CN117757838A - Method for creating sweet corn germplasm and related biological materials thereof - 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

Method for creating sweet corn germplasm and related biological materials thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for creating sweet corn germplasm and a related biological material thereof.

Background

Corn (Zea mays l.) is a major food crop and industrial raw material, is a cereal crop which is most utilized by human processing, has an important role in national production and stable economic development, and along with the improvement of the living standard of people, corn is not only used as food and feed, and the cultivation of multipurpose corn varieties is increasingly valued by breeders. Sweet corn is a subspecies of corn, and is highly appreciated by consumers due to its high content of soluble sugar, intense sweetness, and the unique flavor of corn. In higher plants, the photosynthetic assimilation products are transported mainly in the form of sucrose, which is generally biosynthesized in leaf cells and transferred to bast tissues, and accumulated in fruits by long-distance transport. The kernel is the main place for synthesizing and storing corn nutrient substances and is a direct determining organ for yield, quality and economic value. The key genes involved in the development of the corn kernels are deeply excavated, and the action mechanism of the key genes for regulating the development of the kernels is analyzed, so that the key genes have important theoretical significance for improving the yield and the nutritional value of the corn in the future.

Sweet corn is a species of corn of the genus corn of the family Gramineae. The grain endosperm is more abundant in protein, fat, lysine and tryptophan than ordinary corn, and the eating method is similar to fruits and vegetables, and can be directly eaten, so the grain endosperm is called as 'fruit corn'. Sweet corn is favored by consumers of all classes because of its high content of soluble sugar, intense sweetness, and unique flavor of corn. The upstream industrial chain of sweet corn, especially the cultivation of new varieties, needs to be strengthened. Sweet corn is characterized by delicious taste and sweet but not greasy. Its sweetness is mainly derived from sugars in the kernels, which are synthesized during the ripening of the corn and stored in the kernels. Because sweet corn contains a large amount of vitamins such as vitamin C, vitamin E, beta-carotene and the like, and minerals such as potassium, calcium, magnesium and the like, the sweet corn has rich nutritional value. In addition, the sweet corn is rich in dietary fiber, oligosaccharides and other nutritional ingredients, and is beneficial to promoting intestinal health and reducing blood sugar. Proper amount of sweet corn can play a plurality of health care roles of resisting oxidation, resisting inflammation, reducing blood pressure and the like, so the sweet corn has important breeding and production application values.

Genome editing is a technology for performing directed and precise modification on genome, and mainly comprises Zinc-Finger Nucleases (ZFNs), transcription activator-like effector Nucleases (Transcription Activator-Like Effector Nucleases, TALENs) and regular clustered interval short palindromic repeat systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease, CRISPR/Cas). CRISPR/Cas systems are roughly classified into type I, type II and type III 3, wherein the type II system has relatively simple composition, can function only by three components of Cas9 protein, tracrRNA and crRNA, has high efficiency and easy operability, and becomes the most widely used genome editing system at present. The characteristic protein of the type II CRISPR/Cas system is Cas9, the Cas9 protein has two nuclease domains of RuvC and HNH that are responsible for cleaving both strands of the target DNA, resulting in a double strand break of the target DNA. Wherein the HNH domain is responsible for cleaving the DNA strand complementary to the crRNA, the cleavage site is located 3bp upstream of the protospacer adjacent motif (Protospacer adjacent motif, PAM), and the RuvC domain is responsible for cleaving the non-complementary strand, the cleavage site is located 3-8bp upstream of the PAM. The Cas9 protein also has the function of processing to produce crRNA and cleaving exogenous nucleic acids. The crRNA binds to the tracrRNA by base pairing to form a tracrRNA/crRNA complex, and the researchers can use the tracrRNA and crRNA as two guide RNAs (grnas) or fuse the two together to form a single guide RNA (sgRNA). The sgrnas are able to bind to Cas9 endonucleases and guide Cas9 onto the genome to cleave the target site. The CRISPR/Cas9 system makes the target gene DNA produce double strand break, activates the DNA damage repair mechanism in cell and produces deletion, insertion and other mutation. At present, CRISPR/Cas9 technology is widely applied to a plurality of species such as microorganisms, animals, plants and the like.

