Synthetic gene tandem expression cassette and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a synthesized gene tandem expression cassette and application thereof. According to the invention, the genes cry1Ab, vip3Aa, gr79-epsps and gat are combined into a gene tandem expression cassette form and are integrated into a corn material, so that compared with a single-gene insect-resistant or herbicide-resistant gene adopted in the prior art, the resistance of transgenic corn to lepidoptera pests is remarkably improved, the tolerance to glyphosate also reaches a high resistance level, and the application prospect is wide. Especially, the insect-resistant genes have a synergistic effect, and the effect of quickly killing insects is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a synthetic gene tandem expression frame and application thereof. Background Art

[0002] Corn (Zea mays L.) is an important food crop, feed and industrial raw material, and plays a very important role in the national economy. Corn is affected by a variety of pests throughout its entire growth cycle. There are more than 350 species of corn pests worldwide, among which the main pests are corn borers, fall armyworms, armyworms and cotton bollworms. Corn borers, also known as corn borers, can harm various tissues and parts of corn plants, mainly damaging the heart leaves, stalks and female ears of corn. Specifically, the leaves appear perforated in rows, the stalks are hollow, and the filaments, bracts and young grains are eaten, which ultimately leads to a reduction or complete loss of corn yields. The fall armyworm originated in tropical and subtropical regions of the Americas. It is also known as the false armyworm, fall armyworm, fall armyworm or fall armyworm. It belongs to the genus Spodoptera of the family Noctuidae in the order Lepidoptera. The fall armyworm has a varied diet and a wide range of hosts. It also has a particularly strong migratory ability, a large diffusion range, strong reproductive capacity, a large insect population, and severe overeating damage. It is difficult to control and has the characteristics of being "international, explosive, migratory, and destructive." The armyworm, also known as the armyworm, has a very varied diet of larvae and mainly harms grass plants. When the armyworm breaks out in large numbers, it often causes large-scale notching of leaves, breaking of corn tassels, and large amounts of tassels and grains being eaten, affecting corn yield and quality. The cotton bollworm is most harmful during the corn ear stage. Its larvae feed on corn tassel grains, inducing ear rot, which directly causes corn yield reduction.

[0003] The prevention and control of corn pests is still mainly based on chemical pesticides, but the long-term use of chemical pesticides often has adverse effects on natural enemies of pests and beneficial insects, and there are many disadvantages such as high cost, easy residue, environmental pollution, and destruction of the ecosystem. Bacillus thuringiensis (Bt) is a type of Gram-positive bacteria. The vegetative insecticidal proteins Vip (vegetative insecticidal proteins) expressed in the vegetative growth period and the parasporal crystal proteins Cry (parasporal crystal proteins) in the spore production period have insecticidal activity. Using Bt insecticidal genes to cultivate insect-resistant corn is an effective means of preventing and controlling corn pests. At present, transgenic insect-resistant corn has been widely used in agriculture around the world, greatly reducing the use of pesticides, increasing crop yields, increasing farmers' income, reducing environmental pollution, and providing important guarantees for food security and ecological security. At present, the most commonly used Bt insecticidal genes are cry1Ab, cry1Ac, cry2Ab, cry1F, vip3Aa, etc., and the transformants are mainly transformed with single insect-resistant genes. Different Bt genes target different pests. For example, the cry1Ab gene has a good insecticidal effect on corn borers, and the cry1F and vip3Aa genes have a good insecticidal effect on fall armyworms. However, with the large-scale planting of transgenic corn for many years, insects are exposed to high selection pressure for a long time, which will cause resistance problems. Investigations have found that pest populations resistant to Cry1Ab and Cry1F proteins have appeared in many parts of the world. However, no fall armyworm populations resistant to Vip3Aa proteins have been found in the field. Due to the threat of multiple pests in corn planting, hybridization is currently required to aggregate two or three genes to control multiple corn pests. For example, the insect-resistant and herbicide-resistant corn DBN3601T, which has obtained a production and application safety certificate, aggregates two corn transformants DBN9936 and DBN9501 to control corn borers and fall armyworms. MIR162 and BT11 aggregate cry1Ab genes and vip3Aa genes through hybridization, which increases the breeding cycle and breeding costs.

