Application of SbWOX gene and SbBBM gene in corn breeding and corn genetic transformation method

Abstract

The application discloses application of SbWOX gene and SbBBM gene in corn breeding and a corn genetic transformation method, and relates to a bioengineering technology.The application provides a nucleic acid molecule with co-expression of SbWOX protein gene and SbBBM protein gene, and corresponding gene co-expression vectors and microbial transformants.The application also provides application of the co-expression of the SbWOX gene and the SbBBM gene in the corn genetic transformation method.The application obtains corresponding genes and mutant proteins based on the SbWOX gene and the SbBBM gene of sorghum, the two genes are co-expressed in the corn genetic transformation process, even without adding plant growth hormones in the antibiotic screening medium in the tissue culture process, the plant genetic transformation process can be normally completed, and the transformation efficiency and the regeneration efficiency of the corn can be remarkably improved, the transformation rate reaches 10.42%, and the differentiation rate reaches 32.89%.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of two sorghum genes in maize breeding and methods for maize genetic transformation. Background Technology

[0002] Plant tissue culture is a crucial technique in bioengineering and a vital tool for plant breeding and gene function research. The process of plant genetic transformation typically involves obtaining plant explants, inducing callus, infecting with Agrobacterium, screening for antibiotics, and undergoing dedifferentiation and redifferentiation of the callus to achieve plant cell totipotency, thus laying the foundation for biotechnological breeding and plant functional gene research.

[0003] To address the genotype limitations in plant genetic transformation, current research has begun focusing on the design and application of helper genes or vectors in the field of plant genetic transformation technology. These are generally transcription factors related to plant cell division and differentiation. Regarding maize genetic transformation methods, the paper "Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation" discloses that the maize genes BBM and WUS can significantly improve transformation efficiency in maize genetic transformation research. Overexpression of the BBM and WUS genes as helper plasmids can improve transformation efficiency in some maize inbred lines. Therefore, the use of specific genes as helper plasmids in maize breeding and genetic transformation technology has broad market prospects. Summary of the Invention

[0004] This invention discloses a gene composition and a specific genetic transformation method for promoting callus differentiation in maize and improving the efficiency of maize genetic transformation. This invention constructs a co-expression vector for the SbWOX and SbBBM genes by cloning the SbWOX and SbBBM genes from sorghum. Verification of maize genetic transformation efficiency demonstrates that, without the addition of plant growth hormones in the culture medium, the co-expression composition of the SbWOX and SbBBM genes can significantly improve the transformation efficiency of maize genetic transformation and ensure the normal completion of the maize genetic transformation process. Specifically, this is achieved through the following method.

[0005] In a first aspect, the present invention provides a gene co-expression nucleic acid molecule, comprising a gene sequence encoding an SbWOX protein and a gene sequence encoding an SbBBM protein, wherein a nucleotide sequence encoding a self-splicing peptide is further linked between the gene sequence encoding the SbWOX protein and the gene sequence encoding the SbBBM protein.

[0006] The amino acid sequence of the SbWOX protein is shown in SEQ ID NO.1, or a sequence that has at least 91% homology with the amino acid sequence shown in SEQ ID NO.1 and has the same biological function; the amino acid sequence of the SbBBM protein is shown in SEQ ID NO.2, or a sequence that has at least 91% homology with the amino acid sequence shown in SEQ ID NO.2 and has the same biological function.

[0007] The gene co-expression nucleic acid molecules provided by this invention can be used in the genetic transformation method of explants of plants such as maize.

[0008] The SbWOX and SbBBM proteins provided by this invention are obtained based on "Hongyingzi" sorghum material. It should be noted that:

[0009] (1) The SbWOX protein provided by this invention has a total length of 216 amino acids. Compared with the original amino acid sequence published in the existing sorghum public database annotation (NCBI database gene, ID: XM_002458736.2), it lacks two amino acids at positions 93 and 94—glycine and serine (abbreviation S), and also replaces histidine (abbreviation H) at position 131 with arginine (abbreviation R);

[0010] (2) The SbBBM protein provided by this invention has a total length of 703 amino acids. In comparison, based on the original amino acid sequence published in the existing sorghum public database annotation (NCBI database gene, ID: XM_021457893.1), the proline (abbreviation P) at position 614 is replaced with arginine (abbreviation S).

