WO2017088480A1 - Artificially synthesized insect-resistant gene and application thereof - Google Patents

Abstract

Provided is an artificially synthesized insect-resistant gene and application thereof. The insect-resistant gene is a gene prepared by optimizing a cry1Ah gene according to corn codon preference, and specifically, is one of following (a)-(c): (a) a deoxyribonucleic acid (DNA) molecule represented by a nucleic acid sequence indicated as sequence 3 in a sequence table; (b) a DNA molecule represented by a nucleic acid sequence indicated as positions 1-2007 of sequence 3 in the sequence table; and (c) a protein represented by coding sequence 9 and having a nucleic acid sequence that is 98% similar to sequence 3 or positions 1-2007 of sequence 3 in the sequence table.

Description

Artificial synthetic insect resistance gene and application thereof Technical field

The invention relates to a synthetic insect-resistant gene and application thereof, in particular to an insect-resistant gene designed and synthesized according to a corn preference codon and its application.

Background technique

At present, exogenous genes such as Bt and EPSPS used in plant transgenic breeding are mostly derived from prokaryotes. Due to the characteristics of prokaryotic genes themselves, such as 1) higher AT content, more than 60%, resulting in mRNA expression of genes in plants. The body is easily degraded; 2) there are intron cutpoints and transcription terminator sequences similar to eukaryotic genes, resulting in incomplete transcription, abnormal mRNA cleavage, etc.; 3) codons and plant codons are quite different, resulting in Protein translation efficiency is reduced; 4) its structure is significantly different from eukaryotes such as plants, such as the 5'-UTR sequence without the eukaryotic gene and the polyA tailing sequence at the 3' end, resulting in the expression level of the gene in plants. low. For example, the wild-type insecticidal protein gene from Bacillus thuringiensis is expressed in a very low amount in plants, and its expressed toxic protein accounts for only 0.001% of the total protein or is almost undetectable.

Corn borer is the world's major corn pest, with annual corn loss due to corn borer damage of around 5%. China is a frequent and recurring area of corn borer. Since the 1970s, it has occurred almost every two years, with an annual loss of 3.8-6.4 million tons of corn, equivalent to the output of a medium-sized corn province.

Since the endogenous insect resistance of maize is controlled by multiple genes, and the target pests in the heart leaf stage and the target pest genes in the ear stage are independently inherited, the breeding of the anti-caries hybrids by conventional breeding methods is not only long-term, but also difficult to obtain The parent of two generations of corn borer. At the same time, the maize anti-caries gene is likely to be negatively correlated with high-yield traits. In the past 20 years, no significant progress has been made in the breeding of anti-caries in various countries.

Invention disclosure

It is an object of the present invention to provide a DNA molecule which is an insect resistance gene.

The DNA molecule provided by the present invention, named mcry1Ah, is any of the following (a) to (c):

(a) is a nucleotide sequence such as the DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;

(b) a nucleotide sequence such as the DNA molecule shown in positions 1 to 2007 of SEQ ID NO: 3 in the Sequence Listing;

(c) The nucleotide sequence is at least 98% identical to the position 1 to 2007 of Sequence 3 or Sequence 3 in the Sequence Listing, and encodes the protein of SEQ ID NO: 9.

An expression cassette, recombinant vector, recombinant host strain, recombinant cell line or transgenic plant containing the DNA molecule (mcry1Ah) is also within the scope of the present invention.

The expression cassette comprises the following elements 1)-3):

1) a promoter; 2) the DNA molecule which is transcribed by the promoter; 3) a transcription termination sequence.

Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Such as broccoli mosaic virus constitutive promoter 35S; tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid ester); heat shock promoter; tetracycline-inducible promoter; seed specific Sex promoters, such as the millet seed-specific promoter pF128, seed-loading protein-specific promoters, for example, peaulin, napin, oleosin, and soybean beta conglycin Promoter etc.

In one embodiment of the invention, the promoter is a Ubi promoter derived from maize and is a constitutive expression promoter, the sequence of which is specifically sequence 6 in the sequence listing, or at least 80% identity with sequence 6. And has a promoter function.

Transcriptional terminators useful in the present invention include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and Octoprine synthase terminator.

In one embodiment of the present invention, the transcription termination sequence is specifically a double termination sequence of PolyA plus T-NOS, as shown by sequence 7 in the sequence listing, or at least 80% identical to the position of sequence 7, and A sequence having a transcription termination function.