[0004] Glyphosate is a broad-spectrum, systemic herbicide. With its broad-spectrum, high-efficiency, and environmentally friendly advantages, it has become the most widely used pesticide in the world. The shikimic acid pathway is an important pathway for the synthesis of aromatic amino acids in plants and microorganisms. In the biosynthesis of phenylalanine, tyrosine, and tryptophan, which are aromatic amino acids in higher plants, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is one of the key enzymes. EPSPS catalyzes the condensation of phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) in the shikimic acid metabolic pathway. Glyphosate is a competitive analog of PEP. Its mechanism of action is to competitively inhibit the activity of EPSPS by forming a stable complex EPSPS-S3P-glyphosate with EPSPS and S3P, blocking the conversion of shikimic acid-3-phosphate to 5-enolpyruvate-3-phosphoshikimic acid, hindering the formation of aromatic amino acid compounds, and causing a large amount of shikimic acid accumulation, which promotes the disorder of hormones and key metabolites such as flavonoids and phenolic compounds, disrupts the normal nitrogen metabolism of organisms and leads to the death of organisms. The gr79-epsps and gat genes were isolated and cloned from the soil bacterial metagenome seriously contaminated by glyphosate. The gr79-epsps gene encodes the EPSPS enzyme. Glyphosate cannot block the reaction of GR79-EPSPS synthase catalyzing PEP and S3P to generate EPSP, allowing the anabolism of aromatic amino acids and other compounds in plants to continue, so that plants can acquire herbicide resistance and protect their normal growth. The gat gene encodes glyphosate-N-acetyltransferase, which provides a new mechanism of action for crop resistance to glyphosate that is different from the GR79-EPSPS pathway. Under the action of glyphosate-N-acetyltransferase, acetyl-CoA acts as an acetyl donor, and the secondary amine of the glyphosate molecule acts as an acetyl receptor, causing glyphosate to be acetylated and lose its herbicide activity, making the transgenic plant resistant to herbicides and protecting the normal development and growth of the plant. The gat and gr79-epsps genes with glyphosate herbicide resistance are transferred into crops to make transgenic crops resistant to glyphosate.

[0005] Insect resistance and herbicide tolerance are the two most important traits of transgenic corn. Insect-resistant and herbicide-resistant corn can prevent and control corn pests while simplifying the corn weeding process, and is favored by growers. At present, most of my country's transgenic insect-resistant corn is single-gene insect-resistant, and some double genes are mainly cry1A and cry2A, cry1A and cry1F combinations. There is no research on the combined expression of cry1Ab and vip3Aa genes in series for insect resistance, and at the same time combined with the herbicide-resistant gene gat and gr79-epsps gene. Therefore, a transgenic crop with insect resistance and herbicide tolerance and its cultivation method are urgently needed in production. Summary of the invention

[0006] In response to the needs in production, the present invention provides a synthetic gene tandem expression frame, which comprises cry1Ab, vip3Aa, gr79-epsps and gat genes and has compound resistance to insects and herbicides.

[0007] The present invention also provides the application of a synthetic gene tandem expression frame, which, after transforming corn, enables it to obtain a composite resistance to insects and herbicides, and has higher resistance to corn borer, fall armyworm, armyworm, and cotton bollworm than a single insect-resistant gene, especially has a synergistic effect on fall armyworm and cotton bollworm, and has a rapid insecticidal effect.

[0008] A synthetic gene tandem expression frame comprises four gene expression frames: cry1Ab, vip3Aa, gr79-epsps and gat.

[0009] The synthetic gene tandem expression cassette, wherein the cry1Ab gene expression cassette includes the ZmUbi promoter, the cry1Ab gene and the NOS terminator, the vip3Aa gene expression cassette includes the OsAct promoter, the vip3Aa gene and the nos terminator, the gr79-epsps gene expression cassette includes the ZmUbi promoter, the gr79-epsps gene and the NOS terminator, and the gat gene expression cassette includes the CaMV35S promoter, the gat gene and the CaMV35S polyA terminator. Figure 1 shown.

[0010] The sequence of the synthetic gene tandem expression frame is shown in SEQ ID No.1.

[0011] The plant expression vector containing the synthetic gene tandem expression frame is named pBRI-IRHT5908, wherein the vector backbone is pCAMBIA2300.