Disclosure of Invention

The invention aims to solve the technical problem of how to enable the corn grain shape to have the sweet corn grain shape so as to create sweet corn germplasm. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To solve the above technical problems, the present invention provides, first, any one of the following applications of proteins:

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 name is BT2, and can be any 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).

In the above application, the protein BT2 may be derived from maize (Zea mays).

In order to facilitate purification or detection of the protein of B1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.2 of the sequence Listing.

Such 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 tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The 80% identity or more 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 invention also provides the use of a biological material associated with said protein, said use being 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 may be any of the following:

e1 A nucleic acid molecule encoding said protein BT 2;

e2 A nucleic acid molecule that inhibits or reduces expression of a gene encoding said protein BT 2;

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).

In the above application, the nucleic acid molecule of E1) may be any 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.

Further, E1) the nucleic acid molecule further comprises a nucleic acid molecule having a nucleotide sequence identity of 95% or more with the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.3 and being derived from the same species.

Furthermore, the nucleic acid molecule of E1) may also comprise a nucleic acid molecule which has been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 1.

The DNA molecule shown in SEQ ID No.1 encodes a protein BT2 whose amino acid sequence is SEQ ID No. 2.

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

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

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

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

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

The recombinant vectors described herein can be CRISPR/Cas9 gene editing vectors, which CRISPR/Cas9 gene editing vectors can include the sgrnas (target sequence of SEQ ID No. 4).

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

Further, the nucleotide sequence of the CRISPR/Cas9 gene editing vector can be a sequence formed by sequentially and directly connecting SEQ ID No.5 and SEQ ID No.8 from the N end to the C end.

The recombinant microorganism described herein may specifically be recombinant agrobacterium EHA105/CRISPR/Cas9.

The recombinant agrobacterium tumefaciens EHA105/CRISPR/Cas9 is a recombinant bacterium obtained by introducing the CRISPR/Cas9 gene editing vector into the agrobacterium tumefaciens BMEHA 105.

The invention also provides an sgRNA, wherein the sgRNA targets ZmBT2 genes, and the target sequence of the sgRNA can be SEQ ID No.4.

The invention also provides a gene editing vector containing the sgRNA, and the nucleotide sequence of the gene editing vector can be a sequence formed by sequentially and directly connecting SEQ ID No.5 and SEQ ID No.8 from the N end to the C end.

The invention also provides a method for cultivating transgenic corn, which comprises the steps of utilizing a gene editing technology to reduce or inactivate the activity of a coding gene of the protein in target corn to obtain the transgenic corn, wherein the transgenic corn has any one 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.

In the above method, the decreasing or inactivating the activity of the gene encoding the protein in the corn of interest using the gene editing technique is performed using a CRISPR/Cas9 system, the CRISPR/Cas9 system comprising a vector expressing a sgRNA targeting the gene encoding the protein, and the target sequence of the sgRNA may 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 which are directly linked in sequence from the N-terminus to the C-terminus.

The method for cultivating transgenic corn can comprise the following steps:

(1) Constructing a CRISPR/Cas9 gene editing vector for expressing the sgRNA with the target sequence of SEQ ID No. 4;

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

(3) The transgenic corn is obtained through screening and identification.

Further, the method for cultivating transgenic corn can comprise the steps of introducing the CRISPR/Cas9 gene editing vector (the nucleotide sequence is formed by sequentially and directly connecting SEQ ID No.5 and SEQ ID No.8 from the N end to the C end) into target corn, and screening and identifying to obtain the transgenic corn.

The introduction refers to transformation by recombinant means including, but not limited to, agrobacterium (Agrobacterium) -mediated transformation, biolistic (biolistic) methods, electroporation, or in planta technology.

The invention also provides a mutant gene or any one of the following applications of the 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 can be obtained by deleting 2 nucleotides CT from 1515 to 1516 or 4 nucleotides TTCT from 1513 to 1516 of the ZmBT2 gene shown in SEQ ID No. 3.