In one embodiment of the invention, the expression cassette further comprises an OMK sequence. The OMK sequence consists of a sequent ligation of the Ω sequence and the Kozak sequence, the sequence of which is specifically sequence 5 in the sequence listing, or at least 80% identity to sequence 5, and has enhancer function.

The Ω sequence and the Kozak sequence are derived from tobacco mosaic virus, an enhancer responsible for enhancing expression of the DNA molecule.

In one embodiment of the invention, the expression cassette consists of the Ubi promoter, the OMK sequence, the DNA molecule and the transcription termination sequence, which are sequentially joined, and named Ubi-OMK--mcry1Ah-PolyA -T-NOS. The sequence of Ubi-OMK-mcry1Ah-PolyA-T-NOS is shown in SEQ ID NO: 11 in the Sequence Listing.

The recombinant vector may be either a cloning vector or an expression vector, as needed; in one embodiment of the present invention, the recombinant vector is specifically pS3300-UMG2-UC2A. The sequence of pS3300-UMG2-UC2A is shown as shown in SEQ ID NO: 12 in the Sequence Listing.

Among them, the sequence 3 consists of 2010 nucleotides, and at the end are two stop codons, which encode the cry1Ah protein shown by the sequence 9 in the sequence listing. Sequence 5 consists of 67 nucleotides. Sequence 6 consists of 2009 nucleotides. Sequence 7 consists of 488 nucleotides. Sequence 9 consists of 668 amino acids. Sequence 11 consists of 4605 nucleotides, wherein positions 1-2009 are Ubi promoter sequences, positions 2010-2076 are OMK sequences, positions 2083-4092 are mcry1Ah sequences, and positions 4118-4605 are transcription termination sequences. Sequence 12 consists of 15099 nucleotides, wherein positions 151-2159 are Ubi promoter sequences, positions 2160-2226 are OMK sequences, positions 2233-4242 are mcry1Ah sequences, and positions 4268-4755 are transcription termination sequences.

In one embodiment of the invention, the recombinant host strain is an Agrobacterium carrying the DNA molecule, such as LBA4404.

The recombinant cell line may be a eukaryotic cell or a prokaryotic cell, such as a plant cell line.

The transgenic plants (such as corn) include seeds, callus, whole plants, and cells.

RNA obtained by transcription of the DNA molecule (mcry1Ah) is also within the scope of the present invention.

The use of the DNA molecule (mcry1Ah) or the expression cassette or the recombinant vector for breeding insect-resistant transgenic maize is also within the scope of the present invention. The transgenic corn includes seeds, callus, whole plants, and cells.

The use of the DNA molecule (mcry1Ah) or the expression cassette or the recombinant vector for increasing the expression level of corn cry1Ah protein is also within the scope of the present invention. The cry1Ah protein is a protein represented by SEQ ID NO:9 in the Sequence Listing.

Still another object of the present invention is to provide a method of cultivating insect-resistant transgenic corn.

The method for cultivating the insect-resistant transgenic maize provided by the present invention may specifically comprise the steps of: introducing the DNA molecule (mcry1Ah) into the corn of interest to obtain transgenic corn expressing the DNA molecule (mcry1Ah); the transgenic corn The insect resistance is improved compared to the corn of interest.

Wherein introducing the DNA molecule (mcry1Ah) into the corn of interest is accomplished by introducing the recombinant expression vector into the corn of interest.

The recombinant expression vector can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and the like.

It is also within the scope of the present invention to use the insect-resistant transgenic maize cultivated by the method for breeding insect-resistant transgenic maize provided by the present invention. The transgenic corn includes seeds, callus, whole plants, and cells.

In one embodiment of the invention, the corn is specifically corn Hi-II.

In the present invention, the insect resistance is specifically resistant to corn mash.

DRAWINGS

Figure 1 is a plasmid map of vectors pS3300-UMCT-UMG2, pS3300-UMG2-UCA and pS3300-UMG2-UC2A. Wherein, A is a plasmid map of pS3300-UMCT-UMG2; B is a plasmid map of pS3300-UMG2-UCA; and C is a plasmid map of pS3300-UMG2-UC2A.