[0012] A host cell, comprising the synthetic gene tandem expression frame, or a plant expression vector comprising the synthetic gene tandem expression frame, wherein the host cell is Agrobacterium EHA105.

[0013] The application of the synthetic gene tandem expression frame is to introduce the synthetic gene tandem expression frame into the plant body to improve its insect resistance and herbicide tolerance.

[0014] The plant is corn, the insect resistance is resistance to lepidopteran pests, and the herbicide resistance is resistance to glyphosate.

[0015] Among them, the lepidopteran pests are resistant to corn borer, fall armyworm, armyworm and cotton bollworm.

[0016] A method for cultivating insect-resistant and herbicide-resistant transgenic corn comprises integrating the synthetic gene tandem expression frame, the plant expression vector, or the host cell into a receptor corn material, wherein the insect resistance is resistance to lepidopteran pests, and the herbicide resistance is resistance to glyphosate.

[0017] Among them, the lepidopteran pests are resistant to corn borer, fall armyworm, armyworm and cotton bollworm.

[0018] Compared with the single-gene insect-resistant or herbicide-resistant genes used in the prior art, the present invention significantly improves the resistance of transgenic corn to lepidopteran insects such as corn borer, fall armyworm, armyworm, and cotton bollworm, especially has a synergistic effect on fall armyworm and cotton bollworm, has the effect of rapid insecticidal effect, and also reaches a high level of resistance to glyphosate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the tandem expression cassettes of the genes synthesized in the plant expression vector pBRI-IRHT5908 for maize transformation;

[0020] Figure 2 PCR test results of maize transformed plants;

[0021] Wherein, M is DM5000 DNA Marker; CK+ is a positive control, and the product is amplified using plasmid pBRI-IRHT5908 as a template; CK- is a non-transgenic plant; 0 is a blank control, and the product is amplified using water as a template; 1-5 are products amplified using T1 generation transgenic corn BRI8001-BRI8005 genomic DNA as a template; 6-10 are products amplified using T2 generation transgenic corn BRI8001-BRI8005 genomic DNA as a template; 11-15 are products amplified using T3 generation transgenic corn BRI8001-BRI8005 genomic DNA as a template;

[0022] Figure 3 Southern blot detection diagram of transgenic corn BRI8001, where M is DNA Marker for Southern Blot; CK+ is the positive control, using plasmid pBRI-IRHT5908 as the template hybridization probe; CK- is the non-transgenic plant; 0 is the blank control, using water as the template hybridization probe; 1 is the hybridization of T3 generation transgenic corn BRI8001 genomic DNA / HindⅢ enzyme digestion product, and the legend is the hybridization probe;

[0023] Figure 4 The expression level of target protein of transgenic corn BRI8001 in different tissues of corn;

[0024] Figure A shows the expression level of Cry1Ab protein; Figure B shows the expression level of Vip3Aa protein; Figure C shows the expression level of GR79-EPSPS protein; and Figure D shows the expression level of GAT protein.

[0025] Figure 5 Field identification of glyphosate resistance of transgenic corn BRI8001, where CK- is a non-transgenic B104 plant. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the embodiments, which are intended to illustrate the present invention rather than to limit 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 may be made to the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0027] Example 1. Obtaining transgenic corn

[0028] 1. Gene codon optimization and plant expression vector construction

[0029] According to the codon preference of corn, the codons of cry1Ab, vip3Aa, gat and gr79-epsps genes were optimized to increase the GC content of the gene coding region. At the same time, sequences that improve translation efficiency and positioning signal peptides were added before the gene start codons to enable efficient expression and translation in corn. The initial vector for constructing the plant expression vector was pCAMBIA2300. The target gene cry1Ab, vip3Aa, gr79-epsps and gat gene expression cassettes were inserted into the multi-cloning site of the vector. Each gene expression cassette included promoter, regulatory elements, gene and terminator sequences (expression cassette information is shown in Tables 1-4). The plant expression vector obtained for corn transformation was pBRI-IRHT5908 (stored in the Institute of Biotechnology, Chinese Academy of Agricultural Sciences). The gene expression cassette map is shown in Figure 1 , the nucleic acid sequence is shown in Sequence 1.

