CN113980989A

CN113980989A - Transgenic insect-resistant herbicide-resistant corn and cultivation method thereof

Info

Publication number: CN113980989A
Application number: CN202111420945.5A
Authority: CN
Inventors: 王磊; 张兰; 邹俊杰; 杨德志; 罗彦忠; 刘源; 郑红艳
Original assignee: Beijing Boai Yuanshang Biotechnology Co ltd; Biotechnology Research Institute of CAAS
Current assignee: Beijing Boai Yuanshang Biotechnology Co ltd; Biotechnology Research Institute of CAAS
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-01-28
Anticipated expiration: 2041-11-26
Also published as: WO2023093847A1; CN113980989B

Abstract

The invention belongs to the technical field of plant biology and discloses transgenic insect-resistant herbicide-resistant corn and a cultivation method thereof. The genes mCry1Ab, mCry3Bb and mCP4EPSPS are combined into a gene tandem expression frame form and integrated into a receptor corn material, so that the resistance of the transgenic corn to lepidoptera and coleoptera insects is obviously improved and the resistance to glyphosate also reaches a high resistance level compared with a single-gene insect-resistant or herbicide-resistant gene adopted in the prior art.

Description

Transgenic insect-resistant herbicide-resistant corn and cultivation method thereof

Technical Field

The invention relates to the technical field of plant biology, in particular to transgenic insect-resistant herbicide-resistant corn and a cultivation method thereof.

Background

Corn has become the first large grain crop in China, has wide application, is not only an important feed raw material, but also an important industrial raw material, and plays an important role in national grain safety and national economic development.

During the growth and development of corn, the corn is affected by diseases, insect pests and weeds. Corn pests are more than 350 in the world, and among them, the corn borer with stalk-eating property and leaf-eating property is the most serious. The corn borer is commonly named as borer, belongs to the family of lepidoptera borers, is a worldwide moth-eating pest, seriously affects the yield and quality of corn, and the loss caused by insect pests in the world accounts for more than 15 percent of the yield of crops every year. The corn borers can damage all parts on the ground of corn plants, so that the damaged parts lose functions and the yield of grains is reduced. At present, chemical insecticides are used in a large amount for preventing and controlling the corn borers, so that the ecological environment and the biodiversity are seriously influenced, the production cost and the labor intensity are increased, and the poisoning probability of a human body is increased. Corn armyworms overeat corn leaves with larvae, and when the corn armyworms seriously happen, the corn armyworms eat the leaves in a short period, so that the yield is reduced and even the corn armyworms are harvested absolutely. The symptoms of harm are mainly caused by the fact that larvae bite into the leaves. The harm of the cotton bollworm on the corn is increased year by year, the cotton bollworm becomes an important pest which affects the yield and the quality of the corn, the cotton bollworm larva mainly eats corn grains, the newly hatched larva concentrates on the top of corn ears to bite silks, and the existing prevention and treatment measures mainly use a large amount of chemical pesticides. Spodoptera frugiperda is an important agricultural pest for global early warning of the Food and Agricultural Organization (FAO) of the United nations, is harmful to over 80 plants such as corn, rice, tomato and the like, and has the characteristics of great harm, strong reproductive capacity, high migration speed, large food consumption and the like.

Bt insect-resistant genes from Bacillus thuringiensis are of various types and can be classified into different types according to different classification bases. In 1989, Hofte and Whiteley referred the gene encoding an insecticidal crystal protein to the Cry gene, and represented by the roman numerals I, II, III, IV, V, and english letters in combination. Bt protein genes have 5 major classes and 14 subclasses. The CryI type has the best insect-resistant effect on lepidoptera insects, the CryII type mainly resists lepidoptera and diptera insects, the CryIII type has a better effect on coleoptera insects, the CryIV type mainly aims at the diptera insects, and the CryV type has toxicity on both lepidoptera and coleoptera insects. Cry1Ab and Cry1Ac genes have specific toxicity to lepidoptera pests such as corn borers and the like.

Most of the currently popularized and utilized transgenic corn varieties are single-gene insect-resistant corn, wherein insect-resistant genes Cry1Ab/Ac, Cry3Bb, Cry2Ab and the like are more in application. Domestic field and indoor experiments show that: the Cry1Ab transgenic corn can realize the control of the Asiatic corn borers in the whole growth period and has influence on the feeding of the Asiatic corn borers; the Cry1F transgenic corn artificial feed has a high insecticidal effect on Asiatic corn borer larvae. In addition, Cry1Ab has good insecticidal effect on cotton bollworms and armyworms, and Cry3Bb has good insecticidal effect on coleoptera insects such as corn rootworms and the like. At present, insect-resistant genes adopted by the transgenic insect-resistant corn in China comprise Cry1Ab, Cry1Ah, Cry1Ie and the like, but a single-gene insect-resistant mode is mostly adopted.

Glyphosate (Glyphosate) is an organic phosphine herbicide with systemic property, broad spectrum and application on leaf surfaces, and has the characteristics of high efficiency, low toxicity, easy degradation and the like. It can competitively inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway of plants and bacteria. EPSPS is an enzyme in the aromatic amino acid synthesis pathway that reversibly catalyzes the condensation of S3P (Shikimate-3-phosphate) and pep (phosphoenolpyruvate) to EPSP and inorganic phosphines. The CP4EPSPS gene with glyphosate herbicide resistance is transferred into crops, so that the transgenic crops have glyphosate resistance.

In view of the fact that most of the transgenic insect-resistant corns in China adopt single-gene insect resistance at present, Cry1Ab and Cry3Bb are not researched for combined tandem expression of insect resistance and are used together with herbicide-resistant genes. Therefore, a transgenic crop with lepidoptera and coleoptera resistance combined with insect resistance and herbicide tolerance and a cultivation method thereof are needed in production.

Disclosure of Invention

The invention aims at the urgent need of transgenic corn with lepidoptera and coleoptera resistance and herbicide-resistant composite resistance in production, and provides a method for cultivating transgenic corn with insect resistance and herbicide-resistant composite resistance and the transgenic corn with insect resistance and herbicide-resistant composite resistance obtained by the method.

