Method of Pest Control

The present invention provides a method for controlling Conogethes punctiferalis, which comprises contacting Conogethes punctiferalis with Cry1A protein. The present invention achieves the control of Conogethes punctiferalis by enabling plants to produce Cry1A protein in vivo, which is lethal to Conogethes punctiferalis. In comparison with current agricultural and chemical control methods, the method of the present invention can control Conogethes punctiferalis throughout the growth period of the plants and provide the plants with a full protection. Additionally, the method is stable, complete, simple, convenient, economical, pollution-free and residue-free.

DETAILED DESCRIPTION

Examples

Examples illustrating the method for pest control of the present invention are described as follows.

Acquisition and Synthesis of Cry1A Gene

I. Acquiring the Nucleotide Sequences of Cry1A

The amino acid sequence (818 amino acids) of pesticidal protein Cry1Ab-01 is shown as SEQ ID NO: 1 in the sequence list; the nucleotide sequence (2457 nucleotides) of Cry1Ab-01 encoding said amino acid sequence (818 amino acids) of pesticidal protein Cry1Ab-01 is shown as SEQ ID NO: 4 in the sequence list. The amino acid sequence (615 amino acids) of pesticidal protein Cry1Ab-02 is shown as SEQ ID NO: 2 in the sequence list; the nucleotide sequence (1848 nucleotides) of Cry1Ab-02 encoding the amino acid sequence (615 amino acids) of pesticidal protein Cry1Ab-02 is shown as SEQ ID NO: 5 in the sequence list.

The amino acid sequence (699 amino acids) of pesticidal protein Cry1Ah is shown as SEQ ID NO: 3 in the sequence list; the nucleotide sequence (2100 nucleotides) of Cry1Ah-01 encoding said amino acid sequence (699 amino acids) of pesticidal protein Cry1Ah is shown as SEQ ID NO: 6 in the sequence list; the nucleotide sequence (2100 nucleotides) of Cry1Ah-02 encoding the amino acid sequence (699 amino acids) of pesticidal protein Cry1Ah is shown as SEQ ID NO: 7 in the sequence list.

II. Acquiring Nucleotide Sequences of Cry-Like Genes

The nucleotide sequence (1818 nucleotides) of Cry1Fa encoding the amino acid sequence (605 amino acids) of pesticidal protein Cry1Fa, is shown as SEQ ID NO: 8 in the sequence list; the nucleotide sequence (1947 nucleotides) of Cry1Ie encoding the amino acid sequence (648 amino acids) of pesticidal protein Cry1Ie, is shown as SEQ ID NO: 9 in the sequence list.

III. Synthesizing the Above-Mentioned Nucleotide Sequences

The nucleotide sequences of Cry1Ab-01 (shown as SEQ ID NO: 4 in the sequence list), Cry1Ab-02 (shown as SEQ ID NO: 5 in the sequence list), Cry1Ah-01 (shown as SEQ ID NO: 6 in the sequence list), CryAh-02 (shown as SEQ ID NO: 7 in the sequence list), Cry1Fa (shown as SEQ ID NO: 8 in the sequence list) and Cry1Ie (shown as SEQ ID NO: 9 in the sequence list) are synthesized by Nanjing GenScript Ltd. 5′ end of the synthesized nucleotide sequence of Cry1Ab-01 (SEQ ID NO: 4) is connected to restriction site of NcoI, 3′ end of the synthesized nucleotide sequence of Cry1Ab-01 (SEQ ID NO: 4) is connected to restriction site of SpeI; 5′ end of the synthesized nucleotide sequence of Cry1Ab-02 (SEQ ID NO: 5) is connected to restriction site of NcoI, 3′ end of the synthesized nucleotide sequence of Cry1Ab-02 (SEQ ID NO: 5) is connected to restriction site of SwaI; 5′ end of the synthesized nucleotide sequence of Cry1Ah-01 (SEQ ID NO: 6) is connected to restriction site of AscI, 3′ end of the synthesized nucleotide sequence of Cry1Ah-01 (SEQ ID NO: 6) is connected to restriction site of SpeI; 5′ end of the synthesized nucleotide sequence of Cry1Ah-02 (SEQ ID NO: 7) is connected to restriction site of AscI, 3′ end of the synthesized nucleotide sequence of Cry1Ah-02 (SEQ ID NO: 7) is connected to restriction site of SpeI; 5′ end of the synthesized nucleotide sequence of Cry1Fa (SEQ ID NO: 8) is connected to restriction site of AscI, 3′ end of the synthesized nucleotide sequence of Cry1Fa (SEQ ID NO: 8) is connected to restriction site of BamHI; 5′ end of the synthesized nucleotide sequence of Cry1Ie (SEQ ID NO: 9) is connected to restriction site of NcoI, 3′ end of the synthesized nucleotide sequence of Cry1Ie (SEQ ID NO: 9) is connected to restriction site of SwaI.

