CN117757838A - Methods for creating sweet corn germplasm and related biological materials - Google Patents

Abstract

The invention discloses a method for creating sweet corn germplasm and related biological materials. In particular to an application of a protein BT2 with an amino acid sequence of SEQ ID No.2 in preparing corn with the shape of corn seeds changed to the shape of sweet corn seeds. The invention uses ZmBT2 gene to regulate corn grain shape and CRISPR/Cas9 system genome targeting edit, uses sgRNA (SEQ ID No. 4) and CRISPR/Cas9 gene edit vector to knock out ZmBT2 gene. Experimental results show that the taste sweetness of the corn kernels can be obviously increased by reducing the expression level of the ZmBT2 gene, the yellow intensity of the corn kernels is reduced, the shrinkage degree of endosperm of the corn kernels is increased, the size of the corn kernels embryo is increased, the shape of the corn kernels is changed to that of the sweet corn kernels, and the germplasm of the sweet corn is created. The invention has important significance for cultivating new varieties of sweet corn.

Description

Methods for creating sweet corn germplasm and related biological materials

Technical field

The invention belongs to the field of biotechnology, and specifically relates to methods for creating sweet corn germplasm and related biological materials.

Background technique

Corn (Zea mays L.) is the main food crop and industrial raw material. It is the cereal crop most processed and utilized by humans. It plays an important role in national production and stable economic development. With the improvement of people's living standards, corn is not only used as For grain and feed, the cultivation of multi-purpose corn varieties has attracted increasing attention from breeders. Sweet corn is a subspecies of corn. It is popular among consumers due to its high soluble sugar content, rich sweetness, and unique corn aroma. In higher plants, photosynthetic products are mainly transported in the form of sucrose. Sucrose is generally biosynthesized in leaf cells and transferred to phloem tissue, where it accumulates in fruits through long-distance transportation. The grain is the main place for the synthesis and storage of corn nutrients, and is the organ that directly determines yield, quality and economic value. Deeply digging into the key genes involved in corn grain development and analyzing their mechanisms of regulating grain development will have important theoretical significance for improving corn yield and nutritional value in the future.

Sweet corn is a species of Zea mays in the family Poaceae. Because the content of protein, fat, lysine and tryptophan in the endosperm of the kernel is richer than that of ordinary corn, and its consumption method is similar to that of fruits and vegetables and can be eaten directly, it is known as "fruit corn". Sweet corn is favored by consumers from all walks of life because of its high soluble sugar content, rich sweetness, and unique corn flavor. The upstream industrial chain of sweet corn, especially the cultivation of new varieties, needs to be strengthened urgently. The characteristic of sweet corn is that it tastes delicious, sweet but not greasy. Its sweetness mainly comes from the sugars in the corn kernels, which are synthesized and stored in the corn kernels during the ripening process. Because sweet corn contains a large amount of vitamins such as vitamin C, vitamin E, and beta-carotene, as well as minerals such as potassium, calcium, and magnesium, sweet corn has rich nutritional value. In addition, sweet corn is also rich in nutrients such as dietary fiber and oligosaccharides, which help promote intestinal health and lower blood sugar. Moderate consumption of sweet corn can play a variety of health-care functions such as antioxidant, anti-inflammatory, and lowering blood pressure. Therefore, sweet corn has important breeding and production application value.

Genome editing is a technology for targeted and precise modification of the genome, mainly including Zinc-Finger Nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs) and regular clustering The interspaced short palindromic repeat system (clustered regularly interspaced short palindromic repeats/CRISPR-associatednuclease, CRISPR/Cas). CRISPR/Cas systems are roughly divided into three categories: Type I, Type II and Type III. The composition of Type II system is relatively simple. It only requires three components: Cas9 protein, tracrRNA and crRNA to function, and is highly efficient and easy to operate. sex, becoming the most widely used genome editing system currently. The characteristic protein of the Type II CRISPR/Cas system is Cas9. The Cas9 protein has two nuclease domains, RuvC and HNH, which are responsible for cutting both strands of the target DNA, thereby causing double-stranded breaks in the target DNA. The HNH domain is responsible for cutting the DNA strand complementary to crRNA, and the cleavage site is located 3 bp upstream of the protospacer adjacent motif (PAM). The RuvC domain is responsible for cutting the non-complementary strand, and the cleavage site is located upstream of the PAM. 3–8bp. The Cas9 protein has the function of processing and producing crRNA and cutting exogenous nucleic acids. crRNA combines with tracrRNA through base pairing to form a tracrRNA/crRNA complex. Researchers can use tracrRNA and crRNA as two guide RNAs (gRNA) or fuse the two together to form a single guide RNA (sgRNA). sgRNA can bind to Cas9 endonuclease and guide Cas9 to the genome to cleave the target site. The CRISPR/Cas9 system causes double-stranded breaks in the DNA of the target gene, activating the DNA damage repair mechanism in cells, thereby producing mutations such as deletions and insertions. At present, CRISPR/Cas9 technology has been widely used in many species such as microorganisms, animals and plants.

Contents of the invention

The technical problem to be solved by the present invention is how to make the corn kernel form have the sweet corn kernel form and thereby create sweet corn germplasm. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

In order to solve the above technical problems, the present invention first provides any of the following applications of protein:

A1) Application in regulating endosperm starch content of corn kernels;

A2) Application in preparing sweet corn products;

A3) Application in preparing corn whose kernel morphology changes to sweet corn kernel morphology;

A4) Application in increasing the sweetness of corn kernels;

A5) Application in reducing the yellow intensity of corn kernels;

A6) Application in increasing the degree of endosperm shrinkage of corn kernels;

A7) Application in increasing the size of corn kernel embryos;

The name of the protein is BT2, which can be any of the following:

B1) The amino acid sequence is the protein of SEQ ID No. 2;

B2) A protein with more than 80% identity and the same function as the protein shown in B1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No. 2;

B3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and/or C-terminus of B1) or B2).