To this end, one aspect of the present invention provides a synthetic gene tandem expression cassette comprising mCry1Ab, mCry3Bb and mCP4EPSPS genes.

In a preferred embodiment of the invention, the sequence of the gene of mCry1Ab is shown as sequence 1, the sequence of the gene of mCry3Bb is shown as sequence 2, and the sequence of the gene of mCP4EPSPS is shown as sequence 3.

In another aspect, the invention provides a plant expression vector comprising the gene tandem expression cassette.

In a preferred embodiment of the invention, the plant expression vector is pL 2.

In another aspect, the invention provides a host cell comprising the gene tandem expression cassette described above, or comprising the plant expression vector described above.

In a preferred embodiment of the invention, the host cell is an agrobacterium, more preferably agrobacterium EHA 105.

In another aspect, the invention provides the application of the gene tandem expression cassette, the plant expression vector or the host cell in improving the insect resistance and herbicide tolerance of corn.

In a preferred embodiment of the invention, the insect-resistance is against lepidopteran and coleopteran insects, more preferably against corn borer, armyworm, cotton bollworm and spodoptera frugiperda, more preferably against diabrotica virgifera, and the herbicide-resistance is glyphosate-resistance.

In another aspect, the invention provides a method of breeding transgenic corn with resistance to insects and herbicides, comprising integrating the gene tandem expression cassette, the plant expression vector, or the host cell into recipient corn material.

In another aspect, the invention provides a primer combination for detecting transgenic corn with insect resistance and herbicide tolerance, and the nucleotide sequences are shown as sequences 11 and 12.

In a preferred embodiment of the invention, the transgenic corn comprises the gene tandem expression cassette of the invention, the insect resistance is lepidoptera and coleoptera resistance, the lepidoptera resistance is more preferably corn borer resistance, armyworm resistance, cotton bollworm resistance and spodoptera frugiperda resistance, the coleoptera resistance is more preferably bispodoptera resistance, and the herbicide resistance is preferably glyphosate resistance.

In another aspect, the invention provides a method for detecting insect-resistant herbicide-tolerant transgenic corn, which adopts the primer combination to detect the transgenic corn.

In a final aspect of the invention, a sequence for identifying an exogenous insert of an insect-resistant herbicide-tolerant transgenic maize comprises the exogenous insert and flanking sequences thereof, wherein the sequence is shown as a sequence 10.

In a preferred embodiment of the invention, the exogenous insertion fragment comprises the gene tandem expression cassette, the insect resistance is lepidoptera and coleoptera resistance, the lepidoptera resistance is more preferably corn borer resistance, armyworm resistance, cotton bollworm resistance and spodoptera frugiperda resistance, the coleoptera resistance is more preferably diabrotica biflora resistance, and the herbicide resistance is preferably glyphosate resistance.

From the above description, compared with the single-gene insect-resistant or herbicide-resistant gene adopted in the prior art, the resistance of the transgenic corn to lepidoptera insects such as corn borers, armyworms, cotton bollworms, spodoptera frugiperda and the like, the resistance to coleoptera insects such as diabrotica and the like and the resistance to glyphosate are also improved remarkably.

Drawings

FIG. 1: pL2 plasmid map.

RB: a right border sequence; pVS 1-REF: an agrobacterium replicon;

ColE 1: an E.coli replicon; kan: a kanamycin resistance gene;

LB: a left border sequence; t35 s: CaMV35S terminator;

mCP4 EPSPS: a glyphosate resistant gene; ubi: a maize ubiquitin promoter;

thsp 17: a heat shock protein terminator; mCry3 Bb: an insect-resistant gene;

ZmSP: a signal peptide; OsAct 2: a rice promoter;

E35S: the enhanced CaMV35S promoter;

mCry1 Ab: an insect-resistant gene; tnos: nos terminator.

FIG. 2: the mCry1Ab, mCry3Bb and mCP4EPSPS genes are subjected to PCR detection in BBL2-1 and BBL2-2 strains.

M: a 100bp molecular weight standard; 1: mCry1Ab (BBL 2-1);

2：mCry3Bb(BBL2-1)；3：mCP4EPSPS(BBL2-1)；

4：mCry1Ab(BBL2-2)；5：mCry3Bb(BBL2-2)；

6: mCP4EPSPS (BBL 2-2); 7: mCry1Ab (plasmid, positive control);

8: mCry3Bb (plasmid, positive control); 9: mCP4EPSPS (plasmid, positive control);

10: mCry1Ab (zheng 58, negative control); 11: mCry3Bb (zheng 58, negative control);

12: mCP4EPSPS (Zheng 58, negative control).

FIG. 3: BBL2-2 strain-specific PCR assay.

M: a 100bp molecular weight standard; 1: BBL 2-1;

2: BBL 2-2; 3: zheng 58 (negative control).

FIG. 4: the gene expression level of mCry1Ab, mCry3Bb and mCP4EPSPS gene in BBL2-2 material.

A: detecting the result in the 4-5 leaf stage of the corn; b: and detecting the result in the mature period of the corn.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall into the protection scope of the present invention.

Example 1: obtaining transgenic Material

1. Codon optimization

The method comprises the steps of carrying out comprehensive codon optimization on Cry1Ab, Cry3Bb and CP4EPSPS genes according to the codon usage frequency of the corn, removing sequences which influence mRNA stability, such as AT-rich sequences, namely ATTTA, AATTAA and the like, improving the GC content of a coding region, adding a sequence which improves the translation efficiency in front of a start codon to enable the sequences to be efficiently expressed and translated in the corn, and respectively naming the Cry1Ab, Cry3Bb and CP4EPSPS gene sequences as mCry1Ab, mCry3Bb and mCP4EPSPS genes according to the codon usage frequency of the corn, wherein the nucleotide sequences are shown as sequences 1-3.

2. Expression vector construction

The basic vector is pCAMBIA1300, the selection marker of the pCAMBIA1300 in bacteria is Kan gene which codes aminoglycoside phosphotransferase, and the coding product has no report at present to consider that the coding product has toxic or side effect on animals. It cannot be transferred into plant cells because it is located at the periphery of the T-DNA region.