Figure 2 is a PCR detection map of the transgenic maize cry1Ah gene, mcry1Ah gene and mG2-aroA gene. Among them, A is the PCR detection map of transgenic maize cry1Ah gene; 1: CK ⁺ (plasmid positive control); 2: Marker; 3: CK ^- (no DNA added); 4: CK ^- (non-GM corn); : Turn the cry1Ah gene corn event. B is the PCR detection map of the transgenic maize mcry1Ah gene; 1: CK ⁺ (plasmid positive control); 2: Marker; 3: CK ^- (no DNA added); 4: CK ^- (non-transgenic corn); 5-24: mcry1Ah gene corn event. C is the PCR detection map of the transgenic maize mG2-aroA gene; 1: CK ⁺ (plasmid positive control); 2: Marker; 3: CK ^- (no DNA added); 4: CK ^- (non-transgenic corn); 5-14 : Turn the cry1Ah gene corn event; 15-24: Turn the mcry1Ah gene corn event.

Figure 3 shows the results of tolerance test of glyphosate in transgenic maize seedlings.

Figure 4 shows the results of insect resistance testing in transgenic corn fields. Among them, A is a transgenic mCry1Ah maize line in Hainan, and B is a non-transgenic control line in Hainan.

The best way to implement the invention

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1. Obtainment of codon-optimized insect-resistant genes

This example encodes the first 667 amino acid sequence (the first 667 amino acid sequence) of the protein according to the full-length gene of cry1Ah (nucleotide sequence as shown in SEQ ID NO: 1 in the sequence listing, see Chinese Patent Application: 200410009918.9). As shown in SEQ ID NO:8 in the sequence listing, the corresponding nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing. Under the premise of ensuring the amino acid sequence is unchanged, the corn preference codon is firstly used to artificially optimize the cry1Ah gene. Try to avoid the use of rare corn codons and adjust the frequency of codon usage (Table 1). On this basis, the typical AT-rich sequence present in the DNA sequence which is unstable to the transcript of the plant gene is removed, and the hairpin structure is removed, and the new nucleotide sequence obtained is the sequence 3 in the sequence listing. Sequence 3 shares only 70% homology with the 1-2001 bp (SEQ ID NO: 2) of the cry1Ah gene (SEQ ID NO: 1), while the G+C content increases from 37.4% to 56.3%. At the same time, in order to facilitate cloning, three nucleotides of GGC were added after the start codon. The gene shown in SEQ ID NO:3 is a codon-optimized insect-resistant gene, which is named mcry1Ah. The frequency of use of the codon in the cry1Ah gene and the mcry1Ah gene is shown in Table 1. To facilitate cloning, the inventors introduced a SpeI cleavage site at the 5' end of SEQ ID NO: 3 and introduced an AatII cleavage site at the 3' end. The final sequence is shown in SEQ ID NO: 4 in the Sequence Listing. Sequence 7 is the 7th - 2016th bit of Sequence 4. The position 1-2001 of the sequence 1 (ie, sequence 2) encodes the protein of SEQ ID NO:8 in the sequence listing, and the SEQ ID NO:3 and SEQ ID NO:7-2016 both encode the protein of SEQ ID NO:9 (compared to the protein shown in SEQ ID NO:8). The second amino acid residue of sequence 9 from the N-terminus was added, and the protein was named cry1Ah protein.

Table 1 Corn preferred codon standard

Example 2: Acquisition of mcry1Ah transgenic corn

I. Construction of recombinant expression vectors pS3300-UMG2-UCA and pS3300-UMG2-UC2A

In order to increase the expression level of the mcry1Ah gene (SEQ ID NO: 3) in the recipient organism, the inventors added an Ω sequence and a Kozak sequence at the 5' end of the mcry1Ah gene when constructing a recombinant expression vector for the mcry1Ah gene, Ω/Kozak sequence (abbreviation OMK) is shown in SEQ ID NO: 5 in the Sequence Listing. The Ω sequence is a translation-enhancing sequence derived from the coding region of the plant viral capsid protein gene, consisting of 67 bp, enriched in the TTAAC sequence, and has a UAUUUUUACAACAA sequence at the 5' end and four UUAC sequences, which are constructed during translation of the protein synthesis. Ribosome and rRNA binding sites. The Kozak sequence is a sequence encoding a ribosome-binding protein that facilitates translation of a foreign gene in a plant cell. The promoter uses the constitutive promoter Ubi promoter, the sequence of which is shown in SEQ ID NO:6 in the Sequence Listing. Furthermore, two consecutive stop codons were designed at the 3' end of the coding sequence, and a synthetic PolyA+T-NOS stable termination sequence was added. The PolyA+T-NOS sequence is shown in SEQ ID NO:7 in the Sequence Listing. Among them, PolyA has the function of maintaining mRNA stability, and the T-NOS termination sequence ensures accurate termination of translation.

