Transcriptional enhancer and its use in improving plant resistance

Abstract

This invention discloses a transcription enhancer and its application in improving plant resistance, belonging to the field of variation or genetic engineering. The technical problem solved by this invention is how to increase gene expression levels. The transcription enhancer disclosed in this invention is STE-26, which is the DNA molecule shown in SEQ ID No. 5. STE-26 is inserted site-specifically into an endogenous gene (…). BX1 433 bp upstream of the transcription start site (TSS) of the gene, BX1 The expression level of the gene reached 7.7 times that of the wild type, indicating that STE-26 has enhancer activity, and resistance to Fusarium graminearum, Asian corn borer, and beet armyworm were also significantly improved. This demonstrates that STE-26 of the present invention can not only enhance the expression level of the target gene, but also improve... BX1 Increased gene expression levels enhance maize resistance and regulate its agronomic traits.

Description

Technical Field

[0001] This invention belongs to the field of variation or genetic engineering, and specifically relates to a transcription enhancer and its application in improving plant resistance. Background Technology

[0002] An enhancer is a non-coding DNA sequence that can activate gene transcription independently of its distance and orientation from the target gene by recruiting transcription factors and cofactors. Enhancers can activate target genes across long genomic distances (even millions of base pairs). Utilizing enhancer elements for targeted insertion into the promoter regions of endogenous plant genes through gene editing to significantly increase target gene expression is a crucial strategy for crop trait improvement. Summary of the Invention

[0003] The purpose of this invention is to provide a DNA molecule with enhancer function.

[0004] The DNA molecule provided by this invention is named STE-26, and its sequence is SEQ ID No. 5.

[0005] The present invention also provides biomaterials related to STE-26, which are at least one of the following: D1) An expression cassette containing the DNA molecule; D2) A recombinant vector containing the DNA molecule or the expression cassette described in D1); D3) A recombinant microorganism containing the DNA molecule, the expression cassette of D1), or the recombinant vector of D2); D4) A plant containing the DNA molecule, the expression cassette of D1) or the recombinant vector of D2).

[0006] In this context, D1) refers to an expression cassette containing STE-26 in its promoter. The term "expression cassette" refers to DNA capable of expressing a target protein in a host cell, which may include not only a promoter to initiate transcription of the target gene but also a terminator to terminate transcription. The expression cassette of this invention also includes an enhancer sequence.

[0007] The term "vector" refers to any construct that can introduce heterologous DNA into corn cells.

[0008] The term "construct" refers to any recombinant DNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, or bacteriophages.

[0009] In one embodiment of the present invention, D2) the recombinant vector is a GRAND editing vector, wherein the GRAND editing vector is formed by combining two elements of the p5175-GZ vector. BsaThe recombinant vector was obtained by replacing the sequence between the I restriction sites with the DNA fragment shown in SEQ ID No. 6.

[0010] D4) The plant referred to is a plant cell, plant tissue, plant organ and / or whole plant.

[0011] "Plant cell" is the biological cell of a plant, which is taken from the plant or derived from a culture obtained by culturing cells taken from the plant.

[0012] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0013] The application of STE-26 as a transcriptional enhancer is also within the scope of protection of this invention.

[0014] The term "transcriptional enhancer" (also referred to herein as an enhancer) refers to DNA that enhances the transcription of a target gene to which it is operatively linked. In other words, an enhancer is DNA that has enhanced transcriptional activation capabilities relative to the natural regulatory sequence of the target gene. In one embodiment of the invention, the target gene is... BX1 Genes. In some embodiments of the present invention, the... BX1 The natural regulatory sequence of the gene is as described above. BX1 Gene promoter. In one embodiment of the invention, the... BX1 The promoter of the gene is the DNA molecule shown in SEQ ID No. 2.

[0015] In this application, the term "operable link" refers to an operable connection between segments of a nucleic acid that are functionally related to each other. In some embodiments, the promoter and enhancer of the target gene in an operable link lead to transcription of the open reading frame of the target gene.

[0016] The application of STE-26 in the preparation of strong promoters is also within the scope of protection of this invention.

[0017] The application of STE-26 in improving plant gene expression is also within the scope of protection of this invention.