Construction of Recombinant Expression Vectors and Transformation the Same intoAgrobacterium

As shown inFIG. 1, the synthesized nucleotide sequence of Cry1Ab-01 was ligated with cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600) according to manufacturer's protocol to generate the recombinant cloning vector DBN01-T. (Note: Amp represents Ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the start codon of LacZ; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Cry1Ab-01 is the nucleotide sequence of Cry1Ab-01 (SEQ ID NO: 4); and MCS is a multi-cloning site).

The next step was to transform the recombinant cloning vector DBN01-T into competent cells T1 ofE. coli(Transgen, Beijing, China, CAT: CD501) through a heat-shock method. Specifically, 50 μl competent cells T1 ofE. coliwere mixed with 10 μl plasmid DNA (the recombinant cloning vector DBN01-T), incubated in a water bath at 42° C. for 30 seconds and then in a water bath at 37° C. for 1 hour (in a shaker at 100 rpm). The mixture was then grew overnight on a LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, the pH value was adjusted to 7.5 with NaOH) with Ampicillin (100 mg/l), of which the surface was coated with IPTG (isopropyl-thio-β-D-galactoside) and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). White colonies were picked up and cultured further at 37° C. overnight in LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, Ampicillin 100 mg/L, pH value was adjusted to 7.5 with NaOH). The plasmids were extracted by an alkaline method. Specifically, the cultured bacteria in the medium were centrifuged at 12000 rpm for 1 min. The supernatant was discarded and the precipitated cells were resuspended in 100 μl ice-cold solution 1 (25 mM Tris-HCl, 10 mM EDTA (ethylenediamine tetraacetic acid), 50 mM glucose, pH8.0). Following the addition of 150 μl of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)), the tube was inverted for four times and placed on ice for 3-5 min. 150 μl ice-cold solution III (4 M potassium acetate, 2 M acetic acid) was added to the mixture, mixed immediately and thoroughly and then placed on ice for 5-10 min, followed by a centrifuge at 12000 rpm for 5 min at 4° C. The supernatant was added into 2 volumes of anhydrous ethanol, mixed thoroughly and then incubated for 5 min at room temperature. The mixture was centrifuged at 12000 rpm for 5 min at 4° C. and the supernatant was discarded. The pellet was washed with 70% (V/V) ethanol and then air dried, followed by adding 30 μl of TE (10 mM Tris-HCl, 1 mM EDTA, PH 8.0) containing RNase (20 μg/ml) to dissolve the pellet and digesting RNA in a water bath at 37° C. for 30 min. The plasmids obtained were stored at −20° C. before use.

The extracted plasmids were identified by KpnI and BglI digestion, and positive clones were further verified by sequencing. The results showed that, the nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleotide sequence of Cry1Ab-01 shown as SEQ ID NO: 4 in the sequence list, indicating the proper insertion of the nucleotide sequence of Cry1Ab-01.