In the above applications, the protein BT2 can be derived from Zea mays.

In order to facilitate the purification or detection of the protein in B1), a tag protein can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

The tag proteins include but are not limited to: GST (glutathione sulfhydryl transferase) tag protein, His tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc-tagged protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tagged protein.

The above 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

The present invention also provides applications of biological materials related to the protein, which applications may be any of the following:

D1) Application in regulating endosperm starch content of corn kernels;

D2) Application in preparing sweet corn products;

D3) Application in preparing corn whose kernel morphology changes to sweet corn kernel morphology;

D4) Application in increasing the sweetness of corn kernels;

D5) Application in reducing the yellow intensity of corn kernels;

D6) Application in increasing the degree of endosperm shrinkage of corn kernels;

D7) Application in increasing the size of corn kernel embryos;

The biological material can be any of the following:

E1) a nucleic acid molecule encoding the protein BT2;

E2) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the protein BT2;

E3) an expression cassette containing the nucleic acid molecule described in E1) or E2);

E4) A recombinant vector containing the nucleic acid molecule described in E1) or E2), or a recombinant vector containing the expression cassette described in E3);

E5) A recombinant microorganism containing the nucleic acid molecule described in E1) or E2), or a recombinant microorganism containing the expression cassette described in E3), or a recombinant microorganism containing the recombinant vector described in E4);

E6) A recombinant host cell containing the nucleic acid molecule described in E1) or E2), or a recombinant host cell containing the expression cassette described in E3), or a recombinant host cell containing the recombinant vector described in E4);

E7) Transgenic plant tissue containing the nucleic acid molecule described in E1) or E2), or transgenic plant tissue containing the expression cassette described in E3);

E8) Transgenic plant organs containing the nucleic acid molecule described in E1) or E2), or transgenic plant organs containing the expression cassette described in E3).

In the above application, the nucleic acid molecule described in E1) can be any of the following:

F1) The coding sequence is the DNA molecule of SEQ ID No. 1;

F2) The nucleotide sequence is the DNA molecule of SEQ ID No. 3.

Furthermore, the nucleic acid molecules described in E1) also include nucleic acid molecules that have an identity of more than 95% with the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 3 and originate from the same species.

Furthermore, the nucleic acid molecules described in E1) may also include nucleic acid molecules obtained by codon bias modification based on the nucleotide sequence shown in SEQ ID No. 1.

The amino acid sequence encoded by the DNA molecule shown in SEQ ID No. 1 is the protein BT2 of SEQ ID No. 2.

The nucleotide sequence shown in SEQ ID NO. 1 is the nucleotide sequence of the gene encoding protein BT2 (CDS).

The nucleotide sequence shown in SEQ ID No. 3 is the genomic nucleotide sequence of the ZmBT2 gene.

In the above application, the nucleic acid molecule in E2) can be sgRNA, and the target sequence of the sgRNA can be SEQ ID No. 4.

The expression cassette described herein includes a promoter, a nucleic acid molecule encoding the protein BT2, and a terminator. The promoter may be a U6 promoter.

The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

The recombinant vector described herein may be a CRISPR/Cas9 gene editing vector, and the CRISPR/Cas9 gene editing vector may include the sgRNA (the target sequence is SEQ ID No. 4).

Further, the CRISPR/Cas9 gene editing vector may also include a U6-2 promoter (SEQ ID No. 6).

Further, the nucleotide sequence of the CRISPR/Cas9 gene editing vector may be a sequence consisting of SEQ ID No. 5 and SEQ ID No. 8 directly connected in sequence from the N-terminus to the C-terminus.

The recombinant microorganism described herein can specifically be recombinant Agrobacterium EHA105/CRISPR/Cas9.

The recombinant Agrobacterium EHA105/CRISPR/Cas9 described herein is a recombinant bacterium obtained by introducing the CRISPR/Cas9 gene editing vector into Agrobacterium tumefaciens BMEHA105.

The present invention also provides sgRNA, which targets the ZmBT2 gene, and the target sequence of the sgRNA can be SEQ ID No. 4.

The present invention also provides a gene editing vector containing the sgRNA. The nucleotide sequence of the gene editing vector can be a sequence consisting of SEQ ID No. 5 and SEQ ID No. 8 directly connected in sequence from the N end to the C end. .

The present invention also provides a method for cultivating transgenic corn. The method includes using gene editing technology to reduce or inactivate the activity of the gene encoding the protein in the target corn to obtain the transgenic corn, which has the following characteristics: Any of the above characteristics:

G1) The grain morphology of the transgenic corn changes to the sweet corn grain morphology;

G2) The transgenic corn has a sweeter taste than the target corn;

G3) The grain yellow intensity of the transgenic corn is weaker than that of the target corn;

G4) The degree of shrinkage of the grain endosperm of the transgenic corn is greater than that of the target corn;

G5) The seed embryo of the transgenic corn is larger than the target corn.

In the above method, the use of gene editing technology to reduce or inactivate the activity of the gene encoding the protein in the target corn is performed using the CRISPR/Cas9 system, which includes expression of the encoding gene targeting the protein. The sgRNA vector, the target sequence of the sgRNA can be SEQ ID No. 4.