[0030] Table 1 Expression cassettes containing cry1Ab gene

[0031] name Function ZlUT The promoter sequence derived from the maize ubiquitin protein encoding gene ubiquitin drives the expression of the cry1Ab gene OMK Artificially synthesized enhancer Ω and kozak sequences to enhance gene expression cry1Ab Encodes Cry1Ab protein, highly toxic to lepidopteran pests NOS Artificially synthesized polyA sequence and nos terminator to terminate gene expression

[0032] Table 2 Expression cassettes containing vip3Aa gene

[0033] name Function OsAct The promoter sequence derived from the rice actin gene actin drives the expression of the vip3Aa gene OMK Artificially synthesized enhancer Ω and kozak sequences to enhance gene expression vip3a Encodes Vip3Aa protein, highly toxic to lepidopteran pests nos Artificially synthesized nos terminator terminates gene expression

[0034] Table 3 Expression cassettes containing gr79-epsps gene

[0035]

[0036] Table 4 Expression cassette containing gat gene

[0037]

[0038] 2. Obtaining transformed plants by Agrobacterium transformation

[0039] The vector pBRI-IRHT5908 was transformed into Agrobacterium EHA105 by freeze-thaw method and identified by PCR. Freshly peeled maize embryos of about 1.2 mm were used as materials. After the embryos were placed in the infection medium for one hour, they were washed once with the infection medium and then immersed in the Agrobacterium liquid supplemented with 100 μM acetosyringone for 5 minutes. They were taken out and blotted dry with sterile filter paper, placed on the co-culture medium, and co-cultured at 26°C in the dark for 3 days, with a control. The young embryos are then transferred to the recovery medium and cultured for 10 days until callus tissue is induced. The callus tissue is then removed from the buds and then transferred to the screening medium containing glyphosate. The callus tissue is subcultured every two weeks. After 6 weeks of screening, the resistant callus tissue is transferred to the regeneration medium for light differentiation. After about one week of light exposure, green buds begin to appear. The callus blocks are cut to separate the green buds and transferred to the regeneration medium for culture, which is conducive to the growth of the main stem. When the main stem is extended to 3-4 cm, it is transferred to the regeneration medium for rooting induction. When the corn plant grows strong and has a well-developed root system, it is transferred to a small flower pot in the greenhouse for growth. After two weeks of continuous culture, the transformed seedlings are transferred to the greenhouse ground after they grow well. After the female ear spins silk, it is covered with a paper bag. After the male ear scatters pollen, it is pollinated and the fruit is harvested.

[0040] Example 2: Molecular identification of transgenic corn and acquisition of BRI8001 transformants

[0041] The harvested 90 T0 generation corn seeds were planted, and when the corn grew to the 4-leaf stage, glyphosate (Roundup, active ingredient 240g / mu) at 4 times the medium dose used in the field was sprayed. The leaf genomic DNA was extracted from the resistant plants that were healthy and free of glyphosate damage, and the integration of the target genes cry1Ab, vip3Aa, gat and gr79-epsps in the corn transformants was detected by PCR technology. The target gene PCR primers are shown in Table 5, and the PCR system and reaction conditions are shown in Table 6. The results showed that the target genes cry1Ab, vip3Aa, gat and gr79-epsps could be detected in the T1-T3 generation glyphosate-resistant plants, and the band size was consistent with the expected product size, indicating that the complete expression frame formed by the four target genes was integrated into the corn genome as a whole and stably inherited. The PCR electrophoresis patterns of the target genes of some transgenic plants are shown in the following figure: Figure 2At the same time as the glyphosate resistance identification and PCR molecular detection, the insect resistance of the transformed corn plants was identified, and the second-instar corn borer was inoculated into the heart leaves of the glyphosate-resistant corn plants, and finally the corn transformant BRI8001 with high glyphosate resistance, insect resistance and stable inheritance of four genes was obtained for further analysis.