As the above-mentioned method for the construction of the recombinant cloning vector DBN01-T, the synthesized nucleotide sequence of Cry1Ab-02 (shown as SEQ ID NO: 5) was ligated with cloning vector pGEM-T to generate the recombinant cloning vector DBN02-T. The proper insertion of the nucleotide sequence Cry1Ab-02 in the recombinant vector DBN02-T was verified by enzymatic digestion and sequencing.

As the above method for the construction of the recombinant cloning vector DBN01-T, the synthesized nucleotide sequence of Cry1Ah-01 (shown as SEQ ID NO: 6) was ligated with cloning vector pGEM-T to generate the recombinant cloning vector DBN03-T. The proper insertion of the nucleotide sequence Cry1Ah-01 in the recombinant vector DBN03-T was verified by enzymatic digestion and sequencing.

As the above method for the construction of the recombinant cloning vector DBN01-T, the synthesized nucleotide sequence of Cry1Ah-02 (shown as SEQ ID NO: 7) was ligated with cloning vector pGEM-T to generate the recombinant vector DBN04-T. The proper insertion of the nucleotide sequence Cry1Ah-02 in the recombinant vector DBN04-T was verified by enzymatic digestion and sequencing.

As the above method for the construction of the recombinant cloning vector DBN01-T, the synthesized nucleotide sequence of Cry1Fa (shown as SEQ ID NO: 8) was ligated with cloning vector pGEM-T to generate the recombinant vector DBN05-T. The proper insertion of the nucleotide sequence Cry1Fa in the recombinant vector DBN05-T was verified by enzymatic digestion and sequencing.

As the above method for the construction of the recombinant cloning vector DBN01-T, the synthesized nucleotide sequence of Cry1Ie (shown as SEQ ID NO: 9) was ligated with cloning vector pGEM-T to generate the recombinant vector DBN06-T. The proper insertion of the nucleotide sequence Cry1Ie in the recombinant vector DBN06-T was verified by enzymatic digestion and sequencing.

II. Constructing Recombinant Expression Vectors Comprising Cry1A Gene

Methods for constructing vectors by conventional enzymatic digestion are well known in the art. As shown inFIG. 2, the recombinant cloning vector DBN01-T and expression vector DBNBC-01 (Vector backbone: pCAMBIA2301 (available from CAMBIA institution)) were digested respectively by the restriction enzymes NcoI and SpeI; and the resulting fragment of the nucleotide sequence of Cry1Ab-01 was then inserted into the digested expression vector DBNBC-01 between NcoI and SpeI sites to generate the recombinant expression vector DBN100124. (Note: Kan represents kanamycin gene; RB represents right border; Ubi represents the promoter of maize ubiquitin gene (SEQ ID NO: 10); Cry1Ab-01 represents the nucleotide sequence of Cry1Ab-01 (SEQ ID NO: 4); Nos represents the terminator of nopaline synthase gene (SEQ ID NO: 11); PMI represents phosphomannose isomerase gene (SEQ ID NO: 12); and LB represents left border).

The recombinant expression vector DBN100124 was transformed into competent cells T1 ofE. colithrough a heat-shock method. Specifically, 50 μl competent cells T1 ofE. coliwere mixed with 10 μl plasmid DNA (the recombinant expression vector DBN100124), incubated in a water bath at 42° C. for 30 seconds and then in a water bath at 37° C. for 1 hour (in a shaker at 100 rpm). The mixture was then grew at 37° C. for 12 hours on a LB plate (tryptone 10 g/L, yeast extract 5 g/L. NaCl 10 g/L, agar 15 g/L, the pH value was adjusted to 7.5 with NaOH) with 50 mg/L Kanamycin. White colonies were picked up and cultured further at 37° C. overnight in LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, Kanamycin 50 mg/L, the pH value was adjusted to 7.5 with NaOH). The plasmids were extracted by an alkaline method. Enzymatic digestion with NcoI and SpeI was used to identify the extracted plasmids, and positive clones were further verified by sequencing. The results showed that, the nucleotide sequence inserted into the recombinant expression vector DBN100124 between NcoI and SpeI sites was Cry1Ab-01 shown as SEQ ID NO: 4 in the sequence list.