In the above method, the nucleotide sequence of the vector may be a sequence consisting of SEQ ID No. 5 and SEQ ID No. 8 directly connected in sequence from the N-terminus to the C-terminus.

The method of cultivating transgenic corn according to the present invention may include the following steps:

(1) Construct a CRISPR/Cas9 gene editing vector expressing the sgRNA whose target sequence is SEQ ID No. 4;

(2) Introduce the CRISPR/Cas9 gene editing vector constructed in step (1) into the target corn;

(3) Obtain the transgenic corn through screening and identification.

Further, the method of cultivating transgenic corn according to the present invention may include directly connecting the CRISPR/Cas9 gene editing vector (the nucleotide sequence consists of SEQ ID No. 5 and SEQ ID No. 8 from the N-terminus to the C-terminus). ) is introduced into the target corn, and the transgenic corn is obtained through screening and identification.

The introduction refers to through recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic method, electroporation or in planta technology.

The present invention also provides mutant genes or any of the following applications of said mutant genes:

H1) Application in regulating endosperm starch content of corn kernels;

H2) Application in preparing sweet corn products;

H3) Application in preparing corn whose kernel morphology changes to sweet corn kernel morphology;

H4) Application in increasing the sweetness of corn kernels;

H5) Application in reducing the yellow intensity of corn kernels;

H6) Application in increasing the degree of endosperm shrinkage of corn kernels;

H7) Application in increasing the size of corn kernel embryos;

The mutant gene may be obtained by deleting 2 nucleotides CT at positions 1515-1516 of the ZmBT2 gene shown in SEQ ID No. 3 or deleting 4 nucleotides TTCT at positions 1513-1516.

The ZmBT2 gene and its encoded protein of the present invention can be used to change the morphology of corn kernels and thereby create sweet corn germplasm. By reducing the expression of the ZmBT2 gene (such as knocking out the ZmBT2 gene), the sweetness of the taste of corn kernels can be significantly increased, the yellow intensity of the corn kernels can be reduced, the degree of endosperm shrinkage of the corn kernels can be increased, the embryo size of the corn kernels can be increased, and the morphology of the corn kernels can be improved. Change in kernel morphology to sweet corn. The present invention discovered for the first time the application of the ZmBT2 gene and its encoded protein in regulating corn grain morphology, and based on CRISPR/Cas9 technology, provided a new sgRNA, a gene editing system containing the sgRNA, and a gene editing system using the sgRNA. This method provides a precise, safe and non-exogenous DNA insertion technical method for the creation of corn mutant plants and sweet corn breeding. It is important for cultivating new sweet corn varieties, overcoming the shortcomings of traditional breeding, and promoting the process of commercial sweet corn breeding. significance.

Description of the drawings

Figure 1 shows the constructed gene editing vector construction and mutant types. Among them, A is the vector construction map, B is the gene map and the designed target, and C is the screened pure mutant.

Figure 2. Kernel phenotype results of the homozygous mutant materials screened.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

The nucleotide sequence of the Zm00001d050032 gene in the following examples is shown in SEQ ID No. 3.

The genetic transformation recipient in the following examples is the corn inbred line KN5585. This inbred line has been recorded at: http://www.zeamap.com/organism/5095206. The public can obtain the biological material from the applicant. The biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

The gene editing basic vector in the following examples is CPB-Cas9 (hereinafter referred to as CPB vector). The CPB vector is used as the basic vector. The sequence inserted between the recognition sites of the restriction endonuclease HindIII of the initial CPB vector is SEQ ID No. .7 DNA fragment, keeping the rest of the sequence of the initial CPB vector unchanged, the recombinant vector obtained is a CRISPR/Cas9 gene editing vector. The nucleotide sequence of the CRISPR/Cas9 gene editing vector is composed of SEQ ID No. 5 and SEQ ID No. 8 A sequence composed of direct connections from the N-terminus to the C-terminus.

In the following examples -BluntSimple Cloning Vector (CB111-01), E. coli strain Trans1-T1 (CD501-03), etc. were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

In the following examples, Hind III (R0104V) homologous recombinase (NEB), KOD Plus (KOC-401) and KOD FX (KFX-101) high-fidelity PCR amplification enzyme were purchased from Beijing Bailingke Biotechnology Co., Ltd.

Example 1. Construction of gene editing vector

The coding sequence (CDS) of the ZmBT2 gene in corn variety KN5585 is SEQ ID No. 1, and the coding amino acid sequence is the Glucose-1-phosphate adenylyltransferase protein (BT2 protein for short) of SEQ ID No. 2. In the genomic DNA of maize, the genomic sequence encoding the Glucose-1-phosphate adenylyltransferase protein is shown in SEQ ID No. 3. Positions 6-134 of SEQ ID No.3 are the first exon, positions 1497-1793 are the second exon, positions 3009-3278 are the third exon, and positions 3675-3854 are the fourth exon. Exons, 4146-4249 are the fifth exon, 4367-4478 are the sixth exon, 4599-4697 are the seventh exon, and 4774-4893 are the eighth exon. Exon 5483-5599 is the ninth exon. The sgRNA was designed based on the ZmBT2 (Zm00001d050032) gene in the reference genome sequence B73_RefGen_v5. The sgRNA is located at positions 133-152 of SEQ ID No. 1 (corresponding to positions 1500-1519 of SEQ ID No. 3). The forward and reverse primer sequences of the target site are: 5’-GCACGTTTCGTAGTTGCTGC-3’, 5’-ATGTTGCTCCCGTAGGCTCT-3’. The target sequence of sgRNA was verified by Sanger sequencing in the maize inbred line KN5585.