[0042] Table 5 PCR detection primers for target genes cry1Ab, vip3Aa, gat and gr79-epsps

[0043]

[0044] Table 6 PCR reaction system and reaction conditions

[0045]

[0046] Example 3: Southern blot detection of BRI8001

[0047] The Southern blot detection method was used to identify the target gene copy number of BRI8001 transformants. The specific operation steps were carried out in accordance with the "Molecular Cloning Experiment Guide" and related instructions. The genomic DNA of corn leaves of BRI8001 transformants was extracted and completely digested with restriction endonuclease HindⅢ. The digestion products were separated by 0.7% agarose gel electrophoresis and transferred to the membrane. Four target gene fragments were used as probes. The target gene probe primer sequences are shown in Table 5. The Southern blot detection results are shown in Table 5. Figure 3 As shown in the figure, using vip3Aa, gr79-epsps and gat gene fragments as probes, two bands can be hybridized, indicating that two copies of the gene have been transferred. Since HindⅢ enzyme exists on both sides of the cry1Ab gene and can cut off a fragment of about 10Kb, only one band is hybridized. The Southern blot test results show that the exogenous target gene is inserted into the maize genome in the form of two copies. Through the test cross analysis of BRI8001 and Zheng 58, genetic evidence shows that the genetic compound Mendelian 1:1 segregation of the exogenous gene indicates that the exogenous insertion is a double copy insertion of the single site. The probe sequences of cry1Ab, vip3Aa, gr79-epsps and gat genes are shown in Sequence 2, Sequence 3, Sequence 4 and Sequence 5, respectively.

[0048] Example 4: Detection of the expression level of the target protein of BRI8001 transformant in different tissues

[0049] Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of target proteins Cry1Ab, Vip3Aa, GR79-EPSPS and GAT in maize transformant BRI8001 at the seedling stage (roots and leaves), jointing stage (roots, stems and leaves), silking stage (roots, stems, leaves, tassels, filaments and female ears), and mature stage (roots, stems, leaves and grains). ELISA kits were selected from Shanghai Youlong Biotechnology Co., Ltd. Cry1Ab / Ac ELISA Quantitative Detection Kit (Product No.: AA0341), VIP3A ELISA Quantitative Detection Kit (Product No.: AA1641), AM79 EPSPS ELISA Quantitative Detection Kit (Product No.: AA1941, the amino acids of AM79 protein and GR79 protein are the same) and GAT ELISA Quantitative Detection Kit (Product No.: AA1341) to detect the expression levels of Cry1Ab, Vip3Aa, GR79-EPSPS and GAT, respectively. The experimental method and analysis method of ELISA were carried out according to the instructions of the kit.

[0050] The results of ELISA test showed that the target proteins Cry1Ab, Vip3Aa, GR79-EPSPS and GAT could be detected in different tissues and organs of maize transformant BRI8001, and the content of target proteins in different tissues and organs varied greatly. Figure 4 shown.

[0051] Example 5: Indoor insect resistance test of BRI8001 corn transformants

[0052] The maize transformant BRI8001 (containing cry1Ab, vip3Aa, gat and gr79-epsps), the single gene control maize transformed with cry1Ab and vip3Aa, and the transformant receptor control B104 were sown in the greenhouse. When the maize grew to the heart leaf stage, the young heart leaves were selected, rinsed with tap water, rinsed with distilled water once, and the surface water droplets were absorbed with filter paper and dried. The maize leaves with the main veins removed were cut into leaves with a length and width of about 1 cm, and placed in a 24-well cell culture plate, with 1 to 2 pieces per well. The four major maize pests, Asian corn borer, armyworm, cotton bollworm and fall armyworm, were selected as the test insects, and one newly hatched larva was inoculated into each well. Each plate was a replicate, and repeated 3 times. The cells were raised at 26±1℃, relative humidity of 70% to 80%, and a photoperiod of L16:D8 h.

[0053] Silk growth test at the silking stage: Take young corn ears at the silking stage and bring them back to the room. Select young silks and cut them into small pieces about 1 cm in length. Put them into a 24-well cell culture plate, and insert one newly hatched larvae into each well. Each plate is a replicate, and the test is repeated 3 times. Keep them at 26±1℃, relative humidity 70%-80%, and light cycle L16:D8 h.

[0054] The experiment was conducted once a day. Fresh tissue from the same source was added or replaced at any time according to the consumption of the tissue. The number of surviving larvae was recorded. The experiment was observed for 7 days.