[0018] The present invention also provides a method for preparing maize, the method comprising inserting the DNA molecule shown in SEQ ID No. 5 into the promoter of the maize BX1 gene to be modified to obtain the target maize; The target corn variety possesses the characteristic of enhanced resistance; The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

[0019] The insertion of the DNA molecule shown in SEQ ID No. 5 into the promoter must not affect the original sequence of the natural promoter of the gene, and the inserted DNA molecule must be able to function as an enhancer.

[0020] Furthermore, the method is achieved by replacing the DNA fragment with the nucleotide sequence SEQ ID No. 11 in the promoter of the maize BX1 gene to be improved with a DNA fragment with the nucleotide sequence shown in SEQ ID No. 12.

[0021] The target maize includes at least one of its seeds, hybrid F1 and its offspring.

[0022] In some embodiments of the present invention, the maize to be improved contains the BX1 protein or the gene encoding the BX1 protein (BX1 gene).

[0023] In one embodiment of the present invention, the corn to be improved is sweet waxy corn inbred line N355.

[0024] This invention also provides the application of the aforementioned biomaterial in improving maize resistance; The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

[0025] This invention targets corn through GRAND editing. BX1 The gene was modified by inserting the transcriptional enhancer STE-26 into its promoter region, resulting in homozygous edited maize. BX1 Significantly increased gene expression levels also led to significantly increased resistance to Fusarium graminearum, the Asian corn borer, and the beet armyworm. This indicates that resistance can be improved by increasing gene expression levels. BX1 Genetically bred corn with insect-resistant and disease-resistant traits.

[0026] This invention developed a transcriptional enhancer, STE-26, homologous to a 35S promoter truncation sequence. Identification results in maize showed that STE-26 could be inserted site-specifically into an endogenous gene (…). BX1 433 bp upstream of the transcription start site (TSS) of the gene, BX1The gene expression level reached 7.7 times that of the wild type, indicating that the element has enhancer activity, and resistance to Fusarium graminearum, Asian corn borer, and beet armyworm were also significantly improved. This demonstrates that the transcriptional enhancer STE-26 of this invention can not only enhance the expression level of the target gene, but also improve... BX1 Improving gene expression levels enhances maize resistance and regulates maize agronomic traits, thus holding potential applications in maize breeding. Attached Figure Description

[0027] Figure 1 . Schematic diagram of the GRAND editing media.

[0028] Figure 2 A schematic diagram of GRAND-edited site-specific insertion of the BX1 gene. The scale bar in the upper right corner is 100 bp.

[0029] Figure 3 STE-26 strain BX1 Gene expression levels. Note: t-test used. ****: P <0.0001. STE-26 indicates the STE-26 strain.

[0030] Figure 4 Stalk disease incidence after field inoculation with Fusarium graminearum. Note: The blank control is WT maize plants injected with the same volume of H2O; STE-26 represents the STE-26 strain. The figure shows the injected stem node (the fourth stem node of the maize plant closest to the ground). Scale bar = 5 cm.

[0031] Figure 5 Body weight change in beet armyworm. Note: (a) Body weight change curve; (b) Body weight growth rate. STE-26 indicates the STE-26 strain. Using t-test, *: P <0.05.

[0032] Figure 6 Day 11: Beet armyworm feeding on leaves. STE-26 indicates the STE-26 strain. Note: Scale bar = 1 cm.

[0033] Figure 7 Body weight change in Asian corn borer. Note: (a) Body weight change curve; (b) Body weight growth rate. STE-26 represents the STE-26 strain. Using t-test, ***: P <0.001.

[0034] Figure 8 Day 11: Corn borer leaf-eating situation. STE-26 indicates the STE-26 strain. Note: Scale bar = 1 cm.

[0035] Figure 9Results of maize agronomic traits. Note: (a) ear height; (b) number of rows per ear; (c) number of kernels per row; (d) number of kernels per ear; (e) 100-kernel weight; (f) kernel weight per ear; (g) kernel length; (h) kernel width; (i) tip barrenness length. STE-26 indicates the STE-26 line. t-test was used, ns: no significant difference. Detailed Implementation

[0036] In this document, unless otherwise defined herein, terms should be understood according to their common usage by those skilled in the art. Examples of resources describing many of the molecular biology-related terms used herein can be found in the following references: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0039] In the quantitative experiments described below, at least three replicate experiments were performed.