The target sequence of sgRNA is: 5’-GTTCTTGGAATCATTCTGGG-3’ (SEQ ID No. 4).

The present invention constructs a CRISPR/Cas9 gene editing vector (also known as a CRISPR/Cas9 knockout vector) for knocking out the gene Zm00001d050032 encoding maize adenosine diphosphate glucose pyrophosphorylase. The specific steps are as follows:

1. Design promoter and sgRNA sequences

1.1 Promoter information

The promoter was the maize U6 promoter. According to the sequence comparison, there are multiple U6 promoters in maize. Compared with the U6 promoters in other species, one of the more conservative promoters was adopted and named U6-2. Its sequence As shown in SEQ ID No. 6.

1.2sgRNA sequence

The sgRNA target sequence designed for the ZmBT2 gene is: 5’-GTTCTTGGAATCATTCTGGG-3’ (SEQ ID No. 4).

2.Enzyme digestion system and purification recovery of CPB vector

2.1 Use HindIII to digest the CPB-Cas9 vector (referred to as CPB vector). The reaction system is shown in Table 1.

Table 1. CPB carrier digestion system

The reaction program is 3 hours in a metal bath at 37°C. After the reaction is completed, 1.2% agarose gel electrophoresis is performed to obtain a linearized CPB vector.

2.2 Recovery of enzyme-digested vectors

Use an agarose gel recovery kit to recover the linearized CPB vector, and finally elute it with 40 μl of ddH ₂ O, and store it at -20°C for later use.

3. Primer design

In the process of constructing the vector, the primers that need to be used mainly include the following sequencing primers.

Table 2. Primer sequence list

4. Obtain and purify target fragments

4.1KOD Plus DNA Polymerase

KOD plus is a high-efficiency and high-fidelity PCR enzyme developed based on KOD DNA polymerase. Through buffer optimization and the hot-start method using anti-KOD DNA polymerase antibodies, PCR efficiency and fidelity are higher than the original KOD DNA Polymerase (Code No.: KOD-101). The fidelity is approximately 80 times that of Taq DNApolymerase, making it most suitable for gene cloning. Since the ends of the PCR products have been smoothed, high-efficiency TA cloning can be performed using the dedicated TA cloning kit Target Clone-Plus. Due to the high fidelity of KOD plus, we used KODplus to amplify the target fragment and obtain the required DNA fragment. The reaction system is as follows:

Table 3. KOD plus reaction system

In Table 3, the U6 promoter (U6-2 promoter) was amplified using the KN5585 wild-type genome as a template and 5'-GTCATCTATGTTACTAGATCAAGCTCTAATTGGCCCTTACAAAATAG-3' and 5'-GGAGCGGTGGTCGCAGCTG-3' as primers.

The gene sequence of BGI was synthesized to obtain the coding DNA fragment of sgRNA+scaffold.

The KOD plus reaction program is shown in Table 4:

Table 4. KOD plus reaction program

After PCR, agarose gel electrophoresis was performed to detect the target fragment. The target fragment was cut and recovered using Tiangen's agarose gel recovery kit. Finally, it was dissolved and eluted with 45 μL of ddH ₂ O, and then kept at -20°C for later use.

4.2 Overlap PCR reaction

Gene splicing by overlap extension PCR (SOE PCR) uses primers with complementary ends to form overlapping strands in the PCR products, so that in the subsequent amplification reaction, the overlapping strands are extended to separate the genes from different sources. The amplified fragments overlap and are spliced together. This technology uses PCR technology to perform effective gene recombination in vitro, and does not require endonuclease digestion and ligase treatment. This technology can be used to quickly obtain products that are difficult to obtain by other methods that rely on restriction endonuclease digestion. The key to the success of overlap extension PCR technology is the design of overlapping complementary primers. Overlap extension PCR has extensive and unique applications in site-directed mutation of genes, construction of fusion genes, synthesis of long fragment genes, gene knockout, and amplification of target genes.

Use the overlapping PCR method to connect the target fragment (U6 promoter) obtained in step 4.1 and the coding DNA fragment of sgRNA+scaffold, and finally obtain a fragment containing the U6 promoter, sgRNA and scaffold, named DNA fragment 1, whose core The nucleotide sequence is SEQ ID No. 7. The overlapping PCR reaction system and reaction procedures are shown in Table 5 and Table 6 respectively.

Table 5. Overlap PCR reaction system

In Table 5: DNA Template 1 is the U6 promoter obtained in step 4.1; DNA Template 2 is the coding DNA fragment of sgRNA+scaffold obtained in step 4.1; Primer is: 5'-GTCATCTATGTTACTAGATC-3' and 5'-CGACGGCCAGTGCCAAGCTT- 3'.

