Figure 2
Creating transgenic cotton plants by Agrobactrium-mediated genetic transformation. (A) Callus induction on selective media containing kanamycin. (B) Embryogenic callus emerged from non-embryogenic callus. (C) Somatic embryos and young plantlets developed from embryogenic callus. (D), (E)and(F) Regenerated putative transgenic plants cultured in rooting media, water and soil. (G)Identification the positive and negative plants of T1 progeny on selective media containing kanamycin. (H)Positive T1 transgenic progeny cultivated in greenhouse.
Figure 3
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.
Figure 4
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: null 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.
Figure 5
Figure 5
The relative expression of HMGR and vitellogenin gene in cotton bollworm after feeding on T1-generation positive transgenic and control leaves. (A) The expression level of HMGR in cotton bollworm after feeding on the transgenic leaves from three independent lines and control leaves. (B) The expression of vitellogenin gene in cotton bollworm after feeding on the transgenic leaves from three transgenic lines and control leaves. The expressions of HMGR and Vg gene in cotton bollworm were conspicuously downregulated by the transgenic plant derived dsHMGR, in spite of the variation between lines. 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.
Figure 6
Figure 6
Quantification of larvae lethality after feeding on transgenic dsHMGR cotton leaves. For the insect bioassay, 20 newly-hatched cotton bollworm larvae were reared on the detached fresh leaves from null plants as control and two positive transgenic lines. The lethality of different groups was detected every 12 hours. Leaves of positive plants expressing dsRNA-HMGR bring about higher lethality than those of null plants (CK). 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; *statistically significant at 0.05.
Figure 7
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.
Figure 8
Figure 8
Quantification of HMGR enzyme in the larvae feeding on negative and positive transgenic leaves expressing dsRNAs. Third -instar larvae were reared on negative and positive leaves for more than 168 hours. Three larvae were collected every 24 hours and used to perform protein assay by ELISA. Data of ELISA test showed the decrease of HMGR protein concentration by the ingestion of transgenic leaves expressing dsHMGRs. As is illustrated, the inhibiting effect was not distinct within 48 hours, but became conspicuous when larvae had been fed with transgenic leaves for 96 hours. 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.
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