[0011] It should also be noted that, in addition to the SbWOX protein shown in SEQ ID NO.1 and the SbBBM protein shown in SEQ ID NO.2, the present invention may also select amino acid sequences that have at least 91% homology with the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and these sequences are also required to provide the same biological function.

[0012] Optionally, the homology with the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 is at least 92%. Further optionally, the homology is at least 95%. Further optionally, it is at least 99%.

[0013] The biological functions claimed in this invention refer to functions that can play the same role as SbWOX and SbBBM proteins in this invention. For example, promoting the improvement of transformation efficiency and regeneration efficiency during the genetic transformation of maize plants.

[0014] Furthermore, in the aforementioned gene-co-expressed nucleic acid molecules, the self-cleaving peptide is any one of the self-cleaving peptides T2A, P2A, F2A, and E2A.

[0015] In addition to the specific self-cleaving peptides listed above, other related peptides that can perform the function of self-cleaving peptides can also be used in this invention and fall within the scope of protection of this application.

[0016] Furthermore, among the above-mentioned co-expressed nucleic acid molecules, the gene sequence encoding the SbWOX protein is shown in SEQ ID NO.3, or is a sequence that has at least 91% homology with the gene sequence shown in SEQ ID NO.3 and has the same coding function; the gene sequence encoding the SbBBM protein is shown in SEQ ID NO.4, or is a sequence that has at least 91% homology with the gene sequence shown in SEQ ID NO.4 and has the same coding function.

[0017] Further, optionally, the gene sequence encoding the SbWOX protein may also have at least 92% homology with the gene sequence shown in SEQ ID NO. 3. Even further, the homology may be at least 95%. More preferably, the homology may be at least 99%.

[0018] Similarly, the gene sequence encoding the SbBBM protein may also have at least 92% homology with the gene sequence shown in SEQ ID NO. 4. More preferably, the homology is at least 95%. More preferably, the homology is at least 99%.

[0019] It should be noted that there are two possible cases for homology with the genes shown in SEQ ID NO.3 and SEQ ID NO.4: (1) based on codon diversity, which means that they can encode the SbWOX protein shown in SEQ ID NO.1 and the SbBBM protein shown in SEQ ID NO.2 of the present invention, respectively; (2) other proteins that can encode the same physiological functions as the SbWOX protein and SbBBM protein of the present invention, and can also promote the transformation efficiency and regeneration efficiency of maize plants after co-expression.

[0020] Secondly, the present invention provides a gene co-expression vector comprising the aforementioned gene co-expression nucleic acid molecule.

[0021] Furthermore, the gene co-expression vectors mentioned above were obtained by recombination based on the vector pEGOEPubi-B.

[0022] Furthermore, the nucleotide sequence of the above gene co-expression vector is shown in SEQ ID NO.5.

[0023] It should be noted that, in addition to pEGOEPubi-B, other plasmids or vectors commonly used in this technical field and suitable for this invention can also be selected as the basic vector, such as pCAMBIA3300.

[0024] Thirdly, the present invention provides a method for preparing the above-mentioned gene co-expression vector, comprising the following steps:

[0025] Using the gene encoding the SbWOX protein as a template, PCR amplification was performed using the forward primer shown in SEQ ID NO.6 and the reverse primer shown in SEQ ID NO.7, and the first amplification product was recovered.

[0026] Using the gene encoding the SbBBM protein as a template, PCR amplification was performed using the forward primer shown in SEQ ID NO.8 and the reverse primer shown in SEQ ID NO.9, and the second amplification product was recovered.

[0027] The first and second amplification products were recombined into the base vector to construct the gene co-expression vector.

[0028] It should be noted that, in addition to using the method provided by the present invention, the gene co-expression vector provided by the present invention can also be prepared using other gene co-expression vector construction methods commonly used or known in the art.

[0029] Fourthly, the present invention provides a microbial transformant, which is prepared by transforming the above-mentioned gene co-expression vector into Agrobacterium.

[0030] Generally speaking, microbial transformants refer to the Agrobacterium-derived bacteria obtained by transforming the aforementioned gene co-expression vector into Agrobacterium. These Agrobacterium-derived bacteria, in addition to infecting plant explants during callus preparation, can also express specific proteins in the gene co-expression vector (such as the SbWOX and SbBBM proteins of this invention).