The specific procedures for constructing the recombinant expression vectors pS3300-UMG2-UCA and pS3300-UMG2-UC2A carrying the cry1Ah and mcry1Ah genes are as follows:

a. SpeI and AatII digest the pS3300-UMCT-UMG2 plasmid (the plasmid map is shown as A in Figure 1), and recover large fragments.

The full sequence of the vector of the PS3300-UMCT-UMG2 plasmid is shown in SEQ ID NO: 13 in the Sequence Listing. This plasmid contains the glyphosate-tolerant gene mG2-aroA (SEQ ID NO: 10, see Chinese Patent Application: 201210107071.2).

b. Synthesize the cry1Ah gene (sequence 2) and the mcry1Ah gene (sequence 3), and add SpeI and AatII restriction sites at both ends to obtain the DNA fragment shown by "ACTAGT+sequence 2+TGA+GACGTC" and sequence 4 DNA fragments; the two DNA fragments were digested with SpeI and AatII and ligated with the large fragment recovered in step 1, to obtain recombinant expression vectors pS3300-UMG2-UCA and pS3300-UMG2-UC2A (the plasmid maps of the two plasmids were respectively As shown in B and C in Figure 1).

The structure of the recombinant expression vector pS3300-UMG2-UCA is described as follows: after replacing the small fragment between the restriction sites SpeI and AatII of the pS3300-UMCT-UMG2 plasmid with the DNA fragment shown by the sequence 2+TGA in the sequence listing Recombinant plasmid.

The structure of the recombinant expression vector pS3300-UMG2-UC2A was described as follows: a small fragment between the restriction sites SpeI and AatII of the pS3300-UMCT-UMG2 plasmid was replaced with a recombinant plasmid of the DNA fragment shown in SEQ ID NO:3 in the Sequence Listing. On this vector, the expression cassette containing the mcry1Ah gene was named Ubi-OMK-mCry1Ah-PolyA-T-NOS; the sequence of which is shown in SEQ ID NO: 11 in the Sequence Listing. Full of the carrier The sequence is as shown in SEQ ID NO: 12 in the Sequence Listing.

2. Recombinant expression vector transforms maize to obtain transgenic corn

1. Acquisition of corn conversion starting materials

The young ears of 9 to 13 days after pollination of corn Hi-II were taken, and the leaves were peeled off for surface disinfection. The young embryos are stripped from the sterilized young ears and washed once or twice in the infected culture solution (Formulation: Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1). spare.

2. Recombinant expression vector transforming Agrobacterium

The recombinant expression vectors pS3300-UMG2-UCA and pS3300-UMG2-UC2A prepared in the first step were transformed into Agrobacterium LBA4404, respectively (Reference: Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1).

Agrobacterium LBA4404, which was confirmed to be transferred to the recombinant expression vector pS3300-UMG2-UCA, was named LBA4404/pS3300-UMG2-UCA. Agrobacterium LBA4404, which was confirmed to be transferred to the recombinant expression vector pS3300-UMG2-UC2A, was named LBA4404/pS3300-UMG2-UC2A.

3. Agrobacterium transformation of maize immature embryos

The immature embryo washed with the infected culture solution in the above step 1 is placed in the bacterial solution of the Agrobacterium prepared in the above step 2 with an OD ₆₀₀ of about 0.3-0.5, left for 5 minutes, and then the young embryo is placed in the co-cultivation medium ( References: Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1), co-cultured for 3 days in the dark at about 20 ° C, with young embryos not subjected to Agrobacterium transformation as a control.

A control for transferring the pS3300-UMCT-UMG2 empty vector into the recipient maize immature embryos was also set up in the experiment.

4. Acquisition of genetically modified corn seedlings

The immature embryos co-cultured in the above step 3 were transferred to a selection medium (Reference: Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1), and a final concentration of 1 mM was added to the selection medium. Glyphosate is used as a selection pressure to screen and culture the transformed material, once every two weeks, until a crisp, yellowish and vigorously growing glyphosate resistant callus is grown.