The ZmBT2 gene and the encoding protein thereof can be used for changing the corn grain shape, thereby creating sweet corn germplasm. The sweetness of the taste of the corn kernel can be obviously increased by reducing the expression quantity of the ZmBT2 gene (such as knocking out the ZmBT2 gene), the yellow intensity of the corn kernel is reduced, the shrinkage degree of endosperm of the corn kernel is increased, the size of embryo of the corn kernel is increased, and the shape of the corn kernel is changed to that of the sweet corn kernel. The invention discovers the application of ZmBT2 gene and the encoding protein thereof in regulating and controlling the corn grain shape for the first time, provides a novel sgRNA based on CRISPR/Cas9 technology, a gene editing system containing the sgRNA, and a method for carrying out gene editing by utilizing the sgRNA, provides a precise and safe technical method without exogenous DNA insertion for creating corn mutant plants and breeding of sweet corn, and has important significance for cultivating new varieties of sweet corn, overcoming short plates of traditional breeding, promoting the breeding process of commercial sweet corn.

Drawings

FIG. 1 shows the construction of constructed gene editing vector and the type of mutant. Wherein A is a vector construction map, B is a gene map and a designed target point, and C is a screened pure mutant.

The grain phenotype results for the homozygous mutant material screened in FIG. 2.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available 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 maize inbred line KN5585, which has been described in: http:// www.zeamap.com/organissm/5095206, the biological material being available to the public from the applicant and being used only for repeated experiments of the invention and not for other uses.

The gene editing base vector in the following examples is CPB-Cas9 (hereinafter abbreviated as CPB vector), the CPB vector is used as the base vector, a DNA fragment with SEQ ID No.7 is inserted between recognition sites of restriction enzyme HindIII of the initial CPB vector, the recombinant vector obtained by keeping the rest sequences of the initial CPB vector unchanged is CRISPR/Cas9 gene editing vector, and the nucleotide sequence of the CRISPR/Cas9 gene editing vector is a sequence formed by directly connecting SEQ ID No.5 and SEQ ID No.8 from N end to C end in sequence.

In the following examplesBluntSimple Cloning Vector (CB 111-01), E.coli strain Trans1-T1 (CD 501-03) and the like are purchased from Beijing full gold biotechnology Co.

HindIII (R0104V) homologous recombinase (NEB), KOD Plus (KOC-401) and KOD FX (KFX-101) high-fidelity PCR amplification enzymes were purchased from Beijing-Bailingke Biotechnology Co., ltd in the examples described below.

Example 1 construction of Gene editing vector

The coding sequence (CDS) of ZmBT2 gene in maize KN5585 is SEQ ID No.1, and the coding amino acid sequence is Glucose-1-phosphate adenylyltransferase protein (BT 2 protein for short) of SEQ ID No. 2. In the genomic DNA of corn, the genomic sequence of the coding Glucose-1-phosphate adenylyltransferase protein is shown in SEQ ID No. 3. Positions 6 to 134 of SEQ ID No.3 are the first exon, positions 1497 to 1793 are the second exon, positions 3009 to 3278 are the third exon, positions 3675 to 3854 are the fourth exon, positions 4146 to 4249 are the fifth exon, positions 4367 to 4478 are the sixth exon, positions 4599 to 4697 are the seventh exon, positions 4774 to 4893 are the eighth exon, and positions 5483 to 5599 are the ninth exon. The sgRNA was designed based on the ZmBT2 (Zm 00001d 050032) gene in the reference genome sequence B73_RefGen_v5, and is located at positions 133-152 (corresponding to positions 1500-1519 of SEQ ID No. 3) of SEQ ID No. 1. The sequences of the forward and reverse primers of the target site are respectively as follows: 5'-GCACGTTTCGTAGTTGCTGC-3',5'-ATGTTGCTCCCGTAGGCTCT-3'. The target sequence of sgRNA was verified by Sanger sequencing in maize inbred line KN 5585.

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

The invention constructs a CRISPR/Cas9 gene editing vector (also called CRISPR/Cas9 knockout vector) for knocking out a gene Zm00001d050032 for encoding corn adenosine diphosphate glucose pyrophosphorylase, which comprises the following specific steps:

1. design of promoter and sgRNA sequence

1.1 promoter information

The promoter adopts the U6 promoter of corn. According to sequence alignment, the U6 promoter has a plurality of promoters in corn, is compared with U6 promoters in other species, adopts one of the promoters with higher conservation, is named as U6-2, and has the sequence shown in SEQ ID No. 6.

1.2sgRNA sequences

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

Enzyme digestion system and purification recovery of CPB carrier

2.1 digestion of CPB-Cas9 vector (CPB vector for short) with HindIII, the reaction system is shown in Table 1.