ColE1 is derived from Escherichia coli, mainly has the function of assisting the replication of a vector in Escherichia coli, is widely applied to the construction of engineering vectors, only exists as a replication origin, and does not encode protein. And it is located at the periphery of the T-DNA region and cannot be transferred into plant cells.

pVS1-REP is derived from Pseudomonas, mainly plays a role in helping the plasmid to replicate in Agrobacterium, is widely applied to the construction of engineering vectors, and cannot be transferred into plant cells because it is located at the periphery of the T-DNA region.

The T-DNA is derived from the Ti plasmid, but the tumorigenic genes and sequences unrelated to T-DNA transfer have been removed, leaving only the right border sequence (RB) and the left border sequence (LB) necessary for T-DNA transfer. RB and LB are recognition sequences of exonuclease in T-DNA transfer, and do not code any gene product.

Target genes mCP4EPSPS, mCry3Bb, mCry1Ab and matched promoters and terminators thereof are inserted between RB and LB, a plant expression vector is constructed according to a conventional molecular biological method, and the vector is named as pL2 plasmid, and has the size: 17.8 kb.

The vector map is shown in figure 1. The expression cassettes of the target genes mCP4EPSPS, mCry3Bb and mCry1Ab are shown in tables 1-3.

TABLE 1 mCry3Bb expression cassette

Name (R)	Function(s)
		OsAct2	Rice actin gene promoter.
mCry3Bb	Cry3Bb encoding proteins, resistant to coleopteran insects
		Thsp17	Hsp17 terminator, terminating transcription.

TABLE 2 mCP4EPSPS expression cassette

TABLE 3 mCry1Ab expression cassette

Name (R)	Function(s)
		E35S	Enhanced CaMV35S promoter.
mCry1Ab	Encoding a Cry1Ab protein, against lepidopteran insects.
		Tnos	Nos terminator, terminating transcription.

3. Transgenic material acquisition

The target gene and other gene elements are introduced into the acceptor corn material by means of agrobacterium mediated process.

The transformation process is as follows: taking young ear of corn pollinated for 9-12 days, strippingAnd (4) placing the immature embryos in a liquid medium for infection, and using the immature embryos for agrobacterium infection. Transforming a plasmid containing a target gene into an Agrobacterium EHA105 strain to OD₆₀₀When the ratio is 0.3-0.4, the agrobacterium is resuspended in the infection liquid, and the corn embryo material is placed in the invasion dye liquid for agrobacterium infection. Glyphosate was added to the medium as a selective pressure and the transformed material was subjected to selection culture, subcultured every two weeks. After 2-3 months of selective culture, the resistant callus grows rapidly on the selective culture medium and is fresh in color. Transferring the resistant callus to a regeneration medium to obtain a mature embryoid body. And (4) putting the embryoid on an MS culture medium to grow seedlings and root, so as to obtain the regenerated corn seedlings. And backcrossing the transformed event with Zheng 58 of the maize inbred line by a backcross transformation method to obtain the insect-resistant herbicide-tolerant maize BBL2 with Zheng 58 genetic background containing exogenous genes.

Example 2: detection of genetic stability of target gene in transgenic material

The integration condition and genetic stability of target genes mCry1Ab, mCry3Bb and mCP4EPSPS in corn transformant BBL2 are analyzed by PCR technology.

The result shows that target genes mCry1Ab, mCry3Bb and mCP4EPSPS can be detected in BC4F1, BC5F1 and BC6F1 detection groups, the sizes of the positive specific fragments are respectively consistent with the sizes of the expected fragments, and only one band is provided. Each gene is stably inherited among generations of a transformant, the segregation ratio of the genes accords with a Mendelian single-site inheritance rule, and the three target genes are integrated at one site only.

The PCR band map of the BBL2 target gene is shown in figure 2.

The primers for PCR of the target gene are shown in tables 4 to 6, and the PCR composition and reaction conditions are shown in Table 7.

TABLE 4 mCP4EPSPS specific primers

Primer I(T35s)	5'-ctcaacacatgagcgaaaccc-3' (sequence 4)
		Primer II(CP4)	5'-acccatctcgatcaccgcat-3' (sequence 5)

TABLE 5 specific primers for mCry3Bb

Primer I(C3B)	5'-ctgctcttcctgaaggagtcg-3' (sequence 6)
		Primer II(Hsp)	5'-cagactcgcaagaactcgcac-3' (sequence 7)

TABLE 6 specific primers for mCry1Ab

Primer I(Nos)	5'-gaccggcaacaggattcaatc-3' (sequence 8)
		Primer II(C1A)	5'-ctacttgtaccagaagatcga-3' (SEQ ID NO: 9)

TABLE 7 PCR detection reaction System and reaction conditions

Example 3: BBL2 flanking sequence analysis and specific PCR detection

And (2) carrying out glyphosate resistance identification on 30 obtained transgenic corn events, carrying out insect resistance identification on 13 events with the resistance reaching more than 4 times, screening out 5 events with high resistance to pests such as corn borers, and the like, and further preliminarily determining the integration condition of the target gene in a corn genome by using a whole genome re-sequencing method. The sequence of 1 event expression frame is lost, 2 events are inserted into the corn gene, and 2 events are inserted into the intergenic region, further research finds that the BBL2-2 event has no influence on the growth and development of the corn, the expression frame is complete, the gene expression level is high, and the heredity is stable.

According to the obtained maize genome sequence flanking the BBL2-2 insertion fragment, and further using a PCR amplification sequencing method, maize genome specific sequences near the insertion site of the BBL2-2 event are obtained, the integration position of the target gene in the genome is determined through BLAST analysis in a maize genome database, and the specific sequences on the Tnos side are shown as a sequence 10. The Tnos end specificity PCR detection primer pair consists of a single-stranded DNA molecule shown in a sequence 11 and a single-stranded DNA molecule shown in a sequence 12, and the amplified sequence is shown in a sequence 10.