[0040] The waxy maize inbred line N355 in the following examples is "the waxy maize cultivars N355" disclosed in the following literature. This biological material is available to the public from the applicant and is used solely for replicating the relevant experiments of this invention; it may not be used for any other purpose. (Wang, Y.,) et al. (2021). Creation of aromaticmaize by CRISPR / Cas. J. Integr. Plant Biol. 63: 1664–1670.

[0041] Fusarium graminearum in the following examples ( Fusarium graminearum ): Shandong Shunfeng Biotechnology Co., Ltd.

[0042] Example 1: Improving corn BX1 Gene expression levels can enhance the insect resistance and disease resistance of maize. This embodiment found that improving corn BX1 Gene expression levels can enhance insect resistance and disease resistance in maize. In the sweet waxy maize inbred line N355, BX1 The genome sequence of the gene is SEQ ID No. 1 (exons are positions 669-974, 1112-1421, 1516-1592, 1672-1749, 1881-2018, and 2117-2251 of SEQ ID No. 1), driving... BX1 The sequence of the promoter for gene expression is SEQ ID No. 2. BX1 The CDS sequence of the gene is SEQ ID No. 3, which encodes the BX1 protein shown in SEQ ID No. 4.

[0043] I. Construction of Recombinant Vectors Using GRAND editing methods BX1 The transcriptional enhancer STE-26 (STE: Short Transcriptional Enhancer) was inserted upstream of the gene. This clarified the N355 maize gene. BX1 Gene promoter sequences were analyzed to identify the regulatory elements present within them. BX1 STE-26 was inserted 139 bp upstream of the transcription start site, avoiding important regulatory elements to prevent fragmentation and disruption after insertion. The inserted transcription enhancer STE-26 is 26 bp in length and its sequence is SEQ ID No. 5.

[0044] To construct the GRAND editing vector, the target was first designed and synthesized. BX1DNA fragments of epigRNAs that are gene promoters and inserted into transcription enhancers (named Spacer1-scaffold-RTT1-PBS-linker-evopreQ1-HDV-linker-tRNA (Met)-Spacer2-scaffold-RTT2-PBS2-linker-evopreQ1, sequence SEQ ID No. 6).

[0045] In SEQ ID No. 6, positions 1-20 are the DNA sequence of Spacer 1, positions 21-106 are the DNA sequence of scaffold, positions 107-124 are the DNA sequence of RTT1, positions 125-137 are the DNA sequence of PBS, positions 138-145 are the DNA sequence of linker, positions 146-183 are the DNA sequence of evopreQ1, positions 1-183 are the DNA sequence of epigRNA1; positions 184-251 are the DNA sequence of HDV, and positions 252-259 are the DNA sequence of linker (…). The DNA sequence of the linker is as follows: positions 260-331 are the DNA sequence of tRNA (Met); positions 332-351 are the DNA sequence of Spacer2; positions 352-437 are the DNA sequence of scaffold; positions 438-455 are the DNA sequence of RTT2; positions 456-468 are the DNA sequence of PBS2; positions 469-476 are the DNA sequence of the linker; positions 477-514 are the DNA sequence of evopreQ1; and positions 332-514 are the DNA sequence of epigRNA2.

[0046] The ApR and scaffold sequences of the maize guided editing (PE3) vector p5175 were deleted, and the resulting vector was named p5175-GZ. p5175-GZ is a double-stranded plasmid with a length of 27848 bp. The first 15000 positions of one strand are the first 15000 positions of SEQ ID No. 7, and the first 15001-27848 positions are the first 12848 positions of SEQ ID No. 8.

[0047] Bits 391-1384 of SEQ ID No. 7 represent the promoter. pU6-Com Positions 1392-1462 show the DNA sequence of tRNA (Gly), and positions 1464-1469 and 1478-1483 show... BsaThe recognition sequence for I, positions 1493-1560, is the DNA sequence of HDV.