TABLE 1 CPB vector digestion System

The reaction procedure is that a metal bath is carried out for 3 hours at 37 ℃, and after the reaction is finished, agarose gel electrophoresis with concentration of 1.2 percent is carried out, thus obtaining the linearization CPB carrier.

2.2 recovery of the digestion vector

The linearized CPB vector was recovered using an agarose gel recovery kit, and finally 40. Mu.l of ddH was used ₂ Eluting with O, and storing at-20deg.C.

3. Primer design

In the construction of the vector, the following sequencing primers are mainly used.

TABLE 2 list of primer sequences

4. Obtaining and purifying the desired fragment

4.1KOD Plus DNA polymerase

KOD plus is an enzyme for high-efficiency and high-fidelity PCR developed based on KOD DNA polymerase. By optimization of Buffer and hot start with anti-KOD DNA polymerase antibody, PCR efficiency and fidelity were higher than those of the original KOD DNA Polymerase (Code No.: KOD-101). The fidelity is about 80 times of Taq DNA polymerase, and is most suitable for gene cloning. Since the ends of the PCR products have been smoothed, efficient TA cloning can be performed using a specific TA cloning kit, target Clone-Plus. Because of the high fidelity of KOD plus, we amplified the fragment of interest with KOD plus to obtain the desired DNA fragment. The reaction system is shown in the following table:

TABLE 3 KOD plus reaction System

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

The coding DNA fragment of sgRNA+scaffold is synthesized by the gene sequence of Huada.

KOD plus reaction procedure is shown in Table 4:

TABLE 4 KOD plus reaction procedure

After completion of PCR, agarose gel electrophoresis was performed to cut the target fragment, and then the target fragment was recovered using the reagent kit for agarose gel recovery of tiangen, and finally 45. Mu.L of ddH was used ₂ O is dissolved and eluted, and then is reserved at the temperature of minus 20 ℃.

4.2 overlap PCR reactions

Overlapping extension PCR (gene splicing by overlap extension PCR, SOE PCR) techniques allow the PCR products to form overlapping strands due to the use of primers with complementary ends, thereby overlapping and splicing amplified fragments of different sources together by extension of the overlapping strands in subsequent amplification reactions. The technology can effectively carry out gene recombination in vitro by utilizing the PCR technology, and does not need endonuclease digestion and ligase treatment, and other products which are difficult to obtain by a method relying on restriction endonuclease digestion can be quickly obtained by utilizing the technology. The key to the success of overlap extension PCR techniques is the design of overlapping complementary primers. Overlap extension PCR has wide and unique application in site-directed mutation of genes, construction of fusion genes, synthesis of long fragment genes, gene knockout, amplification of target genes and the like.

And (3) connecting the target fragment (U6 promoter) obtained in the step (4.1) and the coding DNA fragment of the sgRNA+scaffold by using an overlap PCR method, and finally obtaining a fragment containing the U6 promoter, the sgRNA and the scaffold, which is named as a DNA fragment 1, and the nucleotide sequence of the fragment is SEQ ID No.7. The system and procedure of the overlap PCR reaction are shown in tables 5 and 6, respectively.

TABLE 5 overlapping 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+scafold obtained in step 4.1; the Primer is as follows: 5'-GTCATCTATGTTACTAGATC-3' and 5'-CGACGGCCAGTGCCAAGCTT-3'.

TABLE 6 overlapping PCR reaction procedure

NEBuilder ligation and transformation

HiFi DNA Assembly Master Mix Sample Request by utilizing the principle of special recombinase and homologous recombination, the vector linearized by any method and the PCR fragments with 15-25bp overlapping areas at the two ends of the vector can be directionally recombined, and the efficient seamless splicing of 1-5 fragments can be realized at the same time. The linearized CPB vector in step 2 was ligated to DNA fragment 1 (SEQ ID No. 7) in step 4.2 using NEBuilder ligation to give ligation product DNA. The NEBuilder ligation reaction system is shown in Table 7:

TABLE 7 NEBuilder ligation reaction system

NEBuilder ligation protocol is shown in Table 8:

TABLE 8 NEBuilder ligation protocol

The DNA of the connection product is transformed into escherichia coli strain Trans1-T1, and the steps are as follows:

(1) Taking 50 mu L of competent cells melted on ice bath, adding DNA of a connection product, gently mixing, and ice-bathing for 30min;

(2) Heat shock is carried out for 1min in a water bath at the temperature of 42 ℃, and then the centrifuge tube is quickly transferred into an ice bath for 2-5min; (the ice bath process does not shake the centrifuge tube);

(3) 150 mu L of sterile SOC or LB liquid culture medium (without any antibiotics) is added into a centrifuge tube, and the mixture is placed at 37 ℃ for culture at 200rpm for 1h after uniform mixing;

(4) 100. Mu.L of transformed competent cells were pipetted onto LB solid medium plates containing the corresponding antibiotics, spread out evenly with a sterile spreader until the liquid was completely absorbed, and the plates were inverted and incubated in a 37℃incubator for 10-14h.

PCR detection of transformants

LB liquid medium (containing 100 ng/. Mu.L of the corresponding antibiotic) was added to the sterilized PCR tube, 20. Mu.L of each well was added, different single colonies were picked from the LB solid plate with sterilized toothpick or 10. Mu.L gun tip, and the picked single colonies were fully inoculated into the LB liquid medium. 2.0. Mu.L of liquid was removed from 20. Mu.L of liquid medium and transferred to another PCR plate to be used as a DNA template for PCR. Sealing with a silica gel cover of a PCR plate, and culturing in a constant temperature incubator at 37 ℃ for 8-10h.

The PCR system used to detect transformants is shown in the following table:

TABLE 9 PCR detection System (Unit: μL)

In table 9: primers 5'-GGATGTGCGCTCCCTGAATA-3' and 5'-GTAAAACGACGGCCAGT-3'. DNA template is a single colony picked as a template.

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

TABLE 10 PCR detection reaction procedure

The result of the PCR reaction was detected with 1% agarose, and colonies amplified with the target fragment were found to correspond to the colonies according to the number, and the colonies were grown in a centrifuge tube containing 4-6ml of LB liquid medium (100 ng/ml of the corresponding antibiotic) at 37℃for 200rpm overnight.

7. Verification of Positive transformants

Plasmids were extracted from the LB liquid medium (containing 100ng/mL of the corresponding antibiotics) of the expansion culture using a plasmid extraction kit, and were again detected by PCR, and the detection system and PCR procedure were described in step 6. Then, the restriction enzyme is used for enzyme digestion detection, and the system and the reaction conditions are referred to in the step 2. If both tests are expected, the extracted plasmid or bacterial liquid is sent for sequencing analysis. The sequencing analysis method is as follows: first, the quality of the sequencing is confirmed. In the returned sequencing report, two files are respectively used as file suffixes, seq and abi, biokit or snapgene software is used for opening the sequence with good sequencing quality, and the peak is single and no other miscellaneous peaks appear; sequencing a sequence of poor quality, the peaks are chaotic, accompanied by the occurrence of a hybrid peak, and sometimes there is a phenomenon that the peaks are not matched with the sequence. For one generation of sequencing, the first 20-40bp of sequencing is inaccurate and is deleted to ensure sequencing quality. According to the principle of the first generation sequencing, the activity of DNA polymerase used in the sequencing is reduced along with the progress of the reaction, so that the maximum reliable sequence obtained by one sequencing reaction is 650-700bp, if the part to be sequenced exceeds 1Kb, simultaneous sequencing by primers at two ends is recommended, and then Vector: : the Contigexpress software performs splicing, and before splicing, the quality of the sequencing sequence is confirmed. The reliable sequencing sequence was aligned with the template sequence and again verified whether the transferred plasmid was the desired plasmid.

8. Positive transformant strain and plasmid preservation

The positive transformant with correct sequence is inoculated into 350 mu L LB liquid culture medium (containing 100ng/ml corresponding antibiotics), cultured for 8-10h at 200rpm at 37 ℃, and then 350 mu L sterile 50% glycerol (the mixture of glycerol and sterile water is 1:1) is added into an ultra-clean workbench, and the mixture is stored in a refrigerator at-80 ℃ for standby after even mixing; and verifying the plasmid corresponding to the sequencing result without error, writing a corresponding label, and directly storing the label in a refrigerator at the temperature of minus 20 ℃.