As the above method for the construction of the recombinant expression vector DBN100124, the recombinant cloning vector DBN02-T and DBN05-T were enzymatically digested by NcoI and SwaI, AscI and BamHI respectively to generate the nucleotide sequences of Cry1Ab-02 and Cry1Fa, which were inserted into the expression vector DBNBC-01 to obtain the recombinant expression vector DBN100075. As verified by enzymatic digestion and sequencing, the recombinant expression vector DBN100075 included the nucleotide sequences of Cry1Ab-02 and Cry1Fa shown in SEQ ID NO: 5 and SEQ ID NO: 8 in the sequence list.

As the above method for the construction of the recombinant expression vector DBN100124, the recombinant cloning vector DBN03-T was enzymatically digested by AscI and SpeI to generate the nucleotide sequence of Cry1Ah-01, which was inserted into the expression vector DBNBC-01 to obtain the recombinant expression vector DBN100071. As verified by enzymatic digestion and sequencing, the nucleotide sequence inserted into the recombinant expression vector DBN100071 between AscI and SpeI sites was Cry1Ah-01.

As the above method for the construction of the recombinant expression vector DBN100124, the recombinant cloning vector DBN04-T and DBN06-T were enzymatically digested by AscI and SpeI, NcoI and SwaI respectively to generate the nucleotide sequences of Cry1Ah-02 and Cry1Ie, which were inserted into the expression vector DBNBC-01 to obtain the recombinant expression vector DBN100147. As verified by enzymatic digestion and sequencing, the recombinant expression vector DBN100147 included the nucleotide sequences of Cry1Ah-02 and Cry1Ie shown in SEQ ID NO: 7 and SEQ ID NO: 9 in the sequence list.

III. Recombinant Expression Vectors were Transformed intoAgrobacterium

The correctly constructed recombinant expression vectors of DBN100124, DBN100075, DBN100071 and DBN100147 were transformed intoAgrobacteriumLBA4404 (Invitrogen, Chicago, USA; Cat No: 18313-015) through a liquid nitrogen method, respectively. Specifically, 100 μLAgrobacteriumLBA4404 and 3 μL plasmid DNA (the recombinant expression vector from DBN100124, DBN100075, DBN100071 and DBN100147) were placed in liquid nitrogen for 10 minutes, followed by incubation in a water bath at 37° C. for 10 minutes. The transformedAgrobacteriumLBA4404 were inoculated in a LB tube and then cultured at 28° C., 200 rpm for 2 hours. Subsequently, the culture was applied to a LB plate containing 50 mg/L Rifampicin and 100 mg/L Kanamycin until positive individual clonies grew. The individual clonies were picked for further culture to extract plasmids. The recombinant expression vectors were identified by enzymatic digestion, that is, the recombinant expression vector DBN100124 and DBN100075 were digested with restriction enzymes AhdI and XbaI, and the recombinant expression vector DBN100071 and DBN100147 were digested with restriction enzymes StyI and BglII, indicating the correct construction of the recombinant expression vectors, DBN100124, DBN100075, DBN100071 and DBN100147.

Acquisition and Verification of Maize Plants Transformed with Cry1A Gene

I. Generation and Identification of Maize Plants Transformed with Cry1A Gene

According to the conventionalAgrobacteriuminfection method, the sterile cultured immature embryos of Maize Z31 were cultured withAgrobacteriumstrains obtained in III, Example 2, so as to transform T-DNAs in the recombinant expression vectors DBN100124, DBN100075, DBN100071 and DBN100147 (comprising the promoter sequence of maize Ubiquitin gene, the nucleotide sequences of Cry1Ab-01, Cry1Ab-02, Cry1Ah-01, Cry1Ah-02, Cry1Fa and Cry1Ie, PMI gene and the terminator sequence of Nos) into the maize genome, generating the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence of Cry1Ah-02-Cry1Ie. The wild-type maize plants were used as control.