Table 6. Overlap PCR reaction procedure

5.NEBuilder connection reaction and transformation

HiFi DNA Assembly Master Mix Sample Request uses a special recombinase and the principle of homologous recombination to recombine vectors linearized by any method and PCR fragments with an overlapping region of 15-25bp at both ends. It can simultaneously achieve 1-5 Efficient and seamless splicing of fragments. Use the NEBuilder ligation reaction to connect the linearized CPB vector in step 2 to the DNA fragment 1 (SEQ ID No. 7) in step 4.2 to obtain the ligation product DNA. The reaction system connected by NEBuilder is shown in Table 7:

Table 7. NEBuilder connection reaction system

The NEBuilder connection reaction program is shown in Table 8:

Table 8. NEBuilder connection reaction program

Transform the above ligation product DNA into E. coli strain Trans1-T1. The steps are as follows:

(1) Take 50 μL of competent cells melted on ice bath, add the ligation product DNA, mix gently, and keep in ice bath for 30 minutes;

(2) Heat shock in a 42°C water bath for 1 minute, then quickly transfer the centrifuge tube to an ice bath for 2-5 minutes; (Do not shake the centrifuge tube during the ice bath);

(3) Add 150 μL of sterile SOC or LB liquid culture medium (without any antibiotics) to the centrifuge tube, mix well and place it at 37°C and incubate at 200 rpm for 1 hour;

(4) Add 100 μL of transformed competent cells to the LB solid culture medium plate containing the corresponding antibiotics. Use a sterilized spreader to spread the cells evenly until the liquid is completely absorbed. Invert the plate and place it in a 37°C constant temperature incubator. Medium, culture for 10-14h.

6. PCR detection of transformants

Add LB liquid culture medium (containing 100ng/μL of the corresponding antibiotic) to the sterilized PCR tube, add 20μL to each well, use a sterilized toothpick or a 10μL gun tip to pick different single colonies from the LB solid plate, and add 20μL to each well. The picked single colonies were fully inserted into LB liquid culture medium. Take 2.0 μL of liquid from 20 μL of liquid culture medium and transfer it to another PCR plate to be used as a DNA template for PCR. Seal the PCR plate with a silicone cover and place it in a constant temperature incubator at 37°C for 8-10 hours.

The PCR system used to detect transformants is as follows:

Table 9. PCR detection system (unit: μL)

In Table 9: Primers are 5’-GGATGTGCGCTCCCTGAATA-3’ and 5’-GTAAAACGACGGCCAGT-3’. DNAtemplate uses a single colony picked as a template.

The PCR reaction program used to detect transformants is shown in Table 10:

Table 10. PCR detection reaction procedure

The results of the PCR reaction are detected with 1% agarose. For the colonies that have amplified the target fragment, find the corresponding colony according to the number, and expand and culture it into a centrifuge tube containing 4-6ml LB liquid culture medium (100ng/ml corresponding antibiotic) , incubate overnight at 37°C and 200 rpm.

7. Verification of positive transformants

Use a plasmid extraction kit to extract the plasmid from the expanded LB liquid medium (containing 100ng/mL of the corresponding antibiotic), and detect it again with the PCR method. For the detection system and PCR procedure, see step 6. Then use the corresponding restriction endonuclease for enzyme digestion detection. Please refer to step 2 for the system and reaction conditions. If both tests meet expectations, send the extracted plasmid or bacterial fluid for sequencing analysis. The sequencing analysis method is as follows: First, the quality of the sequencing must be confirmed. In the returned sequencing report, there are two files, with .seq and .abi as file suffixes respectively. They can be opened with bioedit or snapgene software. The peaks of sequences with good sequencing quality are relatively single and no other miscellaneous peaks appear; the sequencing quality is poor. The peaks of the sequence are relatively confusing, accompanied by the appearance of miscellaneous peaks, and sometimes the peaks do not match the sequence. For first-generation sequencing, the first 20-40 bp of sequencing are inaccurate and must be deleted to ensure sequencing quality. According to the principle of first-generation sequencing, the activity of the DNA polymerase used in sequencing will also decrease in fidelity as the reaction proceeds. Therefore, the maximum reliable sequence that can be obtained in a sequencing reaction is 650-700bp. If the part to be sequenced exceeds 1Kb, It is recommended to use primers at both ends for sequencing at the same time, and then use Vector::ContigExpress software for splicing. The quality of the sequencing sequence should be confirmed before splicing. Compare the reliable sequencing sequence with the template sequence to verify again whether the transferred plasmid is the expected plasmid.

8. Preservation of positive transformant strains and plasmids

Plug the correctly sequenced positive transformants into 350 μL LB liquid culture medium (containing 100 ng/ml corresponding antibiotics). After culturing for 8-10 hours at 37°C and 200 rpm, add 350 μL sterile 50% glycerol to the ultra-clean workbench. (Mix glycerol and sterile water 1:1), mix well and store in a -80°C refrigerator for later use; for plasmids corresponding to correct sequencing results, write the corresponding label and store directly in a -20°C refrigerator.

The size of the basic CPB vector used in the above construction is about 17.4Kb, which is a medium copy vector. It mainly includes the following elements: Kan+ antibiotic resistance gene; replication element; bar selection gene driven by CaMV 35S promoter; gene sequence encoding Cas9 protein driven by Ubi promoter and DNA sequence of sgRNA driven by maize U6 promoter. Among them, except for the kan+ resistance gene and replication elements, the rest are on the T-DNA fragment, as shown in Figure 1. It carries a Kan resistance gene, which can make the strain Kan+ resistant, so Kan antibiotics can be used for screening. The only HindIII restriction endonuclease site is located between the U6 promoter and RB. After restriction enzyme digestion, CPB can be made into a linear shape for vector construction. In the transgenic positive plants, the Cas9 protein expressed by Ubi::Cas9::Nos on the CPB vector has directional DNA cleavage activity after combining with the sgRNA transcribed by U6::sgRNA::sca ffold; CaMV 35S::bar::CaMV poly (A) The bar protein expressed by signal has herbicide resistance and can be used to screen positive plants.