[0055] Statistical analysis software was used to conduct variance analysis on the survival rates of different pests after feeding on different corn materials.

[0056] 1. Genetically modified corn BRI8001 has high insecticidal activity against Asian corn borer

[0057] The results of the indoor insect resistance of transgenic corn BRI8001 to Asian corn borer showed that all newly hatched larvae of Asian corn borer died after feeding on transgenic corn BRI8001 leaves for 3 days. The mortality rate of cry1Ab transgenic corn was 88.89% and vip3Aa transgenic corn was 44.44% after 3 days, while the mortality rate of corn borer larvae of non-transgenic control B104 was 2.78%, which was a significant difference. All newly hatched larvae of Asian corn borer died after feeding on transgenic corn BRI8001 silks for 4 days. The mortality rate of cry1Ab transgenic corn was 95.83% and vip3Aa transgenic corn was 54.17% after 4 days, while the mortality rate of corn borer larvae of non-transgenic control B104 was 4.17%, which was a significant difference. The above results show that the insect resistance of transgenic corn BRI8001 to corn borer is significantly better than that of transgenic single-gene materials. Especially in the silking stage, judging from the 2-day mortality rate, a synergistic effect was achieved on the newly hatched larvae of the Asian corn borer, indicating that the tandem gene expression frame has a rapid killing effect on the Asian corn borer.

[0058] Table 7 Indoor bioassay results of transgenic corn BRI8001 against Asian corn borer

[0059]

[0060] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between treatments at the α = 0.05 level.

[0061] 2. GM corn BRI8001 has high insecticidal activity against armyworms

[0062] The results of the indoor insect resistance of transgenic corn BRI8001 to armyworms showed that all newly hatched larvae of heartleaf armyworms died after feeding on transgenic corn BRI8001 leaves for 4 days, and the mortality rate of cry1Ab transgenic single-gene corn was 97.22% and 81.94% after 4 days of vip3Aa transgenic single-gene corn, while the survival rate of armyworm larvae in the non-transgenic control B104 reached 100.00%, and no armyworms died, which was a significant difference. All newly hatched larvae of silkworms died after feeding on transgenic corn BRI8001 silk for 4 days, and the mortality rate of cry1Ab transgenic single-gene corn was 94.44% and 66.67% after 4 days of vip3Aa transgenic single-gene corn, while the survival rate of armyworm larvae in the non-transgenic control B104 reached 100.00%, which was a significant difference. The above results show that the insect resistance of transgenic corn BRI8001 to armyworms is significantly better than that of transgenic single-gene materials.

[0063] Table 8 Indoor bioassay results of transgenic corn BRI8001 against armyworms

[0064]

[0065] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between treatments at the α = 0.05 level.

[0066] 3. Genetically modified corn BRI8001 has high insecticidal activity against cotton bollworm

[0067] The results of the indoor insect resistance of transgenic corn BRI8001 to cotton bollworm showed that all newly hatched larvae of cotton bollworm died 4 days after feeding on the silk of transgenic corn BRI8001 during the silking period. The mortality rate of cry1Ab transgenic corn was 94.44% and vip3Aa transgenic corn was 45.83% after 4 days, while the mortality rate of cotton bollworm larvae of the non-transgenic control B104 was 5.56% after 4 days, which was a significant difference. The above results show that the insect resistance of transgenic corn BRI8001 to cotton bollworm is significantly better than that of transgenic single-gene materials. Especially in the silking period, from the perspective of 2d mortality, it has a synergistic effect on newly hatched larvae of cotton bollworm, indicating that this tandem gene expression frame has a rapid killing effect on cotton bollworm.

[0068] Table 9 Indoor bioassay results of transgenic corn BRI8001 against cotton bollworm

[0069]

[0070] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between treatments at the α = 0.05 level.