The obtained glyphosate-resistant callus was transferred to an induction medium (Reference: Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1) to induce differentiation, and matured one month later. Embryoid body. Then, the embryoid bodies were placed on the MS medium to produce roots, that is, the regenerated seedlings of the T ₀ generation transgenic corn were obtained. Seed T ₀ transgenic corn seeds are obtained T ₁ generation transgenic maize mature, T ₁ generation transgenic maize inbred breeding continued obtain T ₂ seeds of transgenic maize. By analogy, seeds of T ₆ generation transgenic corn were obtained. T ₆ generation transgenic maize plants were obtained after seeding the seeds of the T ₆ generation transgenic corn.

5. Identification of T ₆ Generation Transgenic Maize Plants

The T ₆ generation transgenic maize plants were identified by PCR as follows:

First, the genomic DNA of the T ₆ generation transgenic maize plants were extracted separately, as follows:

1) Select 0.1-0.2g of young leaves of transgenic maize regenerated plants, grind in liquid nitrogen, and transfer to 1.5ml Eppendorf tube;

2) Add 0.7ml of CTAB solution (formulation: final concentration of Tris 100mM, final concentration of NaCl 1.4M, final concentration of EDTA 20mM, final concentration of CTAB 2% (w/v), final concentration of mercaptoethanol 0.1% (v/v)) , 60 ° C, 45 points Clock, every 10 minutes, mix upside down.

3) Add 0.7 ml of phenol:chloroform (1:1 ratio by volume), invert several times, centrifuge at 1000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. An equal volume of chloroform:isoamyl alcohol (24:1 by volume) was added, mixed, centrifuged at 1000 rpm for 5 minutes, and the supernatant was transferred to a new centrifuge tube.

4) Add an equal volume of isopropanol in a centrifuge tube, mix by inversion, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, wash once with 70% ethanol, drain, and dissolve in 50 μL of sterile water for PCR detection.

Next, PCR was performed using the genomic DNA of the above-mentioned T ₆ transgenic maize plants as a template. The specific operations are as follows:

1) Detection of the target gene (cry1Ah) in the corn plant transformed with cry1Ah gene (sequence 2), using non-transgenic maize as a negative control, using a reaction system without template addition as a blank control, and positive for pS3300-UMG2-UCA plasmid For the control, corn transferred into the pS3300-UMCT-UMG2 empty vector was used as a no-load control. The PCR amplification primers are as follows:

1Ah_F: 5'-TTAATTGATTTAATATGGGGATTTG-3' (positions 241-265 of sequence 2);

1Ah_R: 5'-ACACGCCCTGACCTAGTTGAG-3' (reverse complement of positions 1118 to 1138 of SEQ ID NO: 2).

The amplified product was 898 bp in length.

Reaction system (20 μL): DNA 1 μL (20-50 ng); 10×buffer 2 μL; MgCl ₂ (2.5 mM) 2 μL; dNTP (2.5 mM) 2 μL; Taq enzyme 0.2 μL; primer 10 μM; and sterile water to 20 μL. The amplification reaction conditions were: 94 ° C, 5 min pre-denaturation; 35 cycles (94 ° C, 1 min; 57 ° C, 1 min; 72 ° C, 1 min); 72 ° C extension for 10 min.

The detection results shown in FIG. 2 A, shown in lanes 5-24 20 cry1Ah gene into T ₆ transgenic corn plants were amplified target band size 898bp; and negative control (non-transgenic maize ), the blank control group and the empty control group did not amplify the target band. This result suggests Cry1Ah gene was integrated into the genome of transfected T ₆ generations cry1Ah maize genes.

2) Detection of the target gene (mcry1Ah) in maize plants transferred to mcry1Ah gene (sequence 3), non-transgenic maize as negative control, blank control system without template addition, and positive expression of pS3300-UMG2-UC2A plasmid For the control, corn transferred into the pS3300-UMCT-UMG2 empty vector was used as a no-load control. The PCR amplification primers are as follows:

1 mAh_F: 5'-CTCAGTCCTCAAGATTCAGACCTTCG-3' (positions 501-526 of SEQ ID NO: 3);

1 mAh_R: 5'-AAAGTTCTCAAGCACTGGGTTGGTGT-3' (reverse complement of positions 869-894 of SEQ ID NO: 3).

The amplified product was 394 bp in length.

The reaction system is the same as above. The amplification reaction conditions were: 94 ° C, 5 min pre-denaturation; 35 cycles (94 ° C, 1 min; 57 ° C, 1 min; 72 ° C, 1 min); 72 ° C extension for 10 min.