The process ofAgrobacterium-mediated transformation of maize was briefly described as follows. The immature embryos isolated from the maize were contacted with theAgrobacteriumsuspension, whereby the nucleotide sequences of Cry1Ab-01, Cry1Ah-02-Cry1Fa, Cry1Ah-01 and/or Cry1Ah-02-Cry1Ie were delivered into at least one cell of either immature embryo byAgrobacterium(step 1: Infection). In this step, the immature embryos were preferably immersed inAgrobacteriumsuspension (OD660=0.4-0.6, infection medium (MS salt 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 68.5 g/L, glucose 36 g/L, Acetosyringone (AS) 40 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, pH 5.3)) to initiate inoculation. The immature embryos were cultured withAgrobacteriumfor a period of time (3 days) (step 2: Co-culture). Preferably, after the step of infection, the immature embryos were cultured on a solid medium (MS salt 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, Acetosyringone (AS) 100 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After the co-culture step, a “recovery” step is optional, wherein there is at least an antibiotic known as inhibiting the growth ofAgrobacterium(Cephalosporins) and no selection agents for plant transformants in the recovery medium (MS salt 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 30 g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8) (step 3: Recovery). Preferably, the immature embryos were cultured on the solid medium with an antibiotic but without selection agents to eliminateAgrobacteriumand provide a recovery period for transformed cells. Next, the inoculated immature embryos were cultured on the medium with a selection agent (mannose) and the growing transformed calluses were selected (step 4: Selection). Preferably, the immature embryos were cultured on a solid selection medium with a selection agent (MS salt 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 5 g/L, mannose 12. 5 g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8), which resulted in a selective growth of transformed cells. Further, the calluses regenerated into plants (step 5: Regeneration). Preferably, the calluses grown on the medium with the selection agent were cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate plants.

The selected resistant calluses were transferred onto the MS differentiation medium (MS salt 4.3 g/L, MS vitamins, casein 300 mg/L, sucrose 30 g/L, 6-benzyladenine 2 mg/L, mannose 5 g/L, agar 8 g/L, pH 5.8), and cultured under 25° C. for differentiation. The differentiated seedlings were transferred onto the MS rooting medium (MS salt 2.15 g/L, MS vitamins, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, agar 8 g/L, pH 5.8), and cultured under 25° C. till the height of about 10 cm. The seedlings were then transferred into a greenhouse and grew to fructify. During the culture in the greenhouse, the seedlings were incubated at 28° C. for 16 hours and then incubated at 20° C. for 8 hours each day.

II. Verification of Maize Plants Transformed with Cry1A Gene by TaqMan Method

Using about 100 mg of the leaves from each of the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence of Cry1Ah-02-Cry1Ie as samples, the genomic DNA was extracted with DNeasy Plant Maxi Kit of Qiagen, and the copy numbers of Cry1A, Cry1F and Cry1Ie genes were determined by a fluorescence quantitative PCR assay with Taqman probe. The wild-type maize plants were analyzed as control according to the above-mentioned method. The experiments were repeated for 3 times and the results were averaged.

The detailed protocol for determining the copy number of Cry1A, Cry1F and Cry1Ie genes was as follows:

Step 11: 100 mg of the leaves from each of the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence of Cry1Ah-02-Cry1Ie, and that of the wild-type maize plants were sampled and homogenized in a mortar with liquid nitrogen. Each sample was in triplicate.

Step 12: The genomic DNA of the above-mentioned samples was extracted with DNeasy Plant Maxi Kit of Qiagen, and the detailed method refers to the manufacturer's protocol.

Step 13: NanoDrop 2000 (Thermo Scientific) was employed to measure genomic DNA concentrations of the above-mentioned samples.