BBL2-2 genomic DNA was extracted for specific PCR amplification. The Tnos end specific amplification primer sequence is as follows:

primer I (BBL2-2n): 5'-ctgtaacgctaactgtagcat-3'; (SEQ ID NO: 11)

Primer II (Tnos): 5'-agagtcccgcaattatacat-3'; (SEQ ID NO: 12)

The theoretical amplification size was 298 bp.

The PCR reaction system and reaction conditions are shown in Table 8.

TABLE 8 PCR detection reaction System and reaction conditions

After the reaction, the PCR product was subjected to agarose gel electrophoresis.

The result shows that different plants of BBL2-2 can detect clear single band with expected size, the detection system is normal, there is no non-specific amplification, no amplified band is existed in backcross transferred receptor Zheng 58 and blank control, and the PCR electrophoresis result is shown in figure 3.

Example 4: BBL2-2 target gene qRT-PCR detection

RNA was extracted from the roots, stems, leaves of seedlings at 4-5 leaf stage of maize BBL2-2 plant, and roots, stems, leaves and seeds of mature plants. RNA extraction was performed by the method of SV Total RNA Isolation System (Promega, USA) and Reverse Transcription by the method of A3500-Reverse Transcription System (Promega, USA). The primers for qRT-PCR detection of the target gene are shown in Table 9.

TABLE 9 foreign Gene qRT-PCR primer information in BBL2-2 transformation Material

Primer name	Sequence of
		mCry1Ab fw	5'-ctacttgtaccagaagatcgac-3' (SEQ ID NO: 13)
mCry1Ab rv	5'-tcagtcctcgttcaggtcggtg-3' (sequence 14)
		mCry3Bb fw	5'-ctgctcttcctgaaggagtcg-3' (sequence 15)
mCry3Bb rv	5'-gatgaactcgatcttgtcgatg-3' (sequence 16)
		mCP4EPSPS fw	5'-acccatctcgatcaccgca-3' (SEQ ID NO: 17)
mCP4EPSPS rv	5'-cagccttcgtatcggagagttc-3' (sequence 18)
		zmActin1 fw	5'-caccttctacaacgagctccg-3' (SEQ ID NO: 19)
zmActin1 rv	5'-taatcaagggcaacgtaggca-3' (sequence 20)

SYBR Premix ExTaq was used^TM(Code DRR041A, TAKARA) fluorescent quantitation kit, PCR instrument Applied Biosystems (http:// www.AppliedBiosystems.com) Prism 7500 analyzer. The expression levels of the genes mCry1Ab, mCry3Bb and mCP4EPSPS of BBL2-2 plant are shown in figure 4.

Example 5: method for measuring BBL2-2 target protein and detection of expression quantity of BBL2-2 target protein in different tissues

The expression of target gene proteins Cry1Ab, Cry3Bb and CP4EPSPS in the root, stem, leaf and kernel tissues of the corn transformant BBL2-2 is analyzed by adopting a double-resistance sandwich enzyme-linked immunosorbent assay (ELISA). ELISA experimental methods and analytical methods were performed according to the kit instructions of EnviroLogoix, Inc.

0.1g of each tissue sample was weighed first, and after grinding with liquid nitrogen, the sample was added to 1mL of sample extraction buffer while diluting the standard with 2mL of sample extraction buffer. And (3) detecting the expression level of the target protein by using an ELISA kit of CP4EPSPS, Cry1Ab/Ac and Cry3 Bb. And analyzing the light absorption values of different samples by a microplate reader, fitting a standard curve equation according to the concentration and optical density value reading of the standard protein, and finally calculating the weight of the protein expressed in each organ in the sample tissue by using Microsoft Excel to express (mu g/g).

ELISA detection results show that the target proteins Cry1Ab, Cry3Bb and CP4EPSPS can be detected in different organ samples of the corn transformant BBL2-2, the content of the target protein in different organs is greatly changed, the content in leaves is higher, and the content of grains is lower. The expression levels of the target protein in different tissues are shown in Table 10.

TABLE 10 expression levels of proteins of interest in different tissues of maize transformant BBL2-2

Note: ± represents the standard deviation; NA indicates that the value is lower than the effective detection range of the standard curve and is not detected.

Example 6: BBL2-2 insect resistance test

1. Test insects

The tested insects of Asiatic corn borer, armyworm, cotton bollworm and Spodoptera frugiperda are all continuously raised on indoor artificial feed for many generations. During the feeding process, the Bt preparation and the Bt insecticidal protein are not contacted. Feeding the seeds under the conditions that the temperature is 27 +/-1 ℃, the relative humidity is 70-80% and the photoperiod is L16: D8 h.

Leaf beetle of Rhagophthalmus bicolor: the insect breeding cage is filled with absorbent cotton, filter paper and corn leaves, and each cage is used for breeding 20 adult diabrotica palmifolia. Temperature 26 +/-1 ℃, light cycle L16: D8h, Relative Humidity (RH) (80 +/-5)%.

2. Test Bt corn

The transgenic corn materials used for the test were: BBL2-2 (containing optimized mCry1Ab, mCry3Bb and mCP4EPSPS expression cassettes), transgenic Cry1Ab corn, transgenic Cry3Bb corn, parental control Zheng 58. Corn material was all planted in greenhouse pots.

3. Insect resistance test

Bioassay of corn borers, cotton bollworms and spodoptera frugiperda: taking 4-6 corn plants in the 4-6 leaf stage, returning the corn plants to the room, selecting the young heart leaves which are just unfolded, washing the young heart leaves with tap water, washing the young heart leaves with distilled water for 1 time, sucking off water drops on the surface of the young heart leaves with filter paper, and drying the young heart leaves. And (3) cutting the corn leaves with the main veins removed into leaves with the length and width of about 1cm, and putting the leaves into a 24-hole cell culture plate with 1-2 leaves in each hole. 1 head of the larva to be initially hatched is inoculated into each hole. Each plate was 1 replicate 4 times. The seeds are raised under the conditions of 27 +/-1 ℃, relative humidity of 70-80% and photoperiod of L16: D8 h.