[0031] Fifthly, the present invention provides an application of the above-mentioned microbial transformant in maize genetic transformation.

[0032] In a sixth aspect, the present invention provides a method for genetic transformation of maize, comprising introducing the gene nucleotide sequence encoding the SbWOX protein shown in SEQ ID NO.1 and the gene nucleotide sequence encoding the SbBBM protein shown in SEQ ID NO.2 into maize explants and co-expressing them.

[0033] Furthermore, in the above-mentioned method for genetic transformation of maize, the co-expression method includes: constructing a gene co-expression vector as described in any one of claims 5-7 by combining the nucleotide sequence of the gene encoding SbWOX protein and the nucleotide sequence of the gene encoding SbBBM protein; or forming a microbial transformant as described in claim 9.

[0034] Furthermore, the above-mentioned method for genetic transformation of maize specifically includes the following steps:

[0035] Preparation of suspensions of microbial transformants;

[0036] Maize explants were obtained by cutting.

[0037] The pretreated maize explants are immersed in a suspension of the microbial transformant, or the pretreated maize explants are mixed with the suspension of the microbial transformant for infection and co-cultured in a co-culture medium.

[0038] The co-cultured maize explants were then subjected to recovery culture, and after callus tissue grew, they were screened to complete the maize genetic transformation.

[0039] Optionally, in the above-mentioned methods for maize genetic transformation, the co-culture method in the co-culture medium is to culture at a constant temperature of 22°C in the dark for 3 days.

[0040] Optionally, in the above-mentioned method of maize genetic transformation, the method of restoring the co-cultured maize explants is as follows: the co-cultured maize explants are transferred to a restoration medium and cultured in the dark at 28°C for 25 days to obtain callus tissue.

[0041] It should be noted that commonly used maize explants can be, but are not limited to, maize ears, stems, and leaves. For example, the husks of a maize ear can be removed, and after pretreatment and sterilization, the embryo can be extracted using an embryo removal knife to serve as the maize explant.

[0042] Through extensive experiments, the applicant of this invention constructed three vectors: pEGOEPubi-B-GFP (expressing GFP protein, green fluorescent protein, and blank control group), pEGOEPubi-B-SbWOX / BBM, and pEGOEPubi-B-ZmWUS / BBM (co-expressing ZmWUS and ZmBBM proteins), and transfected them with Agrobacterium for maize genetic transformation experiments. The results demonstrated that the co-expression combination of SbWOX / SbBBM can improve the transformation and regeneration efficiency of maize explant materials, and that the SbWOX / SbBBM combination is significantly superior to the ZmWUS / BBM combination in promoting maize genetic transformation and regeneration.

[0043] It should be noted that the plant genetic transformation method is a conventional technique in this field. In addition to the method provided by the present invention, any other method that can achieve the co-expression of gene co-expression vectors in plant explants can achieve the effect of enhancing plant genetic transformation and regeneration generated by the present invention.

[0044] It should also be noted that in the genetic transformation process of co-expression of the SbWOX and SbBBM genes in this invention, plant genetic transformation can be completed normally even without the addition of plant growth hormones to the antibiotic screening medium during the tissue culture process during the genetic transformation screening stage. In contrast, in existing technologies, in order to ensure genetic transformation efficiency, most require the addition of plant hormones such as growth hormones to the screening medium to promote the differentiation of plant explants into callus and induce seedling growth.

[0045] Seventhly, the application of the above-mentioned method for genetic transformation of maize in the preparation of maize.

[0046] The genetic transformation recipient material selected in this invention is maize B104. It should be noted that the effect of this invention in improving genetic transformation and regeneration efficiency is not limited to maize B104. Besides maize B104, the above-mentioned technical means of this invention are also applicable to other maize inbred lines and varieties, and can similarly significantly improve the genetic transformation and regeneration efficiency of maize.

[0047] It should also be noted that the gene co-expression vector and microbial transformant provided by this invention can be used not only for the genetic transformation process of maize, but also for other monocotyledonous plants, such as rice, wheat, barley, sorghum, millet, foxtail grass, sugarcane, oats, highland barley, ryegrass, etc.