[0048] Using the p5175-GZ vector as the basic framework, the two vectors in the p5175-GZ vector were separated using the Golden Gate method. Bsa The sequence between the I restriction sites was replaced with DNA fragments of synthesized epigRNAs. The resulting recombinant vector with the correct sequence was designated as the GRAND editing vector. This GRAND editing vector contains a promoter... pU6-Com The transcription of epigRNA is driven by the ZmUBI promoter, which drives the expression of the nCas9 (H840A) recombinant gene bpNLS-nCas9(H840A)-XTEN-NC-NLS-LINKER-M-MLV-bpNLS protein. Figure 1 ).

[0049] The sequence of the nCas9(H840A) recombinant gene is SEQ ID No. 9, encoding the nCas9(H840A) recombinant protein shown in SEQ ID No. 10.

[0050] In SEQ ID No. 9, positions 1-117 are the DNA sequence of bpNLS, positions 118-4218 are the DNA sequence of nCas9 (H840A), positions 4219-4335 are the DNA sequence of XTEN, positions 4336-4503 are the DNA sequence of NC, positions 4504-4524 are the DNA sequence of NLS, positions 4525-4632 are the DNA sequence of LINKER, positions 4633-6174 are the DNA sequence of M-MLV, and positions 6175-6306 are the DNA sequence of bpNLS.

[0051] II. Agrobacterium-mediated genetic transformation and genotype identification of maize Using sweet waxy maize inbred line N355 as the recipient plant, the GRAND editing vector was introduced into Agrobacterium tumefaciens EHA105. The embryos were then infected using Agrobacterium-mediated maize genetic transformation to obtain callus tissue, which was then further cultured to obtain T0 generation transgenic plants.

[0052] To identify the genotype of T0 generation transgenic plants, genomic DNA was extracted from leaves. Two rounds of PCR were used to amplify the target sites, followed by next-generation sequencing (NGS) analysis. Editing efficiency was determined using a 10% threshold in the Hi-TOM assay.

[0053] Sequencing results showed that the editing efficiency of precise insertion of the transcriptional enhancer STE-26 was 13.04% (3 / 23), and the efficiency of non-target editing-induced insertions and deletions was 17.39% (4 / 23) (Table 1). The screened target-edited plants with precise STE-26 insertion (plant numbers are #1, #10, #21 respectively) are all heterozygous genotypes (Table 2), and homozygous plants need to be obtained through further selfing isolation.

[0054] The insertion position of STE-26 in the plant numbered #1 is shown in Figure 2 , the STE-26 enhancer is site-specifically inserted into maize BX1 gene 433 bp upstream of the transcription start site (TSS).

[0055] Table 1. Editing efficiency of GRAND editing in T0 generation transgenic plants

[0056] Table 2. Genotype of target-edited plants in T0 generation

[0057] Note: The bolded part indicates the inserted nucleotide.

[0058] III. Detection of maize BX1 gene expression level by real-time quantitative PCR (qPCR) To identify the change in the expression level of maize BX1 gene after inserting the transcriptional enhancer, RNA was extracted from the leaves of homozygous T3 generation plants containing STE-26 of target-edited plant #1 (recorded as STE-26 line, which is homozygous maize in which the enhancer shown in SEQ ID No.5 replaces the DNA fragment shown by the reverse complementary sequence at positions 881-898 of SEQ ID No.2) and reverse transcribed into cDNA. Primers were designed based on the CDS sequence of maize BX1 gene, with a length of 18-24 nt, a GC content of 40%-60%, a Tm value of 58-62°C, and an amplification product length of 100-200 bp. The qPCR reaction was performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotech), the relative expression level of the gene was calculated according to the obtained ct value, and a differential bar graph was drawn. The results are as follows: Primers used BX1 Gene primers are: q-BX1-F: 5′-CCCGAGCACGTAAAGCAGAT-3′ (SEQ ID No.14); q-BX1-R: 5′-CTTCATGCCCTGGCATACT-3′ (SEQ ID No. 15).

[0059] The internal reference is the GAPDH gene, and the primers are: q-GAPDH-F: 5′-AGCAGGTCGAGCATCTTCG-3′ (SEQ ID No. 16); q-GAPDH-R: 5′-CTGTAGCCCCACTCGTTGTC-3′ (SEQ ID No. 17).