Live testing of armyworm: taking 4-6 corn plants in the 4-6 leaf stage, taking the corn plants back to the room, selecting tender heart leaves, cleaning, airing, putting into a small insect box, and inoculating 20 newly hatched larvae into each box. Each box was 1 replicate 4 times. The seeds are raised under the conditions of 27 +/-1 ℃, relative humidity of 70-80% and photoperiod of L16: D8 h.

The investigation is carried out 1 time every 2 days, fresh leaves of the same source are added or replaced at any time according to the consumption condition that leaf tissues are eaten, the number of the surviving larvae is recorded, and the experiment observation is carried out for 6 days.

Adult diabrotica: feeding corn leaves of different varieties of the double-spot imagoes by using the insect breeding device (4-6 leaf period), and replacing fresh corn leaves every 24 h. 20 adult diabrotica bifida are placed in each insect cage, and 3 insect cages (three repetition) are arranged for each corn variety. The eggs are kept at the temperature of 26 +/-1 ℃, the photoperiod of L16: D8h and the Relative Humidity (RH) (80 +/-5)% and are observed and counted for the death of the double-spots in the insect cage at 24 hours, 48 hours and 96 hours respectively.

And performing variance analysis on the survival rate of different pests eating different corn materials by adopting statistical analysis software.

4. Data statistics and analysis

(1) Indoor insect resistance assay for Asiatic corn borer

The indoor insect resistance result of the transgenic corn BBL2-2 to the Asiatic corn borer shows that the Asiatic corn borer is completely dead after the larva is hatched for 4 days to eat the transgenic material BBL2-2 leaves, 17.7 percent of the transgenic Cry1Ab gene corn still survives for 4 days, and the survival rate of the transgenic corn borer reaches more than 80 percent on the non-transgenic control Zheng 58, so that the obvious difference exists. The BBL2-2 is proved to have obviously better resistance to Asian corn borers than Cry1Ab single-gene transgenic material.

TABLE 11 indoor insect resistance of transgenic maize BBL2-2 to Asiatic corn borer

Note: the data in the table are mean ± sd, and the difference is significant in different lower case letters after the same column of data (P < 0.05).

(2) Indoor insect resistance determination for mythimna larvae

The indoor insect resistance result of the transgenic corn BBL2-2 to the myxozoa shows that the majority of the myxozoa are dead after the larvae which are hatched initially eat the transgenic material BBL2-2 leaves for 6 days, more than 80% of the larvae survive after the Cry1Ab transgenic corn is bred for 6 days, the survival rate of the larvae which are bred by the transgenic corn is more than 90%, and the obvious difference exists. The BBL2-2 is proved to have obvious better resistance to the armyworm than the transgenic Cry1Ab single gene.

TABLE 12 indoor insect resistance to armyworm for transgenic maize BBL2-2

Note: the data in the table are mean ± standard deviation, with different lower case letters indicating significant differences after the same column of data (P < 0.05).

(3) Indoor insect resistance determination for cotton bollworms

The indoor insect resistance result of the transgenic corn BBL2-2 to the cotton bollworm shows that most of the cotton bollworm dies after the cotton bollworm is hatched and eats the transgenic material BBL2-2 leaves for 4 days; 12.5 percent of corn survives after being transformed with Cry1Ab gene for 4 days; the survival rate of non-transgenic control Zheng 58 is more than 70%, and there is a significant difference. The BBL2-2 is proved to have obviously better resistance to cotton bollworm than the transgenic Cry1Ab single gene.

TABLE 13 indoor insect resistance of transgenic maize BBL2-2 to Helicoverpa armigera

(4) Indoor insect resistance assay for spodoptera frugiperda

The indoor insect resistance result of the transgenic corn BBL2-2 to spodoptera frugiperda shows that 42.7 percent of spodoptera frugiperda larvae survive after eating the transgenic material BBL2-2 leaves for 6 days; 59.4% of the corn with the transgenic Cry1Ab gene survives; the survival rate of non-transgenic control Zheng 58 is more than 90%. The BBL2-2 is proved to have obviously better resistance to spodoptera frugiperda than the transgenic Cry1Ab single gene.

TABLE 14 indoor insect resistance of transgenic maize BBL2-2 to Spodoptera frugiperda

(5) Indoor insect resistance determination for Diabrotica biflora

The indoor insect resistance result of the transgenic corn BBL2-2 to Diabrotica bifida shows that the survival rate of the Diabrotica bifida adults eating the transgenic material BBL2-2 leaves for 4 days is 26.7%, 62.5% of the transgenic corn with Cry3Bb gene is survived, and 80% of the transgenic corn with Zheng 58 is survived. The BBL2-2 is proved to have obviously better resistance to Diabrotica bimaculata than Cry3Bb transgenic corn.

TABLE 15 indoor insect resistance of transgenic maize BBL2-2 to Diabrotica

Example 7: BBL2-2 field glyphosate resistance test

1. Test materials

The transgenic corn BBL2-2 (BBL2-2) and the corresponding non-transgenic corn are the control Zheng 58. The target herbicide was noda (41% glyphosate isopropylamine salt) from monsanto.

2. Design of experiments

Random block design was used, 3 replicates. 1.0 m wide isolation belt is arranged among cells, and the area of each cell is 24m²。

The following processing is assumed: (1) the transgenic corn is not sprayed with herbicide; (2) spraying a target herbicide glyphosate on the transgenic corn; (3) the corresponding non-transgenic corn is not sprayed with herbicide; (4) spraying the target herbicide glyphosate on the corresponding non-transgenic corn.

The applied dose of herbicide was set at 4 gradients: (1) medium dosage of pesticide registration label; (2) 2 times of the medium dosage; (3) 4 times of the medium dosage; (4) 6 times of the medium dosage.

3. Investigation method

The seedling rate, plant height (the highest 5 plants were selected), and phytotoxicity symptoms (the lowest 5 plants were selected) were investigated and recorded at 1 week, 2 weeks, and 4 weeks after the application, respectively, including: growth inhibition, chlorosis, blight spots, deformity, etc.