Step 14: The concentrations of genomic DNA of the above-mentioned samples were adjusted to the same in a range of 80-100 ng/μl.

Step 15: The copy numbers of the samples were determined by a fluorescence quantitative PCR method with Taqman probe. A sample that had a known copy number was used as standard, and a sample from the wild-type maize plants was used as control. Each sample was triplicated and the results were averaged. The primers and probes used in the fluorescence quantitative PCR method are as follows.

The following primers and probes were used for detecting the nucleotide sequence of Cry1Ab-01:

Primer 1 (CF1): CGAACTACGACTCCCGCAC, shown as SEQ ID NO: 13 in the sequence list;
Primer 2 (CR1): GTAGATTTCGCGGGTCAGTTG, shown as SEQ ID NO: 14 in the sequence list;
Probe 1 (CP1): CTACCCGATCCGCACCGTGTCC, shown as SEQ ID NO: 15 in the sequence list.

The following primers and probes are used for detecting the nucleotide sequence of Cry1Ab-02:

Primer 3 (CF2): TGCGTATTCAATTCAACGACATG, shown as SEQ ID NO: 16 in the sequence list;
Primer 4 (CR2): CTTGGTAGTTCTGGACTGCGAAC, shown as SEQ ID NO: 17 in the sequence list;
Probe 2 (CP2): CAGCGCCTTGACCACAGCTATCCC, shown as SEQ ID NO: 18 in the sequence list;

The following primers and probes are used for detecting the nucleotide sequence of Cry1Fa:

Primer 5 (CF3): CAGTCAGGAACTACAGTTGTAAGAGGG, shown as SEQ ID NO: 19 in the sequence list;
Primer 6 (CR3): ACGCGAATGGTCCTCCACTAG, shown as SEQ ID NO: 20 in the sequence list;
Probe 3 (CP3): CGTCGAAGAATGTCTCCTCCCGTGAAC, shown as SEQ ID NO: 21 in the sequence list;

The following primers and probes are used for detecting the nucleotide sequence of Cry1Ah-01:

Primer 7 (CF4): ATCGTGAACAACCAGAACCAGTG, shown as SEQ ID NO: 22 in the sequence list;
Primer 8 (CR4): CTCCAGGATCTCGATCTCCG, shown as SEQ ID NO: 23 in the sequence list;
Probe 4 (CP4): CGTGCCGTACAACTGCCTGAACAACC, shown as SEQ ID NO: 24 in the sequence list;

The following primers and probes are used for detecting the nucleotide sequence of Cry1 Ah-02:

Primer 9 (CF5): TCATTTGGGGCTTCGTCG, shown as SEQ ID NO: 25 in the sequence list;
Primer 10 (CR5): TGATTGATCAGCTGCTCAACCT, shown as SEQ ID NO: 26 in the sequence list;
Probe 5 (CP5): CCAGTGGGATGCGTTCCTCGCTC, shown as SEQ ID NO: 27 in the sequence list;

The following primers and probes are used for detecting the nucleotide sequence of Cry1Ie:

Primer 11 (CF6): GAGCATTGATCCTTTCGTCAGTG, shown as SEQ ID NO: 28 in the sequence list;
Primer 12 (CR6): CAAAGTACCGAGGATCTTACCAGC, shown as SEQ ID NO: 29 in the sequence list;
Probe 6 (CP6): CCTCCACAATCCAAACGGGCATCG, shown as SEQ ID NO: 30 in the sequence list.

The 50× mixture of primers/probes, containing 45 μl of 1 mM each primer, 50 μl of 100 μM probe and 860 μl of 1×TE buffer, was stored in an amber tube at 4° C.

PCR Conditions were as Follows:

The data were analyzed by SDS2.3 software (Applied Biosystems).

As shown by the results, the nucleotide sequences of Cry1Ab-01, Cry1Ab-02-Cry1Fa, Cry1Ah-01 and Cry1Ah-02-Cry1Ie were all integrated into the genome of the detected maize plants. The maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 as well as the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie had obtained a single copy of Cry1A, Cry1F and/or Cry1Ie gene in the respective transgenic maize plants.