The basic CPB vector used in the above construction was approximately 17.4Kb in size and was a medium copy vector. Mainly comprises the following components: kan+ antibiotic resistance gene; a replicating member; the bar screening gene driven by CaMV 35S promoter; a gene sequence encoding Cas9 protein driven by the Ub i promoter and a DNA sequence of sgRNA driven by the maize U6 promoter. Wherein, except for the kan+ resistance gene and the replicative element, the rest are on the T-DNA fragment as shown in FIG. 1. The strain can be made to have Kan+ resistance by carrying Kan resistance genes thereon, so that Kan antibiotics can be used for screening. The only HindIII restriction site is located between the U6 promoter and the RB, and CPB can be linearized after cleavage for vector construction. In the transgenic positive plants, the Ubi:: cas9:: cas9 protein expressed by Nos and U6:: sgRNA:: sca ffold transcribed sgRNA on the CPB carrier have directional DNA cleavage activity after being combined; caMV 35S:: bar:: caMV poly (A) signal expressed bar protein has herbicide resistance, and can be used for screening positive plants.

The nucleotide sequence of the constructed CRISPR/Cas9 gene editing vector is a sequence formed by sequentially and directly connecting SEQ ID No.5 and SEQ ID No.8 from the N end to the C end, and the structure is described as follows: a DNA fragment with a sequence of SEQ ID No.7 is inserted between recognition sites of restriction enzyme HindIII of the original vector CPB, and the remaining sequence of the original vector CPB is kept unchanged to obtain the recombinant vector. The CRISPR/Cas9 gene editing vector constructed in the above way is shown in figure 1A, and the CRISPR/Cas9 gene editing vector is formed by directly connecting sequences of SEQ ID No.5 and SEQ ID No.8 from N end to C end in sequence: the 435-4535 th site is Cas9 element, 4658-6629 th site is Ubi promoter element, 6658-8593 th site is 3896 promoter element, 8594-9271 th site is DsRed2, 9272-9524 th site is NOS terminator element, 9927-9946 th site is coding DNA sequence of sgRNA.

Example 2 genetic transformation of maize

1. Acquisition of recombinant EHA105/CRISPR/Cas9

The CRISPR/Cas9 gene editing vector constructed in example 1 was introduced into BMEHA105 Agrobacterium competent cells (Beijing Bomaide Gene technologies Co., ltd., cat# BC 303-01) to obtain recombinant Agrobacterium EHA105/CRISPR/Cas9.

2. Agrobacterium infection

1) N6 liquid medium (2800 mg/L of potassium nitrate, 463mg/L of ammonium sulfate, 400mg/L of monopotassium phosphate, 185mg/L of magnesium sulfate, 165mg/L of calcium chloride, 0.8mg/L of potassium iodide, 1.6mg/L of boric acid, 4.4mg/L of manganese sulfate monohydrate, 1.5mg/L of zinc sulfate heptahydrate, 37.25mg/L of disodium ethylenediamine tetraacetate, 27.85mg/L of ferrous sulfate heptahydrate, 2mg/L of glycine, 11 mg/L of vitamin B, 0.5mg/L of nicotinic acid and 20000mg/L of sucrose) is adopted. The preparation method of the N6 culture medium comprises the following steps: 24.1g of the mixture of the components of the N6 liquid culture medium is weighed, dissolved in 1000mL of distilled water or deionized water under heating and stirring, pH is adjusted to 5.8+ -0.2 (25 ℃), and the mixture is autoclaved at 115 ℃ for 20 minutes. ) Culturing the recombinant Agrobacterium obtained in step 1 in an Agrobacterium shaker at 28deg.C and 200rpm to obtain bacterial liquid OD _600nm 0.8 recombinant Agrobacterium solution, centrifuging at 5000r/min for 10min, collecting thallus, and collecting thallus with prepared infection buffer (1L infection buffer is prepared by mixing 4g N6 culture medium basic salt (product of Phytotech company, cat. No. C416-50L) containing N6vitamin, 2mg 2,4-D,100mg inositol, 0.7g L-proline, 68.4g sucrose, 36g glucose, 1mL AgNO) ₃ (10 mg/mL), 1mL of As (100 mol/L) and water were mixed uniformly to obtain a suspension of cells at pH 5.2, and OD was obtained _600nm The value is about 0.5, and then the dyeing liquor is obtained by shaking for 0.5h at 28 ℃ and 150 r/min.