[0048] Compared with the prior art, the advantages of the present invention are as follows: The present invention obtains the corresponding genes and mutant proteins based on the SbWOX gene and SbBBM gene of sorghum. These two genes are co-expressed in the process of maize genetic transformation. Even if no plant growth hormone is added to the antibiotic screening medium during the tissue culture process, the plant genetic transformation process can still be completed normally. Moreover, it can significantly improve the transformation efficiency and regeneration efficiency of maize, with a transformation rate of 10.42% and a differentiation rate of 32.89%. Attached Figure Description

[0049] Figure 1 This is an electrophoresis image of the five gene fragments recovered by the gel extraction kit in Example 2, amplified by PCR. The markers in the image are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0050] Figure 2 The map shows the vector pEGOEPubi-B-GFP;

[0051] Figure 3 The spectrum of the vector pEGOEPubi-B-ZmWUS / BBM;

[0052] Figure 4 The spectrum of the vector pEGOEPubi-B-SbWOX / BBM;

[0053] Figure 5 , 6 These are histological diagrams of the screening and seedling induction stages during the genetic transformation of maize, representing the blank control group, positive control group, and experimental group, respectively. Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] The following specific implementation method uses the "Hongyingzi" sorghum variety as the material for cloning sorghum genes. The sorghum variety was provided by Professor Shao Mingbo of the Dryland Grain Research Institute of the Guizhou Academy of Agricultural Sciences.

[0056] Maize variety B104 was selected as the transformation recipient material, and maize variety B73 was selected as the maize gene cloning material. Both were bred by Wuhan Aidijing Biotechnology Co., Ltd.

[0057] Experimental Example 1: Cloning of the Target Gene

[0058] This implementation plan sets up 3 groups of vectors for experiments, selects maize as the genetic transformation recipient, and further selects the maize ubiquitin promoter UBI to promote the target gene with better results.

[0059] Blank control group: expressed GFP (green fluorescent protein), and publicly available data show that GFP has no effect on maize conversion efficiency;

[0060] Positive control group: co-overexpression of maize ZmWUS and ZmBBM, these two genes have been shown in many studies to have a significant promoting effect on maize genetic transformation;

[0061] Experimental group: The sorghum SbWOX gene and SbBBM gene were co-overexpressed, and these two genes were used as the target genes of this invention;

[0062] The SbWOX gene is based on the NCBI database gene (ID: XM_002458736.2), but it lacks two amino acids at positions 93 and 94—glycine and serine (abbreviation S), and replaces histidine (abbreviation H) at position 131 with arginine (abbreviation R).

[0063] The SbBBM gene is based on the NCBI database gene ID: XM_021457893.1, with the proline (abbreviation P) at position 614 replaced by arginine (abbreviation S).

[0064] For ease of comparison, both the positive control group and the experimental group used a self-cleaving peptide T2A as the linker in their co-expressed structure. Furthermore, the self-cleaving peptide can also be any one of the self-cleaving peptides P2A, F2A, or E2A.

[0065] The cloning primer sequences for the blank control group are as follows (as shown in SEQ ID NO.10 and SEQ ID NO.11, respectively):

[0066] GFP-F: 5'-actaggtctcgcaccatggtgagcaagggcgaggagc-3'

[0067] GFP-R: 5'-actaggtctctaccgtcacttgtacagctcgtccatg-3'

[0068] The cloning primer sequences for the positive control group are as follows (as shown in SEQ ID NO.12 to SEQ ID NO.15 respectively):

[0069] ZmWUS-1F: 5'-actaggtctcgcaccatggcggccaatgcgggcggcggt-3'

[0070] ZmWUS-1R: 5'-actaggtctctgccaacttcaacaaatcaaaattcaacaattgcatactccctgcagca-3'

[0071] ZmBBM-2F: 5'-actaggtctcgtggctggagatgttgaatctaatcctggacctatggccactgtgaaca-3'

[0072] ZmBBM-2R: 5'-actaggtctctaccgttaagtgtcgttccagacactga-3'

[0073] The cloning primer sequences for the experimental group are as follows (as shown in SEQ ID NO.6 to SEQ ID NO.9 respectively):

[0074] SbWOX-1F: 5'-actaggtctcgcaccatggaggcgctgagcgggc-3'

[0075] SbWOX-1R: 5'-actaggtctctgccaacttcaacaaatcaaaattcaacaattggtagggaccgaagctg-3'

[0076] SbBBM-2F: 5'-actaggtctcgtggctggagatgttgaatctaatcctggacctatggctactgtgaaca-3'

[0077] SbBBM-2R: 5'-actaggtctctaccgttaagtatcgttccagacact-3'

[0078] The gene amplification process is as follows:

[0079] Using the primers SbWOX-1F and SbWOX-1R, the SbWOX gene of sorghum was amplified from the cDNA of the "Hongyingzi" material. The actual amplified sequence (as shown in SEQ ID NO.3) and the translated amino acid sequence (as shown in SEQ ID NO.1) were obtained.