Detection of Pesticidal Proteins in the Transgenic Maize Plants

I. Detection of the Pesticidal Protein Contents in the Transgenic Maize Plants

Solutions involved in this experiment are as follows:

3 mg of fresh leaves from each of the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 as well as the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie were sampled and homogenized with liquid nitrogen, followed by the addition of 800 μl extraction buffer. The mixture was centrifuged at 4000 rpm for 10 min, then the supernatant was diluted 40-fold with the extraction buffer and 80 μl of diluted supernatant was used for ELISA test. Due to the high identity between Cry1Ah amino acid sequence and Cry1Ab amino acid sequence, the antibody against Cry1Ab can be applied for pesticidal protein Cry1Ah. ELISA (enzyme-linked immunosorbent assay) kit (ENVIRLOGIX Company, Cry1Ab/Cry1Ac kit) was employed to determine the ratio of the pesticidal protein (Cry1Ab and Cry1Ah) content divided by the weight of the fresh leaves. The detailed method refers to the manufacturer's protocol.

Meanwhile, the wild-type maize plants and the non-transgenic maize plants identified by Taqman were used as controls, and the determination followed the above-described methods. For three lines transformed with Cry1Ab-01 (S1, S2 and S3), three lines transformed with Cry1Ab-01-Cry1Fa (S4, S5 and S6), three lines transformed with Cry1Ah-01 (S7, S8 and S9), three lines transformed with Cry1Ah-02-Cry1Ie (S10, S11 and S12), one line identified as non-transgenic plant (NGM) by Taqman and one line as wild type (CK), three plants for each line were used and each plant was repeated six times.

Experimental results of the pesticidal protein (Cry1Ab protein) contents in the transgenic plants were shown in Table 1. Experimental results of the pesticidal protein (Cry1Ah protein) contents in the transgenic plants were shown in Table 2. The ratios of the averaged expressions of the pesticidal protein (Cry1Ab) divided by the weight of the fresh leaves in the maize plants transformed with the nucleotide sequence of Cry1Ab-01 and Cry1Ab-01-Cry1Fa were determined as 8536.2 and 8234.7, respectively; the ratios of the averaged expressions of the pesticidal protein (Cry1Ah) divided by the weight of the fresh leaves in the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and Cry1Ah-02-Cry1Ie were determined as 5374.3 and 5382.2, respectively. These results indicate that Cry1Ab and Cry1Ah proteins present a high and stable expression in the transgenic maize plants.

TABLE 1Average amount of Cry1Ab protein expressedin the transgenic maize plantsAmount of Cry1Ab proteinAmount of Cry1Ab proteinexpressed in each plant (ng/g)expressed in each kind(repeated six times per plant)of lines (ng/g)Line123Average amount (ng/g)S17160.210444.49080.88536.2S28534.48581.27330.2S38817.49185.77691.2S47088.49837.510626.48234.7S59866.76863.34222.4S69912.17724.17970.9NGM−1.70−1.00CK0−4.22.30

TABLE 2Average amount of Cry1Ah protein expressedin the transgenic maize plantsAmount of Cry 1 Ah proteinAmount of Cry1Ah proteinexpressed in single plant (ng/g)expressed in each kind(repeated six times per plant)of lines (ng/g)line123Average amount (ng/g)S75220.55520.25550.65374.3S85130.45249.35486.5S95205.45437.55568.1S105305.35374.95653.85382.2S115136.05229.55546.8S125240.35502.25450.7NGM−12.680−11.260CK0−15.13−13.210

II. Detection of Pest Resistance of the Transgenic Maize Plants

The maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01, the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie, the wild-type maize plants and the non-transgenic maize plants identified by Taqman were detected for their resistance toConogethes punctiferalis.