Phytotoxicity symptom grading is performed according to GB/T19780.42, which is as follows:

level 1: the corn grows normally without any damage symptom;

and 2, stage: slight phytotoxicity of corn is less than 10%;

and 3, level: the corn can be recovered later due to medium phytotoxicity, and the yield is not influenced;

4, level: the corn has serious phytotoxicity and is difficult to recover, thereby causing the reduction of yield;

and 5, stage: the corn has serious phytotoxicity and can not be recovered, thereby causing obvious yield reduction or no yield.

The herbicide damage rate is calculated according to the following formula: x [ Σ (N × S)/(T × M) ] × 100

Wherein: x is the damage rate in percent (%); n is the number of the same-stage damaged strains; s is the number of grades; t is the total number of plants; m is the highest level.

4. Herbicide application dosage and method

The applied dose of herbicide was set at 4 gradients: the medium dose, 2 times, 4 times and 6 times of the medium dose of the pesticide registration label were 900g a.i./ha, 1800g a.i./ha, 3600g a.i./ha and 5400g a.i./ha, respectively.

The application method comprises the following steps: and (4) treating stems and leaves, wherein the liquid spraying amount is 450L/ha.

5. Analysis of results

The BBL2-2 does not spray medicine and sprays 900g a.i./ha, 1800g a.i./ha, 3600g a.i./ha and 5400g a.i./ha, and the seedling rate is 100% after 1, 2 and 4 weeks of treatment of glyphosate with four concentrations. The seedling rate of the non-transgenic corn is 100% after 1, 2 and 4 weeks of non-spraying treatment, and the seedling rate is 0 after the non-transgenic corn is sprayed for 1 week with 900, 1800, 3600 and 5400g of a.i./ha glyphosate.

TABLE 16 seedling percentage (%)

Note: the values in the table are the average values of 3 cells, and the seedling rate is 100% or 0%, so that no significance analysis is performed.

No pesticide damage was observed in BBL2-2 after spraying glyphosate at four concentrations of 900g a.i./ha, 1800g a.i./ha, 3600g a.i./ha and 5400g a.i./ha for 1, 2 and 4 weeks. The non-transgenic corn control had a damage rate of 98.67% after spraying 900g of a.i./ha glyphosate for 1 week, with individual plants still alive and all plants dead after spraying for 2 weeks. The non-transgenic corn controls all died after spraying 1800, 3600 and 5400g a.i./ha glyphosate for 1 week, with 100% damage.

TABLE 17 Damage (%)

Note: the values in the table are the average of 3 cells. The damage rates were 0% or 100%, respectively, and therefore no significance analysis was performed.

SEQUENCE LISTING

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> transgenic insect-resistant herbicide-tolerant corn and cultivation method thereof