[0080] The SbBBM gene of sorghum was amplified from the cDNA of “Hongyingzi” material using the above primers SbBBM-2F and SbBBM-2R. The actual amplified sequence (as shown in SEQ ID NO.4) and the translated amino acid sequence (as shown in SEQ ID NO.2) are shown.

[0081] Using the primers ZmWUS-1F and ZmWUS-1R, the ZmWUS gene of maize was amplified from the cDNA of the “B73” material. The nucleotide sequence encoding the ZmWUS protein that was actually amplified is shown in SEQ ID NO.16.

[0082] Using the primers ZmBBM-2F and ZmBBM-2R, the ZmBBM gene of maize was amplified from the cDNA of the "B73" material. The nucleotide sequence encoding the ZmBBM protein that was actually amplified is shown in SEQ ID NO.17.

[0083] The GFP gene was amplified using the above primers GFP-F / GFP-R, and the actual amplified nucleotide sequence encoding the GFP protein is shown in SEQ ID NO.18.

[0084] The amplification system (the high-fidelity enzyme KOD-FX for amplification was purchased from Toyobo Biotechnology Co., Ltd.) is detailed in Table 1 below.

[0085] Table 1 Amplification System

[0086]

[0087] The PCR amplification reaction procedure is shown in Table 2 below.

[0088] Table 2 PCR amplification reaction procedure

[0089]

[0090] Experiment Example 2: Construction of three test vectors;

[0091] The five gene fragments mentioned above were recovered using a gel extraction and recovery kit (purchased from Axygen). Figure 1 As shown. Enzyme digestion and ligation were performed using the Golden Gate method.

[0092] The enzyme digestion and ligation system (both BsaI-HF and T4 DNA ligase used for ligation were purchased from NEB) is detailed in Table 3 below.

[0093] Table 3 Enzyme digestion and ligation system

[0094]

[0095] The PCR amplification process was performed at 37°C for 5 minutes, then at 20°C for 5 minutes, for a total of 10 cycles. The resulting mixture was then transformed into DH5α Escherichia coli competent peptide cells, and then confirmed by bacterial testing and sequencing.

[0096] Finally, three vectors were assembled, named pEGOEPubi-B-GFP, as shown in the diagram. Figure 1 As shown, the vector nucleotide sequence is shown in SEQ ID NO.19; pEGOEPubi-B-ZmWUS / BBM, and the spectrum is shown in... Figure 2 As shown, the vector nucleotide sequence is shown in SEQ ID NO.20; pEGOEPubi-B-SbWOX / BBM, and the spectrum is shown in... Figure 3 As shown, the vector nucleotide sequence is shown in SEQ ID NO. 5.

[0097] Experiment Example 3: Maize genetic transformation process of three test vectors

[0098] The reference "Agrobacterium-mediated high-frequency transformation of anelite commercial maize (Zea mays L.) inbred line" (Myeong-Je Cho, Plant Cell, DOI 10.1007 / s00299-014-1656-x) describes a genetic transformation method mediated by Agrobacterium EHA105 to infect maize immature embryos. Three vectors, pEGOEPubi-B-GFP, pEGOEPubi-B-SbWOX / BBM, and pEGOEPubi-B-ZmWUS / BBM, were introduced into the genome of maize B104 material to enable them to function.

[0099] The detailed process is as follows:

[0100] 1. Preparation of bacterial culture

[0101] Three vectors were introduced into Agrobacterium EHA105 using chemical transformation. After cold shock and heat shock, the vectors were incubated at 28°C for 1 hour and then plated on LB agar plates containing Rif / Kan antibiotics (antibiotics purchased from Sangon Biotech Co., Ltd.). The plates were then incubated in the dark at 28°C for 48 hours.

[0102] The cultured Agrobacterium was collected using an inoculation loop into a 15 ml sterile centrifuge tube containing suspension. The Agrobacterium was then evenly dispersed in the suspension using a vortex mixer, and diluted with the suspension until the OD value was reached. 600 =0.3 and keep it in reserve.