The nucleotide sequence of the CRISPR/Cas9 gene editing vector constructed above is a sequence consisting of SEQ ID No. 5 and SEQ ID No. 8 directly connected in sequence from the N-terminus to the C-terminus. Its structure is described as follows: In the restriction of the initial vector CPB A recombinant vector is obtained by inserting a DNA fragment with the sequence SEQ ID No. 7 between the recognition sites of the sexual endonuclease HindIII and keeping the rest of the sequence of the initial vector CPB unchanged. The CRISPR/Cas9 gene editing vector constructed above is shown in Figure 1A. In the sequence consisting of SEQ ID No. 5 and SEQ ID No. 8 directly connected from the N end to the C end: positions 435-4535 are Cas9 elements. Positions 4658-6629 are Ubi promoter elements, positions 6658-8593 are 3896 promoter elements, positions 8594-9271 are DsRed2, positions 9272-9524 are NOS terminator elements, and positions 9927-9946 are the coding of sgRNA DNA sequence.

Example 2. Genetic transformation of corn

1. Obtaining the recombinant strain EHA105/CRISPR/Cas9

The CRISPR/Cas9 gene editing vector constructed in Example 1 was introduced into BMEHA105 Agrobacterium competent cells (Beijing Bomeide Gene Technology Co., Ltd., product number: BC303-01) to obtain recombinant Agrobacterium EHA105/CRISPR/Cas9.

2. Agrobacterium infection

1) Use N6 liquid culture medium (potassium nitrate 2800mg/L, ammonium sulfate 463mg/L, potassium dihydrogen phosphate 400mg/L, magnesium sulfate 185mg/L, calcium chloride 165mg/L, potassium iodide 0.8mg/L, boric acid 1.6mg /L, manganese sulfate monohydrate 4.4mg/L, zinc sulfate heptahydrate 1.5mg/L, disodium ethylenediaminetetraacetate 37.25mg/L, ferrous sulfate heptahydrate 27.85mg/L, glycine 2mg/L, vitamin B1 1mg/L, vitamin B6 0.5mg/L, niacin 0.5mg/L, sucrose 20000mg/L. N6 medium preparation method: weigh 24.1g of the mixture of the above N6 liquid medium components, add it to 1000mL distilled water or deionized water In water, heat and stir to dissolve, adjust the pH to 5.8±0.2 (25°C), and autoclave at 115°C for 20 minutes.) Cultivate the recombinant Agrobacterium obtained in step 1 in an Agrobacterium shaker at 28°C and 200 rpm to obtain the bacteria The liquid OD _600nm is 0.8 recombinant Agrobacterium bacteria liquid, centrifuge at 5000r/min for 10 minutes, collect the cells, and use the prepared infection buffer (1L infection buffer is to mix 4g of N6 culture medium basic salt containing N6 vitamin (Phytotech company product) , Cat. No. C416-50L), 2mg 2,4-D, 100mg inositol, 0.7g L-proline, 68.4g sucrose, 36g glucose, 1mL AgNO ₃ (10mg/mL), 1mL As (100mol/L) and The bacterial cells obtained by mixing with water (pH 5.2) were suspended until the OD _600nm value was about 0.5, and then shaken at 28°C and 150r/min for 0.5h to obtain the infection solution.

2) Soak the well-growing calli of the corn inbred line KN5585 in the infection buffer for 1 hour, then transfer to the infection solution prepared in step 1), soak for 15 minutes, and dry.

3) Place the infected callus in step 2) into a co-culture medium (1L co-culture medium is 4g N6 salt containing N6vitamin, 2mg 2,4-D, 30g sucrose, 8g agar, 1mL AgNO ₃ (10mg /mL), 1mL As (100mol/L), 3mL L-cysteine (100mg/mL) and water, pH5.8), cultured at 20°C for 3 days, transferred to recovery medium (1L The recovery medium is a mixture of 4g N6 salt, 1ml N6 vitamin 1000×, 1.5mg 2,4-D, 0.7gL-proline, 30g sucrose, 5μM AgNO ₃ , 0.5g MES, 100mg cefotaxime, 100mg vancomycin , obtained by mixing 8g agar and water, pH 5.8), cultured at 28°C for 10 days, then transferred to recovery medium containing 1.5mg/L glufosinate ammonium, cultivated in the dark at 28°C for 7 days, and screened for positive calli.

4) Transfer the positive callus obtained in step 3) to the embryoid body induction medium (1L embryoid body induction medium is to mix 4.43g MS salt containing MS vitamin (containing myo-inositol), 0.25mg2,4-D , 30g sucrose, 5mg 6-BA, 4g plant gel, 1mL Cefo (250mg/mL) and water, pH 5.8), cultured in the dark for 2 weeks, then transferred to differentiation medium (1L differentiation medium The medium is obtained by mixing 4.43g MS salt containing MS vitamin (containing inositol), 30g sucrose, 4g plant gel, 1mL Cefo (250mg/mL) and water, pH 5.8), and transfer it after green seedlings grow. To rooting medium (1L rooting medium is obtained by mixing 2.215g 1/2MS, 30g sucrose, 51.55mg MSvitamin, 4g plant gel and water, pH 5.8), grow to a certain height, and culture in the air After 3 days, transplant the seedlings.

5) Take about 3 cm of plant leaves, put them into a tube and grind them thoroughly, add 500 μl of buffer (from bar gene detection test strips), and insert the bar gene detection test strips (Beijing Altron Gold Label Biotechnology Co., Ltd., product number: A07-13-413), the plants with positive strips are T ₀ generation positive plants.

