Cotton plants expressing CYP6AE14 double-stranded RNA show enhanced resistance to bollworms

Abstract

RNA interference (RNAi) plays an important role in regulating gene expression in eukaryotes. Previously, we generated Arabidopsis and tobacco plants expressing double-stranded RNA (dsRNA) targeting a cotton bollworm (Helicoverpa armigera) P450 gene, CYP6AE14. Bollworms fed on transgenic dsCYP6AE14 plants showed suppressed CYP6AE14 expression and reduced growth on gossypol-containing diet (Mao et al., in Nat Biotechnol 25: 1307-1313, 2007). Here we report generation and analysis of dsRNA-expressing cotton (Gossypium hirsutum) plants. Bollworm larvae reared on T2 plants of the ds6-3 line exhibited drastically retarded growth, and the transgenic plants were less damaged by bollworms than the control. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that the CYP6AE14 expression level was reduced in the larvae as early as 4 h after feeding on the transgenic plants; accordingly, the CYP6AE14 protein level dropped. These results demonstrated that transgenic cotton plants expressing dsCYP6AE14 acquired enhanced resistance to cotton bollworms, and that RNAi technology can be used for engineering insect-proof cotton cultivar.

Fig. 1

Effect of T1 transgenic cotton on larvae growth. a The dsRNA construct pBI121-dsCYP6AE14 contained a 35S promoter, a sense fragment of CYP6AE14 cDNA from +472 to +940, a 120-nucleotide intron of Arabidopsis RTM1 gene (Johansen and Carrington 2001), the CYP6AE14 fragment in antisense orientation, and a NOS terminator. b northern blot detection of dsRNA homologues to CYP6AE14 in the leaves of transgenic (ds1-ds6) and nontransgenic control (R15) plants. c, d Net weight increase of larvae reared on leaves of T1 transgenic cotton plants. Third-instar larvae previously grown on artificial diet were transferred to nontransgenic (R15) or T1 transgenic cotton plant leaves for 4 days, respectively. Values are means ± standard deviation (SD). *P < 0.05; **P < 0.01

Fig. 2

northern and Southern blots of dsCYP6AE14 in T1 and T2 lines of ds6-3. a northern blot to detect dsRNA of CYP6AE14 in leaves of T2 plants of ds6-3. b Southern blot assay to determine copies of CYP6AE14 in T1 and T2 plants of ds6-3. Genomic DNA of ds6-3 and three individuals of T2 generation (ds6-3-2, ds6-3-3, and ds6-3-4) were digested by enzymes as indicated

Fig. 3

Suppression of CYP6AE14 expression in larvae fed on ds6-3 T2 plants. a northern blot of the small RNAs of CYP6AE14 in midgut of third-instar larvae fed on nontransgenic control R15 (lane 2) and ds6-3 T2 plants (lane 3) for 3 days. Lane 1: total RNAs from ds6-3 T2 plants as positive control. b and c northern blot (b) and qRT-PCR (c) analysis of CYP6AE14 transcripts in midgut of second-instar larvae fed on control (R15) or ds6-3 T2 (ds) plants for indicated time. d western blot detection of CYP6AE14 proteins in midgut of second-instar larvae fed on nontransgenic control R15 or ds6-3 T2 plants for 3 days. Values are means ± SD

Fig. 4

Effect of ds6-3 T2 plants on larvae growth. a Net weight increase of larvae fed on leaves of nontransgenic control R15 (blue) or ds6-3 T2 (ds) plants (red) for indicated days. b Images of larvae that were fed on leaves of nontransgenic control R15 or ds6-3 T2 plants for 4 and 6 days, respectively. c Gossypol equivalents in leaves of nontransgenic control R15 or ds6-3 T2 plants. d Gossypol equivalents in midgut of the larvae fed on leaves of nontransgenic control R15 and ds6-3 T2 plants, respectively, for 6 days. Values are means ± SD. *P < 0.05; **P < 0.01

Fig. 5

Transgenic cotton plants were less damaged by bollworms than the control. Second-instar larvae were divided into 2 groups. Each group contained 40 individual larvae and were fed on control (R15) and ds6-3 T2 (ds) leaves with similar conditions. a Consumption of leaves of control and ds6-3 T2 plants by second-instar larvae for the first 3 days. b Leaf consumption from the 4th to 6th day was recorded. Values are means ± SD. *P < 0.05. c Image of larvae on cotton boll. Larvae previously reared on leaves of nontransgenic control R15 or ds6-3 T2 plants for 10 days were transferred to cotton boll for another day