Fresh leaves of the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01, the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie, the wild-type maize plants and the maize plants identified as non-transgenic plants (V3-V4 stage) by Taqman were sampled, respectively. The leaves were rinsed with sterile water and water on the leaves was dried up by gauze. The veins of the leaves were removed, and the leaves were cut into stripes of approximately 1 cm×4 cm or 1 cm×2 cm. One or two stripes of the leaves were placed on filter paper wetted with distilled water on the bottom of round plastic Petri dishes. Ten heads ofConogethes punctiferalis(newly hatched larvae) were putted into each dish, and the dishes with pests were covered with lids and placed at 25-28° C., relative humidity of 70%-80% and photoperiod (light/dark) 16:8 for 3 days. According to three indicators, the developmental progress, mortality and leaf damage rate of theConogethes punctiferalis's larvae, the resistance score was acquired: score=100×mortality+[100×mortality+90×(the number of newly hatched pests/the total number of inoculated pests)+60×(the number of newly hatched−the number of negative control pests/the total number of inoculated pests)+10×(the number of negative control pests/the total number of inoculated pests)]+100×(1−leaf damage rate). For three lines transformed with the nucleotide sequence Cry1Ab-01 (S1, S2 and S3), three lines transformed with the nucleotide sequence Cry1Ab-02-Cry1Fa (S4, S5 and S6), three lines transformed with the nucleotide sequence Cry1Ah-01 (S7, S8 and S9), three lines transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie (S10, S11 and S12), one line identified as non-transgenic plants (NGM) by Taqman and one line as wild type (CK), three plants for each line were used and each plant was repeated six times. The results were shown in Table 3 as well asFIGS. 3 and 4.

As shown in Table 3, the scores of the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01, the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie were all around 280 or above, while the score of the wild-type maize plants was generally about 120 or less.

As shown inFIGS. 3 and 4, compared with the wild-type maize plants, the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie killed great amount of the newly hatchedConogethes punctiferalislarvae, and greatly suppressed the growth of small amount of survived larvae so that the larvae still remained in the newly hatched state after 3 days. Additionally, the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie only had a minor damage, presenting a very small amount of pinhole-like damages; the leaf damage rates were all about 3% or less.

Thus, it is proved that the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie were all showed high resistance toConogethes punctiferalis, which are sufficient to cause adverse effects on the growth ofConogethes punctiferalisso that they can be controlled.

The above results also showed that, the effective control ofConogethes punctiferaliswas resulted from Cry1A protein produced in the maize plants transformed with the nucleotide sequence of Cry1Ab-01, the maize plants transformed with the nucleotide sequence of Cry1Ab-02-Cry1Fa, the maize plants transformed with the nucleotide sequence of Cry1Ah-01 and the maize plants transformed with the nucleotide sequence Cry1Ah-02-Cry1Ie. Similar transgenic plants capable of expressing Cry1A could be produced to controlConogethes punctiferalis, based on the same toxic effect of Cry1A protein onConogethes punctiferalis. Cry1Ab proteins described in the present invention include, but not limited to, the Cry1A proteins shown in the specific embodiments by the specific sequences. The transgenic plants can also generate at least one kind of a second pesticidal protein that is different from Cry1A, e.g., Cry1Ie, Cry1Fa, Vip3A and Cry1Ba proteins.

In conclusion, the present invention can controlConogethes punctiferalisby enabling the plants to produce Cry1A protein in vivo, which is toxic toConogethes punctiferalis. In comparison with current agricultural and chemical control methods, the method described by the present invention can controlConogethes punctiferalisthroughout the growth period of the plants and provide a full protection to the plants. Additionally, the method is stable, complete, simple, convenient, economical, pollution-free and residue-free.

Finally it should be noted that, the above embodiments merely illustrate the technical solutions of the present invention and it is not limited to those, although the preferred embodiments with reference to the present invention have been described in detail, people of ordinary skill in the art should appreciate that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the invention.