[0071] 4. GM corn BRI8001 is highly resistant to fall armyworm

[0072] The results of the indoor insect resistance of transgenic corn BRI8001 to fall armyworm showed that all newly hatched larvae of fall armyworm at the heart leaf stage fed on the leaves of transgenic corn BRI8001 for 2 days, the mortality rate of cry1Ab transgenic corn was 25.00% after 2 days, and the mortality rate of vip3Aa transgenic corn was 51.39% after 2 days, while the survival rate of fall armyworm larvae of the non-transgenic control B104 reached 100.00%, with significant differences. All newly hatched larvae of fall armyworm at the silking stage fed on the silk of transgenic corn BRI8001 for 3 days, the mortality rate of cry1Ab transgenic corn was 18.06% after 3 days, and the mortality rate of vip3Aa transgenic corn was 90.28% after 3 days, while the survival rate of fall armyworm larvae of the non-transgenic control B104 reached 100.00%, with significant differences. The above results show that the insect resistance of transgenic corn BRI8001 to fall armyworm is significantly better than that of transgenic single-gene materials. Especially in the silking stage and heart leaf stage, judging from the 2-day mortality rate, a synergistic effect was achieved on the newly hatched larvae of the fall armyworm, indicating that the tandem gene expression frame has a rapid killing effect on the fall armyworm.

[0073] Table 10 Indoor bioassay results of transgenic corn BRI8001 against fall armyworm

[0074]

[0075] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between treatments at the α = 0.05 level.

[0076] The indoor biological test results of the above four major corn pests show that BRI8001, which is transgenic with two insect-resistant genes cry1Ab and vip3Aa, has high insecticidal activity against the four pests tested, while single-gene transgenic corn does not have high insecticidal activity against the four corn pests. For example, transgenic corn with cry1Ab gene has insecticidal activity against corn borer, cotton bollworm and armyworm, but has lower activity against fall armyworm. Similarly, transgenic corn with vip3Aa gene has insecticidal activity against fall armyworm, but its insecticidal activity against corn borer, armyworm and cotton bollworm is lower than that of BRI8001.

[0077] Example 6: High tolerance of transgenic corn BRI8001 to glyphosate in the field

[0078] 1. Test materials

[0079] The genetically modified corn BRI8001 and the non-genetically modified corn control B104 were used as the target herbicide. Roundup (41% glyphosate isopropylamine salt) produced by Monsanto Company was used as the target herbicide.

[0080] 2. Experimental Design

[0081] Block design, 3 replications, plot size 30m 2 (5m×6m), row spacing 60cm, plant spacing 25cm. A 1.0m wide isolation belt was set between the plots. The treatments included no herbicide spraying on GM corn, spraying the target herbicide (glyphosate) on GM corn, no herbicide spraying on non-GM corn, and spraying the target herbicide (glyphosate) on non-GM corn. The herbicide application rates were 1, 2, and 4 times the dose in the pesticide registration label (active ingredient 60g / mu) (Table 11), and the watering rate was 450L / hectare. Application was carried out according to the recommended time for herbicide-resistant corn.

[0082] Table 11 Glyphosate herbicide dosage

[0083]

[0084] 3. Survey Methods

[0085] The survival rate, plant height, and pesticide damage symptoms were investigated and recorded at 1, 2, and 4 weeks after the application of the drug. The method was based on "Environmental Safety Testing of Transgenic Plants and Their Products - Herbicide-Resistant Corn Part 1: Herbicide Tolerance" (Ministry of Agriculture and Rural Affairs Announcement No. 953-11.1-2007). The herbicide damage rate was calculated as follows.

[0086]

[0087] In the formula: X is the damage rate, in percentage (%); N is the number of damaged plants at the same level; S is the level number; T is the total number of plants; M is the highest level.

[0088] Grading of pesticide damage symptoms: Level 1: Corn grows normally without any harmful symptoms; Level 2: Corn is slightly damaged by pesticides, with damage less than 10%; Level 3: Corn is moderately damaged by pesticides and can recover later without affecting yield; Level 4: Corn is severely damaged by pesticides and difficult to recover, resulting in reduced yield; Level 5: Corn is severely damaged by pesticides and cannot recover, resulting in a significant reduction in yield or even a complete loss of yield.

[0089] 4. Results Analysis

[0090] BRI8001 had a 100% seedling rate after 1, 2, and 4 weeks without herbicide spraying and after 1, 2, and 4 times of medium dose glyphosate spraying. The non-transgenic corn control B104 had a 100% seedling rate after 1, 2, and 4 weeks without herbicide spraying. After 1, 2, and 4 times of medium dose glyphosate spraying, the plants died and the seedling rate was 0. Figure 5 shown.