[0060] qPCR results showed that the edited maize line with STE-26 insertion... BX1 Gene expression levels were 7.7 times higher than those of wild-type maize N355 (WT). Figure 3 This indicates that the transcriptional enhancer effectively upregulated... BX1 Gene expression levels.

[0061] IV. Field inoculation to identify maize disease resistance The maize to be tested was the STE-26 line, wild-type maize N355 (WT).

[0062] Fusarium graminearum ( Fusarium graminearum The bacterial strain was inoculated onto PDA medium and cultured at 28°C for approximately 5 days. Mycelial discs (approximately 0.5 cm in diameter) were pressed out from the medium and transferred into mung bean soup medium using a sterile syringe needle, 20 discs per 100 mL culture bottle. The culture bottles were then placed in a shaker at 28°C and 200 rpm to induce sporulation. After 4 days of culture, the bacterial suspension was filtered through double-layered sterile gauze, and the spores were collected by centrifugation. The spores were resuspended in sterile water to prepare a spore suspension, and the concentration was adjusted to 10. 6 / mL. Inoculation was performed during the flowering stage of the corn to be tested. A cone was inserted at a 45° angle downwards into the middle of the fourth stem node closest to the ground, ensuring the cone reached the middle of the stem without penetrating it. 1.5 mL of spore suspension was injected into the wound using a syringe. Simultaneously, an equal volume of sterile water was injected into wild-type corn as a blank control. 25 plants were inoculated for each line. The field was watered once before inoculation, followed by normal fertilizer and water management. No pesticides were applied before or after inoculation. Twelve days later, each plant was identified. The injected stem node was cut off with a sickle, and the stem was longitudinally sectioned along the wound. The percentage of lesions on the cross-section was counted for each plant, photographed, and the disease severity grade of each plant was recorded. The grading criteria are shown in Table 3. The disease severity grade of each line was calculated to evaluate the disease resistance and susceptibility level.

[0063] Table 3. Disease severity classification of maize inoculated with Fusarium graminearum

[0064] Disease severity of each strain = (M1 + M2 + … + M)R ) / R, where R is the total number of replicates for the strain; M1, M2...M R These represent the disease severity of the 1st, 2nd...Rth repeating plants, respectively.

[0065] Compared with wild-type maize, the lesions on the stems of the STE-26 line were significantly reduced. Figure 4 Based on this assessment, the disease grade of the STE-26 strain was also significantly reduced (Table 4), decreasing from grade 5.67 for wild-type maize to grade 4.52. (Note: This likely refers to a separate point in the original text.) BX1 Maize with increased gene expression levels exhibits enhanced resistance to Fusarium graminearum.

[0066] Table 4. Disease severity of plants after field inoculation with Fusarium graminearum

[0067] Note: Data is presented as mean ± standard deviation. **: P <0.01.

[0068] V. Indoor Insect Inoculation Test to Determine Corn's Insect Resistance The maize to be tested was the STE-26 line, wild-type maize N355 (WT).

[0069] beet armyworm ( Spodoptera exigua ) or Asian corn borer ( Ostrinia furnacalis (Guenée) Insect eggs were placed in a culture box and cultured at 26℃ with 16 h light / 8 h dark to obtain newly hatched larvae. Young heart leaves of the test corn (four to five leaves and one heart stage) grown in a greenhouse were taken, cut into appropriate sizes, and placed in a 12-well culture plate. One newly hatched larva was inoculated into each well using a paintbrush. Each strain had one 12-well plate, inoculated with 12 larvae, and cultured at 26℃ with 16 h light / 8 h dark. The day of inoculation was recorded as day 0 (0d). Three days after inoculation, the leaves were photographed to show signs of larval damage, the larvae's weight was measured, and fresh leaves were replaced. Fresh leaves were then replaced every other day, and the larvae's weight was recorded for 11 days. The larvae's growth curve was statistically analyzed, and the weight growth rate was calculated. The weight on day 3 after inoculation was taken as the initial weight (W0), and the weight on day 11 (t=11) was taken as the final weight (W). t ), calculate the weight gain rate, weight gain rate = (W t -W0) / W0. During the experiment, it is necessary to keep edible leaves in the culture plate. Therefore, the frequency of replacing fresh leaves can be adjusted according to the grazing situation after 8-11 days.