[0103] 2. Preparation and treatment of immature embryos

[0104] After removing the husks from corn ears of B104 corn grown in the greenhouse, the ears were soaked in a 20% sodium hypochlorite solution for about 30 minutes and rinsed 5 times with sterile water. Then, the sterilized corn ears were placed on a clean bench, and the embryos were removed with an embryo removal knife and placed in a 2ml EP tube containing PHI-I infection solution (Zhao et al. 2000) for later use.

[0105] 3. Infection and co-culture of Agrobacterium EHA105

[0106] The embryos prepared in step 2 were heated in a 46°C water bath for 3 minutes, followed by an ice bath for 1 minute. The suspension in the embryos was aspirated. The Agrobacterium EHA105 suspension prepared in step 1 was mixed with the embryos and infected for 5 minutes. The embryos were gently shaken to suspend them and then allowed to stand to allow the bacterial solution to fully contact the embryos.

[0107] The infected embryos and bacterial solution were poured onto the prepared PHI-T co-culture medium (Zhao et al. 2000) to ensure even distribution of the embryos and bacterial solution. The bacterial solution was dried on a clean bench until no obvious bacterial solution was observed. The embryos with the scutellum facing down were then turned over and placed in a 22°C constant temperature incubator for 3 days in the dark.

[0108] 4. Recovery and Filtering

[0109] After co-culture, the maize B104 embryos were transferred to DBC3 recovery medium, 50 embryos / plate, and cultured in the dark at 28°C for 7 days to allow callus tissue to grow.

[0110] This specific implementation method takes the screening of immature embryos with glufosinate resistance as an example. The immature embryos that have grown callus tissue are transferred from the recovery medium to the glufosinate resistance screening medium (without hormones), 30 embryos / plate, and cultured in the dark at 28°C for 25 days to obtain resistant callus tissue.

[0111] The screening culture medium formula used in this invention is as follows: MS salt (containing vitamins) 4.43 g / L; VB1 0.5 mg / L; L-proline 1.38 g / L; sucrose 30 g / L; acid-hydrolyzed casein 0.5 g / L; plant gel 3 g / L; AgNO3 5 mg / L; termethin 400 mg / L; glufosinate (10%) 0.4 ml / L.

[0112] In existing normal maize genetic transformation processes, thiamethoxam (a growth hormone) is essential for the normal proliferation of plant callus tissue; otherwise, the callus tissue will exhibit slow growth and poor efficacy. In this application, by transforming pEGOEPubi-B-SbWOX / BBM into maize immature embryos and co-expressing it during callus tissue culture and differentiation, it was found that even without the addition of thiamethoxam or any other growth hormones, high genetic transformation and differentiation efficiencies could still be achieved.

[0113] 5. Differentiation and regeneration of maize callus tissue;

[0114] The resistant callus tissue was placed on differentiation medium, 9 tissues / plate, and incubated at 28℃ with a photoperiod of 16h light culture / 8h dark culture. After 14 days of culture, the callus differentiated and sprouted.

[0115] Experiment Example 4: Statistical Analysis of Maize Genetic Transformation Efficiency in Three Experiments

[0116] The screening and regeneration stages of maize genetic transformation in Experiment Example 3, the number of calluses in the screening stage, the number of calluses in the differentiation stage, the number of calluses that differentiated and turned green, and the number of induced seedlings were statistically analyzed.

[0117] In this invention, the formulas for calculating the conversion rate and differentiation rate are as follows:

[0118] Conversion efficiency = Number of induced seedlings divided by the number of screened calluses;

[0119] Differentiation efficiency = Number of induced seedlings divided by the number of differentiated and green callus;

[0120] Upper selection callus refers to the proliferation and expansion of callus tissue caused by positive cells on a selection medium with glufosinate selection pressure, where there is no glufosinate selection pressure on the recovery medium.

[0121] Updifferentiation and greening refers to callus tissue that turns green and begins to differentiate after screening and is cultured under light.

[0122] Corn callus tissue that does not turn green during the differentiation light culture stage essentially loses its regeneration ability and is therefore not included in the differentiation efficiency statistics. The statistical results of the three sets of experiments in this invention are shown in Table 4 below, and the screening of callus state and induced seedling state are shown below. Figure 4 , 5 The two figures, from left to right, show the callus tissue screening and seedling induction stages of the blank group, positive control group, and experimental group, respectively.