[0091] Table 12 Seedling rate of corn materials after spraying glyphosate

[0092]

[0093] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between the treatments at the α = 0.05 level. 1×, 2×, 4× respectively represent spraying of medium-dose herbicide, spraying of 2 times the medium-dose herbicide, and spraying of 4 times the medium-dose herbicide.

[0094] BRI8001 had no damage after being sprayed with no herbicide or 1, 2, or 4 times the medium dose of glyphosate for 1, 2, or 4 weeks. The non-transgenic corn control B104 had no damage after being sprayed with no herbicide for 1, 2, or 4 weeks, but all the plants died after being sprayed with 1, 2, or 4 times the medium dose of glyphosate for 1 week, with a damage rate of 100%.

[0095] Table 13 Damage rate of corn materials after spraying glyphosate

[0096]

[0097] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between the treatments at the α = 0.05 level. 1×, 2×, 4× respectively represent spraying of medium-dose herbicide, spraying of 2 times the medium-dose herbicide, and spraying of 4 times the medium-dose herbicide.

[0098] There was no significant difference in plant height between BRI8001 without herbicide spraying and spraying with 1, 2, and 4 times the medium dose of glyphosate for 1, 2, and 4 weeks and the non-transgenic corn control B104 without herbicide spraying.

[0099] Table 14 Plant height survey results under different glyphosate spraying concentrations

[0100]

[0101] Note: The data in the table are mean ± standard error. The lowercase letters after the data indicate the significant difference between the treatments at the α = 0.05 level. 1×, 2×, 4× respectively represent spraying of medium-dose herbicide, spraying of 2 times the medium-dose herbicide, and spraying of 4 times the medium-dose herbicide.

[0102] The above experiments show that after the four gene tandem expression frames of cry1Ab, vip3Aa, gr79-epsps and gat were transferred into corn, its herbicide resistance was enhanced, and the insect resistance was not affected by the herbicide resistance genes. Moreover, a synergistic effect occurred between the insect-resistant genes, which had the effect of rapid insecticidal effect.

Claims

1. A synthetic gene tandem expression cassette comprising four gene expression cassettes: cry1Ab, vip3Aa, gr79-epsps and gat.

2. The synthetic gene tandem expression cassette according to claim 1, wherein the cry1Ab gene expression cassette includes a ZmUbi promoter, a cry1Ab gene and a NOS terminator, the vip3Aa gene expression cassette includes an OsAct promoter, a vip3Aa gene and a nos terminator, the gr79-epsps gene expression cassette includes a ZmUbi promoter, a gr79-epsps gene and a NOS terminator, and the gat gene expression cassette includes a CaMV35S promoter, a gat gene and a CaMV35S polyA terminator.

3. The synthetic gene tandem expression cassette according to claim 2, whose sequence is shown in SEQ ID No.

1.

4. A plant expression vector comprising the synthetic gene tandem expression cassette according to any one of claims 1 to 3, named pBRI-IRHT5908, wherein the vector backbone is pCAMBIA2300.

5. A host cell comprising the synthetic gene tandem expression cassette according to any one of claims 1 to 3, or a plant expression vector comprising the synthetic gene tandem expression cassette according to claim 4, wherein the host cell is Agrobacterium EHA105.

6. Use of the synthetic gene tandem expression cassette according to any one of claims 1 to 3, for introducing the synthetic gene tandem expression cassette into a plant to improve its insect resistance and herbicide tolerance.

7. The use according to claim 6, wherein the plant is corn, the insect resistance is resistance to lepidopteran pests, and the herbicide resistance is resistance to glyphosate.

8. The use according to claim 7, wherein the lepidopteran pests are resistant to corn borer, fall armyworm, armyworm and cotton bollworm.

9. A method for cultivating insect-resistant and herbicide-resistant transgenic corn, comprising integrating the synthetic gene tandem expression frame described in any one of claims 1 to 3, the plant expression vector described in claim 4, or the host cell described in claim 5 into a recipient corn material, wherein the insect resistance is resistance to lepidopteran pests, and the herbicide resistance is resistance to glyphosate.

10. The method according to claim 9, wherein the lepidopteran pests are resistant to corn borer, fall armyworm, armyworm and cotton bollworm.

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