<130> 20210722

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 2448

<212> DNA

<213> Artificial Synthesis

<400> 1

atggacaaca acccgaacat caacgagtgc atcccgtaca actgcctcag caaccctgag 60

gtcgaggtgc tcggcggtga gcgcatcgag accggttaca cgcccatcga catctcgctc 120

tccctcacgc agttcctgct cagcgagttc gtgccaggcg ctggcttcgt cctggggctc 180

gtggacatca tctggggcat ctttggcccc tcccagtggg acgccttcct ggtgcaaatc 240

gagcagctca tcaaccagag gatcgaggag ttcgccagga accaggccat cagccgcctg 300

gagggcctca gcaacctcta ccaaatctac gctgagagct tccgcgagtg ggaggccgac 360

cccactaacc cagctctccg cgaggagatg cgcatccagt tcaacgacat gaacagcgcc 420

ctgaccacgg ccatcccact cttcgccgtc cagaactacc aagtcccgct cctgtcggtg 480

tacgtccagg ccgccaacct gcacctcagc gtgctgaggg acgtgagcgt gtttggccag 540

aggtggggct tcgacgccgc caccatcaac agccgctaca acgacctcac caggctgatc 600

ggcaactaca ccgaccacgc tgtccgctgg tacaacactg ggctggagcg cgtctgggga 660

cctgattcta gggactggat tcgctacaac cagttcaggc gcgagctgac cctcacggtc 720

ctggacattg tgtcgctctt cccgaactac gactcccgga cctacccgat ccgcacggtg 780

tcgcaactga cccgcgaaat ctacacgaac cccgtcctgg agaacttcga cggtagcttc 840

aggggcagcg cccagggcat cgagggctcc atcaggagcc cgcacctgat ggacatcctc 900

aacagcatca ctatctacac cgatgcccac cgcggcgagt actattggtc cgggcaccag 960

atcatggcct ccccggtcgg gttcagcggc cccgagttta cctttcctct ctacggcacg 1020

atgggcaacg ccgctccgca acaacgcatc gtcgcacagc tgggccaggg cgtctaccgc 1080

accctcagct ccacgctgta ccgcaggccc ttcaacatcg gtatcaacaa ccagcagctg 1140

tccgtcctgg atggcactga gttcgcctac ggcacctcct cgaacctgcc ctccgctgtc 1200

taccgcaaga gcggcacggt ggattcgctg gacgagatcc cgccacagaa caacaatgtg 1260

ccccccaggc agggtttttc ccacaggctc agccacgtgt ccatgttccg ctcgggcttc 1320

agcaactcgt cggtgagcat catcagggct cctatgttct cctggattca tcgcagcgcg 1380

gagttcaaca atatcattcc gtcctcgcaa atcacccaaa tccccctcac gaagtccacc 1440

aacctgggca gcgggacctc cgtggtgaag ggtccaggct tcacgggcgg cgacatcctg 1500

cgcaggacct cgccgggcca gatcagcacc ctccgcgtca acatcacggc tcccctgtcc 1560

cagagatacc gcgtcaggat tcggtacgct agcaccacga acctgcaatt ccacacctcc 1620

atcgacggca ggccgatcaa tcagggtaac ttctcggcca ccatgtccag cggcagcaac 1680

ctccaatccg gcagcttccg caccgtgggt ttcacgaccc ccttcaactt ctccaacggc 1740

tccagcgttt tcaccctgag cgcccacgtg ttcaattccg gcaatgaggt ctacattgac 1800

cgcattgagt tcgtgccagc cgaggtcacc ttcgaggccg agtacgacct ggagagagcc 1860

cagaaggctg tcaatgagct gttcacgtcc agcaatcaga tcggcctgaa gaccgacgtc 1920

actgactacc acatcgacca agtctccaac ctcgtggagt gcctctccga tgagttctgc 1980

ctcgacgaga agaaggagct gtccgagaag gtgaagcacg ccaagcgtct cagcgacgag 2040

aggaatctcc tccaggaccc caatttccgg ggcatcaaca ggcagctcga ccgcggctgg 2100

cgcgggagca ccgacatcac gatccagggc ggcgacgatg tgttcaagga gaactacgtg 2160

actctcctgg gcactttcga cgagtgctat cctacctact tgtaccagaa gatcgacgag 2220

tccaagctca aggcttacac tcgctaccag ctccgcggct acatcgaaga cagccaagac 2280

ctggagattt acctgatccg ctacaacgcc aagcacgaga ccgtcaacgt gcccggtact 2340

ggttccctct ggccgctgag cgccccctcg ccgatcggca agtgtgccca ccacagccac 2400

cacttctcct tggacatcga cgtgggctgc accgacctga acgaggac 2448

<210> 2

<211> 1959

<212> DNA

<213> Artificial Synthesis

<400> 2

atggccaacc ccaacaatcg ctccgagcac gacacgatca aggtcacccc gaactccgag 60

ctgcagacca accacaacca gtacccgctg gccgacaacc ccaactccac cctcgaagag 120

ctgaactaca aggagttcct ccgcatgacc gaggactcgt ccacggaggt cctcgacaac 180

tccaccgtga aggacgccgt cgggaccggc atctccgtgg ttgggcagat cctgggcgtc 240

gttggcgtcc ccttcgcagg tgctctcacc tccttctacc agtcgttcct caacaccatc 300

tggccctccg acgccgaccc gtggaaggcc ttcatggcgc aagtggaagt cctcatcgac 360

aagaagatcg aggagtacgc caagtccaag gcgctcgccg agctgcaagg cctgcaaaac 420

aacttcgagg actacgtcaa cgcgctgaac tcctggaaga agacgcctct ctccctgcgc 480

tcgaagcggt cccaggaccg catccgggag ctgttctcgc aggccgagtc ccacttccgc 540

aactccatgc cgtcgttcgc cgtctccaag ttcgaggtgc tcttcctgcc cacctacgcg 600

caggctgcca acacccacct cctgttgctc aaggacgccc aggtcttcgg cgaggaatgg 660

ggctactcct cggaggacgt cgccgagttc taccgtcgcc agctgaagct cacgcaacag 720

tacaccgacc actgcgtgaa ctggtacaac gtcggcctga acggcctcag gggctccacc 780

tacgacgcat gggtcaagtt caaccgcttc cgcagggaga tgacgctgac cgtcctcgac 840

ctgatcgtgc tcttcccctt ctacgacatc cgcctgtact ccaagggcgt caagaccgag 900

ctgacgcgcg acatcttcac ggacccgatc ttcctgctca cgaccctcca gaagtacggt 960

cccaccttcc tgtccatcga gaactcgatc cgcaagcccc acctcttcga ctacctccag 1020

ggcatcgagt tccacacgcg cctgaggcca ggctacttcg gcaaggactc cttcaactac 1080

tggtccggca actacgtcga gaccaggccc tcgatcggct cctcgaagac gatcacctcc 1140

cctttctacg gcgacaagtc gaccgagccc gtccagaagc tgtccttcga cggccagaag 1200

gtgtaccgca ccatcgccaa cacggacgtc gcggcttggc cgaacggcaa ggtgtacctc 1260

ggcgtcacga aggtggactt ctcccagtac gatgaccaga agaacgagac ctccacgcag 1320

acctacgact ccaagcgcaa caatggccac gtctcggccc aggactccat cgaccagctg 1380

ccgcctgaga ccactgacga gcccctggag aaggcctact cccaccagct caactacgcg 1440

gagtgcttcc tgatgcaaga ccgcaggggc accatcccct tcttcacgtg gacccaccgg 1500

tccgtcgact tcttcaacac catcgacgcc gagaagatca cccagctgcc cgtggtcaag 1560

gcctacgcgc tctcctcggg tgcctccatc attgagggtc caggcttcac cggtggcaac 1620

ctgctcttcc tgaaggagtc gtcgaactcc atcgccaagt tcaaggtcac cctcaactcc 1680

gctgccttgc tgcaacgcta ccgggtccgc atccggtacg cctccaccac gaacctgcgc 1740

ctcttcgtcc agaactccaa caatgacttc ctcgtgatct acatcaacaa gaccatgaac 1800

aaggacgatg acctgacgta ccagaccttc gacctcgcca ccacgaactc caacatgggc 1860

ttctcgggcg acaagaatga actgatcatt ggtgctgagt ccttcgtctc gaacgagaag 1920

atctacatcg acaagatcga gttcatcccg gtgcagctc 1959

<210> 3

<211> 1365

<212> DNA

<213> Artificial Synthesis

<400> 3

atgcttcacg gtgcaagcag ccggcccgca accgcccgca aatcctctgg cctttcggga 60

accgtccgca ttcccggcga caagtcgatc tcccaccggt ccttcatgtt cggcggtctc 120

gcgagcggtg aaacgcgcat caccggcctt ctggaaggcg aggacgtcat caatacgggc 180

aaggccatgc aggcgatggg cgcccgcatc aggaaggaag gcgacacctg gatcatcgac 240

ggcgtcggca atggcggcct cctggcgcct gaggcgccgc tcgatttcgg caatgccgcc 300

acgggctgcc gcctgacgat gggcctcgtc ggggtctacg atttcgacag caccttcatc 360

ggcgacgcct cgctcacaaa gcgcccgatg ggccgcgtgt tgaacccgct gcgcgaaatg 420