The detection results shown in FIG. 2 B, Lane 20 shown mcry1Ah 5-24 gene into T ₆ transgenic corn plants were amplified target band size 394bp; and negative control (non-transgenic maize ), the blank control group and the empty control group did not amplify the target band. This result suggests mCry1Ah gene was integrated into the genome of T ₆ Substitute mcry1Ah maize gene.

3) The maize gene transformed into the cry1Ah gene (sequence 2) and the mcry1Ah gene (sequence 3) were tested for the resistance gene mG2-aroA (sequence 10), and the non-transgenic maize was used as a negative control, with no template added. The system was a blank control, and the pS3300-UMG2-UC2A plasmid was used as a positive control, and the maize transformed into the pS3300-UMCT-UMG2 empty vector was used as a no-load control. The PCR amplification primers are as follows:

mG2_F: 5'-CCACCTGGCTCCAAGTCTATCA-3' (positions 142-163 of sequence 10);

mG2_R: 5'-GCGTCAACCTGTGCTCCAAA-3' (reverse complement of positions 715-734 of SEQ ID NO: 10).

The amplified product was 593 bp in length.

The reaction system is the same as above. The amplification reaction conditions were: pre-denaturation at 95 ° C for 5 min, denaturation at 94 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 40 min, 35 cycles, and finally extension at 72 ° C for 10 min.

As shown in Lanes 5-14 10 shown cry1Ah transferred gene detection result C as shown in FIG. 2 T ₆ transgenic corn plants and lanes 15-24 10 shown mcry1Ah gene into T ₆ transgenic maize The plants were amplified to obtain a target band of 593 bp; while the negative control group (non-transgenic maize) and the blank control group did not amplify the target band (the empty control group was amplified by carrying the mG2-aroA gene). Target strip). This result suggests mG2-aroA gene has been integrated into the T ₆ group cry1Ah transgenic maize genes or mcry1Ah.

Example 3: ELISA detection of cry1Ah protein expression in transgenic maize plants

Experimental Materials: Select 12 transgenic events were the same growth stage (five-leaf stage) T ₆ transgenic corn (cry1Ah gene transfer, gene transfer mcry1Ah, and transfer pS3300-UMCT-UMG2 empty vector) 10 each. At the same time, untransgenic corn was used as a negative control.

Sample treatment: 1g (fresh weight) leaves of each functional leaf (top three leaves) were mixed. After liquid nitrogen was ground, transfer to a 10ml centrifuge tube and add 3ml sample extract, shake vigorously, centrifuge at 4 °C for 1h, and take the supernatant. For the sample to be tested, spare.

The fresh weight is obtained by grinding the leaves with liquid nitrogen into a powder sample and adding the weight of the centrifuge tube containing the extract minus the weight of the centrifuge tube containing the extract before adding the powder sample.

The sample determination method is as follows:

1. Add the sample: Add 50 μL of the enzyme-labeled secondary antibody to each well of the ELISA plate (EnviroLogix product, product number: AP 003CRBS), and then quickly add the standard: dilute the cry1Ah protein standard sample solution to 3 μg/ml, then 2 Dilute 11 gradients (including 0 wells) and add 50 μL per well, repeating 3 times. 50 μL of the sample extract to be tested was repeated 3 times.

The method for preparing the cry1Ah protein standard is as follows: the DNA fragment shown in SEQ ID NO: 1 is ligated into the multiple cloning site of the prokaryotic expression vector pET-28a(+) to obtain the prokaryotic expression vector pET-28a-Cry1Ah expressing the Cry1Ah protein. . pET-28a-Cry1Ah was transformed into E. coli BL21, and its expression of cry1Ah protein was induced by IPTG. After collecting the cells, the cells were disrupted by ultrasonication, and the expressed protein was released into the extract. After purification with His tag protein purification kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0009A), cry1Ah protein was purified and subjected to SDS-PAGE electrophoresis. SDS-PAGE electrophoresis showed that the purity was about 95%, and the concentration of cry1Ah protein was determined to be 3.0 mg/ml by eppendorf protein nucleic acid analyzer biophotometer plus.

2, gently shake the sample per hole, to avoid sample spillage.

3. Incubate for 1-2 hours at room temperature while gently shaking at 200 rpm on a shaker.

4. Wash the plate: smash the sample, drop it twice in the newspaper, and then put it in the washing machine for 5 times.

5. Add 100 μl of substrate buffer to each well.

6. Protect from light and incubate for 15 to 30 minutes.

7. Add 100 μl of stop solution.

8. Colorimetric: Read on the plate, using a wavelength of 450 nm.

9. The concentration of the cry1Ah protein standard solution (ng/mL) was used as the X-axis, and the OD value of the obtained cry1Ah protein standard was measured as the Y-axis in step 8. The standard curve was drawn by EXCEL.