2) Soaking the callus of the maize inbred line KN5585 with good growth in an infection buffer for 1h, transferring to the infection solution prepared in the step 1), soaking for 15min, and airing.

3) Placing the infected callus of the step 2) in a co-culture medium (1L co-culture medium is prepared by mixing 4g N6 salt containing N6vitamin, 2mg 2,4-D,30g sucrose, 8g agar, 1mL AgNO) ₃ (10 mg/mL), 1mL of As (100 mo L), 3mL of L-cysteine (100 mg/mL) and water were mixed, and the mixture was incubated at 20℃for 3 days, and transferred to a recovery medium (1L recovery medium was prepared by mixing 4g of N6 salt, 1ml N6vitamin 1000X, 1.5mg of 2,4-D,0.7g of L-proline, 30g of sucrose, 5. Mu.M AgNO) ₃ 0.5g MES,100mg cefotaxime, 100mg vancomycin, 8g agar and water, pH 5.8), cultured at 28℃for 10 days, then transferred to a recovery medium containing 1.5mg/L glufosinate, and cultured at 28℃in dark for 7 days, and positive calli were selected.

4) Transferring the positive callus obtained in the step 3) to an embryoid-inducing medium (1L embryoid-inducing medium is obtained by uniformly mixing 4.43g MS salt containing MS vitamin (containing inositol), 0.25mg2,4-D,30g sucrose, 5mg 6-BA,4g plant gel, 1mL cefuroxime (250 mg/mL) and water, pH 5.8), culturing in dark for 2 weeks, transferring to a differentiation medium (1L differentiation medium is obtained by uniformly mixing 4.43g MS salt containing MS vitamin (containing inositol), 30g sucrose, 4g plant gel, 1mL Cefo (250 mg/mL) and water, pH 5.8), transferring to a rooting medium (1L rooting medium is obtained by uniformly mixing 2.215g 1/2MS,30g sucrose, 51.55mg MSvitamin,4g plant gel and water, pH 5.8), rooting, growing to a certain height, and culturing in air for 3 days.

5) Taking plant leaves of about 3 cm, putting into a tube, grinding thoroughly, adding 500 μl buffer (from bar gene test strip), and inserting bar gene test strip (Beijing Aoque gold label biotechnology Co., ltd., product number: a07-13-413), the plants with positive bands are T ₀ And (5) replacing positive plants.

Example 3 identification of maize genetic transformation results

1. Transgenic component verification

DNA was extracted and purified using plant genomic DNA extraction kit (Tiangen, china). T-DNA was detected by PCR of Bar dipsticks (Agdia, cat. # STX14200/0012, US) and SpCas9 genes. SpCas9 PCR amplification uses a forward primer of sequence 5'-CAACCGGAAAGTGACCGTGA-3' and a reverse primer of sequence 5'-CACCACCTTC ACTGTCTGCA-3'. The PCR procedure included 94℃for 3min;95 ℃ 30s,58 ℃ 30s,68 ℃ 20s,35 cyclesThe method comprises the steps of carrying out a first treatment on the surface of the Finally, the extension is carried out for 10min at 68 ℃. The PCR reaction system is as follows: 10 XBuffer 5. Mu.L; 2mM dNTP 5. Mu.L; 25mM MgSO ₄ 2μL；Primer(10μM)1.5μL；DNA Template 2μL；KOD plus(1U/μL)1μL；ddH ₂ O 32μL。

2. Mutation detection

Primers 5'-GCACGTTTCGTAGTTGCTGC-3' and 5'-ATGTTGCTCCCGTAGGCTCT-3' are designed according to the target position of the CRISPR/Cas9 gene editing vector, target gene amplification is carried out on corresponding plants through PCR, and after sequencing, the mutation types are analyzed by DSDecode software. And (3) verifying a PCR reaction system and a reaction program by using the transgenic components in the step (1).