ggcgtgcagg tgaaatcgga agacggtgac aggcttcccg ttaccttgcg cgggccgaag 480

acgccgacgc cgatcaccta ccgcgtgccg atggcctccg cacaggtgaa gtccgccgtg 540

ctgctcgccg gcctcaacac gcccggcatc acgacggtca tcgagccgat catgacgcgc 600

gatcatacgg agaagatgct ccagggcttc ggcgccaacc ttaccgtcga gacggatgcg 660

gacggcgtga ggaccatccg gctggaaggc cgcggcaagc tcaccggcca agtcatcgac 720

gtgccgggcg acccgtcctc gacggccttc ccgctggttg cggccctgct tgttccgggc 780

tccgacgtca ccatcctcaa cgtgctgatg aaccccaccc gcaccggcct catcctgacg 840

ctccaggaaa tgggcgccga catcgaagtc atcaacccgc gccttgccgg cggcgaagac 900

gtggcggacc tgcgcgttcg gtcctccacg ctgaagggcg tcacggtgcc ggaagaccgc 960

gcgccttcga tgatcgacga atatccgatt ctcgctgtcg ccgccgcctt cgcggaaggg 1020

gcgaccgtga tgaacggtct ggaagaactc cgcgtcaagg aaagcgaccg gctctcggcc 1080

gtcgccaatg gcctcaagct caatggcgtg gattgcgatg agggcgagac gtcgctcgtc 1140

gtgaggggcc gccctgacgg caaggggctc ggcaacgcct cgggcgccgc cgtcgccacc 1200

catctcgatc accgcatcgc catgagcttc ctcgtcatgg gcctcgtgtc ggagaaccct 1260

gtcacggtgg acgatgccac gatgatcgcc acgagcttcc cggagttcat ggacctgatg 1320

gccgggctgg gcgcgaagat cgaactctcc gatacgaagg ctgcc 1365

<210> 4

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 4

ctcaacacat gagcgaaacc c 21

<210> 5

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 5

acccatctcg atcaccgcat 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 6

ctgctcttcc tgaaggagtc g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 7

cagactcgca agaactcgca c 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 8

gaccggcaac aggattcaat c 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 9

ctacttgtac cagaagatcg a 21

<210> 10

<211> 298

<212> DNA

<213> Artificial Synthesis

<400> 10

agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 60

gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattaaa ctatcagtgt 120

ttgcgattcc taattaggtc aacaaaagct gcaccgacct tttgcgatac tagtctcaat 180

catttaattt taacctggtc taaaatcata tgggccgggt gcatatatac tagctagctc 240

tatgtgtgct gtgccaggtt aagagaccta tacatacatg ctacagttag cgttacag 298

<210> 11

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 11

ctgtaacgct aactgtagca t 21

<210> 12

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 12

agagtcccgc aattatacat 20

<210> 13

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 13

ctacttgtac cagaagatcg ac 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 14

tcagtcctcg ttcaggtcgg tg 22

<210> 15

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 15

ctgctcttcc tgaaggagtc g 21

<210> 16

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 16

gatgaactcg atcttgtcga tg 22

<210> 17

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 17

acccatctcg atcaccgca 19

<210> 18

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 18

cagccttcgt atcggagagt tc 22

<210> 19

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 19

caccttctac aacgagctcc g 21

<210> 20

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 20

taatcaaggg caacgtaggc a 21

Claims

1. A synthetic gene tandem expression cassette comprises mCry1Ab, mCry3Bb and mCP4EPSPS genes, wherein the sequence of the mCry1Ab gene is preferably shown as a sequence 1, the sequence of the mCry3Bb gene is preferably shown as a sequence 2, and the sequence of the mCP4EPSPS gene is preferably shown as a sequence 3.

2. Plant expression vector comprising a gene tandem expression cassette according to claim 1, preferably the expression vector pL 2.

3. A host cell comprising the gene tandem expression cassette of claim 1, or comprising the plant expression vector of claim 2, preferably agrobacterium, more preferably agrobacterium EHA 105.

4. Use of the tandem gene expression cassette of claim 1, the plant expression vector of claim 2, or the host cell of claim 3 for increasing insect and herbicide resistance in maize, wherein the insect resistance is preferably against lepidopteran and coleopteran insects, more preferably against corn borer, armyworm, cotton bollworm and spodoptera frugiperda, more preferably against diabrotica spp, and the herbicide resistance is preferably glyphosate resistance.

5. A method of breeding transgenic corn resistant to an insect and herbicide, comprising incorporating the gene tandem expression cassette of claim 1, the plant expression vector of claim 2, or the host cell of claim 3 into recipient corn material, wherein the insect resistance is preferably against lepidopteran and coleopteran insects, more preferably against corn borer, armyworm, cotton bollworm, and spodoptera frugiperda, more preferably against coleopteran diabrotica, and the herbicide resistance is preferably glyphosate resistance.

6. A primer combination for detecting transgenic corn with insect resistance and herbicide tolerance has nucleotide sequences shown as sequences 11 and 12.

7. A method for detecting transgenic corn with insect resistance and herbicide tolerance, which adopts the primer combination of claim 6 to detect the transgenic corn.

8. The primer combination of claim 6 or the method of claim 7, wherein the transgenic maize comprises the gene tandem expression cassette of claim 1, is resistant to insects, preferably to lepidoptera and coleopteran insects, more preferably to corn borer, armyworm, cotton bollworm and spodoptera frugiperda, more preferably to diabrotica biflora, and is resistant to herbicides, preferably to glyphosate.

9. A sequence for identifying an exogenous insert of an insect-resistant herbicide-resistant transgenic corn, comprising an exogenous insert and sequences flanking the exogenous insert, the sequence being shown in sequence 10.

10. The sequence according to claim 9, wherein the foreign insert comprises the gene tandem expression cassette of claim 1, is resistant to insects, preferably lepidopteran and coleopteran, more preferably to corn borer, armyworm, cotton bollworm and spodoptera frugiperda, more preferably to diabrotica, and is resistant to herbicides, preferably to glyphosate.