10. Substituting the OD value of the sample to be tested measured in step 8 into the standard curve equation drawn in the above step 9, calculating the cry1Ah protein content in the sample to be tested.

11. The content of cry1Ah protein in fresh weight = cry1Ah protein content in the sample to be tested × volume of sample to be tested / fresh weight of sample, unit: μg/g

12. The experiment was set up with 3 repetitions, taking the average of three experimental results, and the obtained standard curve has a standard curve equation of y=9035.2x+198.75 (R ² =0.9997).

13. The test results of the samples to be tested are shown in Table 2. It can be seen that the expression level of cry1Ah protein in T ₆ transgenic maize was significantly higher than that in non-transgenic maize (P<0.01), and the expression of cry1Ah protein in transgenic mcry1Ah maize. The amount was significantly higher than that of the trans-Cry1Ah gene (P<0.05). Into pS3300-UMCT-UMG2 empty vector T ₆ generations cry1Ah maize plant and corn protein expression in transgenic plants are not substantially the same, no significant difference. This result indicated that the expression of cry1Ah protein in transgenic maize was significantly improved after codon optimization.

Table 2 ELISA results of cry1Ah protein expression in transgenic plants

Note: The cry1Ah protein concentration unit is μg/g, which represents the micrograms of cry1Ah protein measured per gram of leaf (fresh weight).

Example 4: Field Herbicide Tolerance and Insect Resistance Test of Transgenic Maize Plants

I. Field herbicide tolerance test of transgenic corn plants

1, the test material: T ₆ and Substitute cry1Ah mcry1Ah gene maize plants, as well as into pS3300-UMCT-UMG2 empty vector T _6-generation corn plants, while setting the control non-transgenic corn plants.

2, test design: 5M line length, 3 lines, 3 repetitions, density: 60 × 35cm

3. Test treatment: Spray Roundup (containing 41% glyphosate, the recommended dosage in the field is 150-250ml/mu) according to the dosage of 800ml/mu.

4. Test treatment period: 5-6 leaf stage. The results of the experiment were observed after 7 days.

5, test results

As shown in Figure 3 (after 15 days of spraying the farm), (1) the mcry1Ah gene plant was treated with 800 ml/mu of Roundup, and no obvious phytotoxicity was observed. (2) After transfecting the cry1Ah gene plant, after 800ml/mu of Roundup treatment, there was no obvious phytotoxicity and the performance was normal. (3) Plants transferred to pS3300-UMCT-UMG2 empty vector, after 800ml/mu of Roundup treatment, had no obvious phytotoxicity and showed normal performance. (4) All non-transgenic plants have died.

2. Detection of insect resistance in transgenic corn plants in the field

1, the test material: T ₆ and Substitute cry1Ah mcry1Ah gene maize plants, as well as into pS3300-UMCT-UMG2 empty vector T _6-generation corn plants, while setting the control non-transgenic corn plants.

2, test design: 5M line length, 3 lines, 3 repetitions, density: 60 × 35cm

3. Test treatment: the transgenic plants that survived the spraying of glyphosate in step 1 (non-transgenic corn plants were not sprayed with glyphosate), and the newly hatched larvae (corn mash) per plant were 30-40. The head was inoculated into the corn heart with a brush, and after 3 days of inoculation, the second insect was caught, and the number of insects was the same as the first time. After 14 days, the damage degree of maize leaves and the survival rate of larvae were investigated. The investigation was carried out according to the technical specification of corn resistance identification NY/T1248.5.

4. Test treatment period: Artificial insects are carried out when the corn plants develop to the 8-10 leaf stage (small trumpet period), and the time of insect collection is selected in the morning or evening.

5, test results

As shown in Figure 4 and Table 3: (1) Transgenic mcry1Ah gene plants showed normal performance and no pest response during the late growth period; (2) plants transfected with cry1Ah gene and transferred into pS3300-UMCT-UMG2 empty vector and Non-transgenic plants are severely pests. This result indicated that the insect resistance of the corn transformed into the codon-optimized mcry1Ah gene was significantly improved.