Example 3. Identification of maize genetic transformation results

1. Verification of genetically modified ingredients

DNA was extracted and purified using a plant genomic DNA extraction kit (Tiangen, China). T-DNA was detected by Bar test strips (Agdia, Cat. #STX14200/0012, US) and PCR of the SpCas9 gene. SpCas9 PCR amplification uses the forward primer with the sequence 5'-CAACCGGAAAGTGACCGTGA-3' and the reverse primer with the sequence 5'-CACCACCTTC ACTGTCTGCA-3'. The PCR program includes 94°C for 3 minutes; 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 68°C for 20 seconds; and a final extension of 10 minutes at 68°C. The PCR reaction system is: 10x Buffer 5μL; 2mM dN TP 5μL; 25mM MgSO ₄ 2μL; Primer (10μM) 1.5μL; DNA Template 2μL; KOD plus (1U/μL) 1μL; ddH ₂ O 32μL.

2. Mutation detection

Design primers 5'-GCACGTTTCGTAGTTGCTGC-3' and 5'-ATGTTGCTCCCGTAGGCTCT-3' according to the target position of the CRISPR/Cas9 gene editing vector, amplify the target gene of the corresponding plant through PCR, and use DSDecode software to analyze the mutation type after sequencing. The PCR reaction system and reaction procedures are the same as the genetically modified component verification in step 1.

Through Agrobacterium-mediated genetic transformation, 10 transformation events were obtained. Through PCR amplification and Sanger sequencing, it was found that a mutation occurred in the sgRNA binding region (C in Figure 1), and a frameshift mutation was obtained. The types of mutations include ZmBT2-1: reduced by two bases; ZmBT2-2: reduced by four bases, etc. The specific mutation types are as follows:

Compared with the wild type, ZmBT2-1 has the following mutations in the ZmBT2 genes in the two homologous chromosomes: "5'-AGTGTTCTTGGAATCATTCTGGGAGGTGG-3' in the ZmBT2 gene (corresponding to positions 130-158 of SEQ ID No. 1, 1497-1525 of SEQID No. 3)" was mutated to "5'-AGTGTTCTTGGAATCATTG GGAGGTGG-3'". This mutation causes the deletion of two nucleotides "CT" at positions 1515-1516 of SEQ ID No. 3 in the sequence listing. The deletion of these two nucleotides causes a frameshift mutation, leading to early termination of translation, resulting in Glucose- The function of the 1-phospha te adenylyltransferase protein is lost, thereby knocking out the ZmBT2 gene. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 1, C.

Compared with the wild type, ZmBT2-2 has the following mutations in the ZmBT2 genes in the two homologous chromosomes: "5'-AGTGTTCTTGGAATCATTCTGGGAGGTGG-3' in the ZmBT2 gene (corresponding to positions 130-158 of SEQ ID No. 1, 1497-1525 of SEQID No. 3)" was mutated to "5'-AGTGTTCTTGGAATCATTC TGGGAGGTGG-3'". This mutation causes the deletion of four nucleotides "TTCT" at positions 1513-1516 of SEQ ID No. 3 in the sequence listing. The deletion of these four nucleotides causes a frameshift mutation, leading to early termination of translation, resulting in Glucose- The function of the 1-ph osphate adenylyltransferase protein is lost, thereby knocking out the ZmBT2 gene. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 1, C.

3. Self-cross or test-cross the T0 generation positive plants obtained by genetic transformation to obtain T0 generation seeds. After planting, ZmBT2 gene knockout heterozygous mutants are screened from the T1 generation positive plants, and then self-crossed to obtain seeds, and the T2 generation is planted. Go on for genotype testing and genetically modified component testing. Finally, homozygous negative mutants were selected for self-breeding.

The results are shown in Figure 1. Chromosome 1 and chromosome 2 respectively represent the DNA double strands of the tested plants. The present invention successfully constructed a knockout vector for knocking out the adenosine diphosphate glucose pyrophosphorylase gene Zm00001d050032 (Fig. 1A, B). Through Agrobacterium-mediated genetic transformation, 10 transformation events were obtained. Taking the sgRNA binding position as the center, design the upstream primer and downstream primer of this site. Through PCR amplification and Sanger sequencing, it was found that mutations occurred in the sgRNA binding region (Figure 1C), and frameshift mutations were obtained. Finally, ZmBT2 homozygous loss-of-function mutant materials were selected for identification of sweet corn kernel morphology.

Example 4. Phenotypic identification of transgenic corn

Grain morphology identification

Kernel morphology index is a geometric measurement that describes the size and shape of the kernel. The structure of corn kernel mainly includes three parts: embryo, endosperm and seed coat. The seed coat accounts for about 6% to 8% of the grain mass and protects the grain from abiotic and biotic stress. The embryo accounts for 10% to 15% of the grain mass. It is a necessary tissue for seed germination and contains most of the fat in the grain. The endosperm is located around the embryo, accounting for 80% to 85% of the grain mass, and contains nutrients needed for the development of the embryo. The mutants selected fruit ears with normal and uniform growth, and randomly selected grains in the fruit ears for photographing and observation.