[0070] Feeding trials involving beet armyworm showed that beet armyworms fed STE-26 strain leaves experienced significantly slower weight gain compared to those fed wild-type corn. Figure 5In the middle (a) group, the weight gain rate on day 11 (d) was significantly reduced, with a decrease of 26.81%. Figure 5 (b) indicates that insect growth was inhibited. Observing the leaf erosion, both leaves of the wild-type corn were eaten, while in the STE-26 strain, most leaves had only one obvious insect hole or neither leaf had obvious insect holes. Figure 6 ).

[0071] Feeding trials involving Asian corn borers showed that the weight change curves of corn borers fed with STE-26 strain leaves were significantly slower than those of wild-type corn. Figure 7 In the middle (a) group, the weight gain rate on day 11 (d) decreased significantly, with a decrease of 39.36%. Figure 7 (b)). Regarding leaf erosion, wild-type corn leaves typically show insect holes the size of match heads, while the STE-26 strain leaves show more pinhole-like insect holes. Figure 8 These results illustrate BX1 Increased gene expression levels can enhance corn's resistance to insects.

[0072] VI. Field Agronomic Traits Survey The maize to be tested was the STE-26 line, wild-type maize N355 (WT).

[0073] A randomized block design was used, with three replicate plots for each maize variety. Ridges were prepared in the field, 1.15 m wide, with two rows per ridge. The standard plot size was 5 m² with four rows, plant spacing of 24 cm, and 20 plants per row, with single or double seeding depending on seed quantity. Four protective rows were placed around the perimeter of the maize. Before sowing, 25 kg of 15-15-15 Stanley compound fertilizer and 20 kg of microbial fertilizer were applied per mu (approximately 0.067 hectares). One week after pollination, 10 plants from the middle two rows of each plot were surveyed, and ear height was measured and recorded. Forty-five days after pollination, the middle two rows of ears were harvested from each plot, and yield-related agronomical traits such as number of rows per ear, number of kernels per row, number of kernels per ear, 100-kernel weight, ear weight, kernel length, kernel width, and tip barrenness length were measured, along with yield per mu. Kernel moisture content was measured, and yield calculations were based on a standard moisture content of 14%.

[0074] The results showed that homozygous T3 generation edited maize did not differ significantly from wild-type maize in traits such as ear height, number of rows per ear, number of kernels per row, number of kernels per ear, 100-kernel weight, kernel weight per ear, tip barrenness length, and kernel size (including kernel length and kernel width). Figure 9 In terms of yield, there was no significant difference between homozygous T3 generation edited maize and wild-type maize, indicating that the improved resistance did not have a negative impact on yield (Table 5).

[0075] Table 5. T3 generation maize yield

[0076] Note: Data is presented as mean ± standard deviation.

[0077] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing corn, characterized in that: The method involves inserting the DNA molecule shown in SEQ ID No. 5 into the promoter of the maize BX1 gene to be modified to obtain the target maize; The target corn variety possesses the characteristic of enhanced resistance; The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

2. The method according to claim 1, characterized in that: The method is achieved by replacing the DNA fragment with the nucleotide sequence SEQ ID No. 11 in the promoter of the maize BX1 gene to be improved with a DNA fragment with the nucleotide sequence shown in SEQ ID No.

12.

3. Application of biomaterials in improving maize resistance; characterized by: The biomaterial is at least one of the following: D1) An expression cassette containing the DNA molecule shown in SEQ ID No. 5; D2) A recombinant vector containing the DNA molecule shown in SEQ ID No. 5 or the expression cassette described in D1); D3) A recombinant microorganism containing the DNA molecule shown in SEQ ID No. 5, the expression cassette described in D1), or the recombinant vector described in D2); D4) A plant containing the DNA molecule shown in SEQ ID No. 5, the expression cassette described in D1), or the recombinant vector described in D2); The resistance is insect resistance and / or disease resistance, wherein the insect resistance is resistance to beet armyworm and / or Asian corn borer; and the disease resistance is resistance to Fusarium graminearum.

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