Table 3 Identification results of insect resistance of transgenic plants

Industrial application

The present invention is directed to the problem that the expression of the prokaryotic insect resistance gene cry1Ah (SEQ ID NO: 1) is low in plants, and is optimized by using maize preference codons to maintain the stability of the RNA while maintaining the original amino acid sequence unchanged. Structures (such as polyA, repeats, AT and GC tandem repeats, RNA secondary structure, ribosome binding sites, etc.), increase GC content, etc., so that it is highly efficient and stable expression in maize. The experiment confirmed that the transgenic maize with the synthetic insect resistance gene (sequence 3) provided by the invention has a significantly increased expression level of cry1Ah protein, and the expression level of cry1Ah protein per gram (fresh weight) leaves can reach 3.18 μg. The original gene expression was only 1.01 μg. At the same time, the transgenic maize of the artificially-resistant insect resistance gene (sequence 3) provided by the present invention also has obvious resistance to target pests, and is transferred to the T6 generation plant of the mcry1Ah gene, and after being infected by the 5-6 leaf stage of the corn, No obvious pests, normal performance; and transgenic cry1Ah gene (pre-optimization, sequence 2) and non-transgenic plants, after the insects, the insect pests are very serious.

Claims (18)

1. The DNA molecule is any of the following (a)-(c):

   (a) is a nucleotide sequence such as the DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;

   (b) a nucleotide sequence such as the DNA molecule shown in positions 1 to 2007 of SEQ ID NO: 3 in the Sequence Listing;

   (c) The nucleotide sequence is at least 98% identical to the position 1 to 2007 of Sequence 3 or Sequence 3 in the Sequence Listing, and encodes the protein of SEQ ID NO: 9.
2. An expression cassette, recombinant vector, recombinant host strain, recombinant cell line or transgenic plant comprising the DNA molecule of claim 1 or 2 or 3.
3. The expression cassette according to claim 2, wherein said expression cassette comprises the following elements 1) to 3):

   1) a promoter; 2) the DNA molecule which is transcribed by the promoter; 3) a transcription termination sequence.
4. The expression cassette according to claim 3, wherein the promoter is a Ubi promoter.
5. The expression cassette according to claim 4, wherein the sequence of the Ubi promoter is as shown in SEQ ID NO: 6 in the Sequence Listing, or at least 80% identical to SEQ ID NO: 6 and has a promoter function.
6. The expression cassette according to any one of claims 3 to 5, wherein the transcription termination sequence is as shown in SEQ ID NO: 7, or at least 80% identical to SEQ ID NO: 7 and has a transcription termination function. .
7. The recombinant DNA fragment according to any one of claims 3 to 6, wherein the expression cassette further comprises an OMK sequence; the OMK sequence consists of a Ω sequence and a Kozak sequence.
8. The expression cassette according to claim 7, wherein the OMK sequence is represented by the sequence 5 in the sequence listing or has at least 80% identity with the sequence 5 and has an enhancer function.
9. The recombinant DNA fragment according to claim 8, wherein the expression cassette is composed of the Ubi promoter, the OMK sequence, the DNA molecule, and the transcription termination sequence.
10. The expression cassette according to claim 9, wherein the sequence of the expression cassette is sequence 11 in the sequence listing.
11. The recombinant vector according to claim 2, wherein the recombinant vector is a recombinant cloning vector or a recombinant expression vector.
12. The recombinant vector according to claim 2 or 11, wherein the sequence of the recombinant vector is the sequence 12 in the sequence listing.
13. An RNA obtained by transcription of the DNA molecule of claim 1.
14. Use of the DNA molecule of claim 1 or the expression cassette of any of claims 2-12 or the recombinant vector for breeding insect-resistant transgenic maize.
15. Use of the DNA molecule of claim 1 or the expression cassette of any one of claims 2 to 12 or the recombinant vector for increasing the expression level of maize cry1Ah protein; the cry1Ah protein is sequence 9 of the sequence listing Show protein.
16. A method for cultivating an insect-resistant transgenic corn, comprising the steps of: introducing the DNA molecule of claim 1 into a corn of interest, and obtaining a transgenic corn expressing the DNA molecule; the transgenic corn The insect resistance is improved compared to the corn of interest.
17. The method according to claim 16, wherein the introduction of the DNA molecule of claim 1 into the corn of interest is carried out by introducing the recombinant vector of claim 2, 11 or 12 into the corn of interest. .
18. The insect-resistant transgenic maize cultivated by the method of claim 16 or 17.

Images