The results are shown in Figure 2. The mutant plant tasted significantly sweeter than the wild type 30 days after pollination. The seeds of mutant plants (loss-of-function mutants with ZmBT 2 gene frameshift mutations) are lighter in color than the wild type, and the endosperm of the mutant seeds is shrunken, and the embryos are larger than those of the wild type. These characteristics indicate that the mutant plants Comply with the characteristics of sweet corn kernels. The above experimental results show that knocking out the corn adenosine diphosphate glucose pyrophosphorylase gene Zm00001d050032 through gene editing can change the grain morphology and make the grain have a sweet corn grain phenotype (for example: make the grain color lighter; make the grain endosperm more shrinkage; enlarging the grain embryo), thereby quickly creating sweet corn germplasm.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

Claims (10)

1. Any of the following uses of the protein:

a1 Application in regulating starch content of corn kernel endosperm;

a2 Use in the preparation of sweet corn products;

a3 The application of the corn seeds in preparing the corn with the shape changed from the shape of the corn seeds to the shape of the sweet corn seeds;

a4 Application in increasing the sweetness of the taste of corn kernels;

a5 Use in reducing the yellow intensity of corn kernels;

a6 Use of a composition for increasing the degree of shrinkage of the endosperm of a corn kernel;

a7 Use in increasing the size of a maize kernel embryo;

the protein is any one of the following:

b1 A protein having an amino acid sequence of SEQ ID No. 2;

b2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residues in the amino acid sequence shown in SEQ ID No.2, has more than 80 percent of identity with the protein shown in B1) and has the same function;

b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).

2. Use of a biological material related to a protein as claimed in claim 1, wherein the use is any of the following:

d1 Application in regulating starch content of corn kernel endosperm;

d2 Use in the preparation of sweet corn products;

d3 The application of the corn seeds in preparing the corn with the shape changed from the shape of the corn seeds to the shape of the sweet corn seeds;

d4 Application in increasing the sweetness of the taste of corn kernels;

d5 Use in reducing the yellow intensity of corn kernels;

d6 Use of a composition for increasing the degree of shrinkage of the endosperm of a corn kernel;

d7 Use in increasing the size of a maize kernel embryo;

the biological material is any one of the following:

e1 A nucleic acid molecule encoding the protein of claim 1;

e2 A nucleic acid molecule which inhibits or reduces the expression of a gene encoding the protein of claim 1;

e3 An expression cassette comprising the nucleic acid molecule of E1) or E2);

e4 A recombinant vector comprising the nucleic acid molecule of E1) or E2), or a recombinant vector comprising the expression cassette of E3);

e5 A recombinant microorganism comprising the nucleic acid molecule of E1) or E2), or a recombinant microorganism comprising the expression cassette of E3), or a recombinant microorganism comprising the recombinant vector of E4);

e6 A recombinant host cell comprising the nucleic acid molecule of E1) or E2), or a recombinant host cell comprising the expression cassette of E3), or a recombinant host cell comprising the recombinant vector of E4);

e7 A transgenic plant tissue comprising the nucleic acid molecule of E1) or E2), or a transgenic plant tissue comprising the expression cassette of E3);

e8 A transgenic plant organ comprising the nucleic acid molecule of E1) or E2), or a transgenic plant organ comprising the expression cassette of E3).

3. The use according to claim 2, wherein E1) the nucleic acid molecule is any one of the following:

f1 A DNA molecule whose coding sequence is SEQ ID No. 1;

f2 A DNA molecule with a nucleotide sequence of SEQ ID No. 3.

4. The use according to claim 2, characterized in that E2) the nucleic acid molecule is an sgRNA whose target sequence is SEQ ID No.4.

An sgRNA, wherein the sgRNA targets the ZmBT2 gene and the target sequence of the sgRNA is SEQ ID No.4.

6. A gene editing vector comprising the sgRNA of claim 5, wherein the nucleotide sequence of the gene editing vector is a sequence consisting of SEQ ID No.5 and SEQ ID No.8 which are directly connected in sequence from the N-terminus to the C-terminus.

7. A method of breeding transgenic corn comprising decreasing or inactivating the activity of a gene encoding the protein of claim 1 in a corn of interest using gene editing techniques to produce said transgenic corn, said transgenic corn having any of the following characteristics:

g1 The grain morphology of the transgenic corn changes to the sweet corn grain morphology;

g2 The taste sweetness of the transgenic corn is higher than that of the target corn;

g3 The grain yellow intensity of the transgenic corn is weaker than that of the target corn;

g4 A grain endosperm crimp level of said transgenic corn is greater than that of said target corn;

g5 The grain embryo of the transgenic corn is larger than the corn of interest.

8. The method of claim 7, wherein the decreasing or inactivating activity of the gene encoding the protein of claim 1 in the maize of interest using gene editing techniques is performed using a CRISPR/Cas9 system comprising a vector expressing a sgRNA targeting the gene encoding the protein, the target sequence of the sgRNA being SEQ ID No.4.

9. The method according to claim 8, wherein the nucleotide sequence of the vector is a sequence consisting of SEQ ID No.5 and SEQ ID No.8 directly linked in sequence from the N-terminus to the C-terminus.

10. A mutant gene or any one of the following uses of said mutant gene:

h1 Application in regulating starch content of corn kernel endosperm;

h2 Use in the preparation of sweet corn products;

h3 The application of the corn seeds in preparing the corn with the shape changed from the shape of the corn seeds to the shape of the sweet corn seeds;

h4 Application in increasing the sweetness of the taste of corn kernels;

h5 Use in reducing the yellow intensity of corn kernels;

h6 Use of a composition for increasing the degree of shrinkage of the endosperm of a corn kernel;

h7 Use in increasing the size of a maize kernel embryo;

the mutant gene is obtained by deleting 2 nucleotides CT at 1515-1516 positions or 4 nucleotides TTCT at 1513-1516 positions of ZmBT2 gene shown in SEQ ID No. 3.