[0123] Table 4. Statistical table of the number of callus or regenerated seedlings at each stage of the three experimental groups.

[0124]

[0125] from Figure 4 As can be seen in the screening stage, without the addition of plant hormones, the callus proliferation in the experimental group was significantly better than that in the blank group and the control group. Figure 5 As can be seen, during the differentiation and regeneration stage, the callus differentiation efficiency of the experimental group was significantly higher than that of the control group.

[0126] In summary, through statistical analysis of data from the screening to the differentiation stages during maize genetic transformation, this invention found that both the SbWOX / SbBBM co-expression composition and the ZmWUS / BBM co-expression composition can significantly improve callus transformation and differentiation efficiency.

[0127] Compared with the blank control group, the positive control group had a maize genetic transformation efficiency of 2.5 times and a differentiation efficiency of 1.5 times.

[0128] Compared with the blank control group, the genetic transformation efficiency of maize in the experimental group was 3.4 times that of the blank control group, and the differentiation efficiency was 2.3 times that of the blank control group.

[0129] This demonstrates that the SbWOX / SbBBM co-expression combination in the experimental group significantly enhanced maize callus transformation and differentiation compared to the ZmWUS / BBM co-expression combination in the positive control group. The transformation efficiency of the experimental group was 1.4 times that of the positive control group, and the differentiation efficiency was 1.5 times that of the positive control group.

[0130] Therefore, this invention demonstrates that co-expression of SbWOX and SbBBM proteins significantly improves transformation efficiency during maize genetic transformation, and this efficiency is significantly higher than that achieved with co-expression of ZmWUS and ZmBBM proteins. This lays the foundation for addressing key issues in genetic transformation production applications limited by genotype and explores the feasibility of utilizing sorghum SbWOX and SbBBM genes to improve systemic genetic transformation efficiency.

[0131] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for genetic transformation of maize, characterized in that, The invention includes introducing the gene sequence encoding the SbWOX protein shown in SEQ ID NO.1 and the gene sequence encoding the SbBBM protein shown in SEQ ID NO.2 into maize explants and co-expressing them. The SbWOX and SbBBM gene co-expression composition is used to improve the transformation efficiency of maize genetic transformation.

2. The application of the maize genetic transformation method according to claim 1 in maize breeding.

3. A gene co-expression nucleic acid molecule, characterized in that, It includes a gene sequence encoding the SbWOX protein and a gene sequence encoding the SbBBM protein, and a nucleotide sequence encoding a self-splicing peptide is also linked between the gene sequence encoding the SbWOX protein and the gene sequence encoding the SbBBM protein. The amino acid sequence of the SbWOX protein is shown in SEQ ID NO.1; the amino acid sequence of the SbBBM protein is shown in SEQ ID NO.

2.

4. The gene co-expression nucleic acid molecule according to claim 3, characterized in that, The self-cleaving peptide is any one of the self-cleaving peptides T2A, P2A, F2A, and E2A.

5. The gene co-expression nucleic acid molecule according to claim 3, characterized in that, The gene sequence encoding the SbWOX protein is shown in SEQ ID NO.3; the gene sequence encoding the SbBBM protein is shown in SEQ ID NO.

4.

6. A gene co-expression vector, characterized in that, Includes the gene co-expression nucleic acid molecule as described in any one of claims 3-5.

7. The gene co-expression vector according to claim 6, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

5.

8. A method for preparing a gene co-expression vector as described in claim 6 or 7, characterized in that, Includes the following steps: Using the gene encoding the SbWOX protein as a template, PCR amplification was performed using the forward primer shown in SEQ ID NO.6 and the reverse primer shown in SEQ ID NO.7, and the first amplification product was recovered. Using the gene encoding the SbBBM protein as a template, PCR amplification was performed using the forward primer shown in SEQ ID NO.8 and the reverse primer shown in SEQ ID NO.9, and the second amplification product was recovered. The first and second amplification products were recombined into the base vector to construct the gene co-expression vector.

9. A microbial transformant, characterized in that, The gene co-expression vector described in claim 6 or 7 is prepared by transforming Agrobacterium tumefaciens.

10. The application of the microbial transformant of claim 9 in maize genetic transformation.

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