By agrobacterium-mediated genetic transformation, 10 transformation events were obtained. The findings were by PCR amplification and Sanger sequencing: mutations were made in the region where sgrnas bind (C in fig. 1), and frameshift mutations were obtained. Types of mutations include Zm BT2-1: two bases are reduced; zmBT2-2: four base reduction, etc. Specific mutation types are as follows:

the ZmBT2 genes in 2 homologous chromosomes are mutated as follows compared with the wild type ZmBT 2-1: "5'-AGTGTTCTTGGAATCATTCTGGGAGGTGG-3' (corresponding to positions 130-158 of SEQ ID No.1 and positions 1497-1525 of SEQ ID No. 3)" in the ZmBT2 gene is mutated to "5'-AGTGTTCTTGGAATCATTG GGAGGTGG-3'". The mutation causes the 1515-1516 position of the SEQ ID No.3 sequence in the sequence table to delete two nucleotides CT, the deletion of the two nucleotides causes frame shift mutation, the translation is stopped in advance, and the function of the Glucose-1-phospha te adenylyltransferase protein is deleted, so that the ZmBT2 gene is knocked out, and the sequencing result of the mutation site and the peripheral nucleotides is shown in the figure 1C.

The ZmBT2 gene of 2 homologous chromosomes has the following mutation compared with the wild type: "5'-AGTGTTCTTGGAATCATTCTGGGAGGTGG-3' (corresponding to positions 130-158 of SEQ ID No.1 and positions 1497-1525 of SEQ ID No. 3)" in the ZmBT2 gene is mutated to "5'-AGTGTTCTTGGAATCATTC TGGGAGGTGG-3'". The mutation causes the 1513-1516 position of the SEQ ID No.3 in the sequence table to delete four nucleotides TTCT, the deletion of the four nucleotides causes frame shift mutation, the translation is stopped in advance, and the function of the Glucose-1-ph osphate adenylyltransferase protein is lost, so that the ZmBT2 gene is knocked out, and the sequencing result of the mutation site and the peripheral nucleotides is shown in the figure 1C.

3. Selfing or testing the T0 generation positive plants obtained by genetic transformation to obtain T0 generation seeds, screening ZmBT2 gene knockout heterozygous mutants from the T1 generation positive plants after planting, carrying out selfing to obtain the seeds, and carrying out genotype detection and transgene component detection after the T2 generation planting. And finally, screening out homozygous negative mutants for selfing propagation.

As a result, as shown in FIG. 1, chromosome 1 and chromosome 2 represent DNA double strand of the test plant, respectively. The invention successfully constructs a knockout vector for knocking out the adenosine diphosphate glucose pyrophosphorylase gene Zm00001d050032 (FIGS. 1A and B). By agrobacterium-mediated genetic transformation, 10 transformation events were obtained. The upstream primer and the downstream primer of the site were designed centered around the location of sgRNA binding. By PCR amplification and Sanger sequencing, it was found that mutations occurred in the region where sgRNA bound (fig. 1C), and frame shift mutations were obtained. Finally, zmBT2 functional-deleted homozygous mutant material was selected for sweet corn kernel morphology identification.

Example 4 phenotype identification of transgenic maize

Grain shape identification

The kernel shape index is a geometric measure describing the size and shape of the kernel, and the corn kernel structure mainly comprises an embryo, endosperm and seed coat. The seed coat accounts for about 6% -8% of the mass of the seed grains, and the seed grains are protected from abiotic and biotic stress. The embryo accounts for 10% -15% of the mass of the seed, is a tissue necessary for seed germination, and contains most of fat in the seed. The endosperm is positioned around the embryo and accounts for 80-85% of the mass of the seed grain, and contains nutrients required by embryo development. The mutants are selected from clusters with normal, uniform and consistent growth, and grains are randomly selected from the clusters for photographing and observation.

The results are shown in FIG. 2. The mutant plants were significantly sweeter than the wild type in mouthfeel 30 days after pollination. The mutant plant (the mutant with the function of ZmBT2 gene frame shift mutation) has lighter color and light yellow than wild type seeds, the endosperm of the mutant seeds is shrunken, and the embryo is larger than wild type seeds, and the characteristics indicate that the mutant accords with the characteristics of the sweet corn seeds. The test results show that the corn adenosine diphosphate glucose pyrophosphorylase gene Zm00001d050032 is knocked out by a gene editing means, so that the grain shape can be changed, and the grain has a sweet corn grain phenotype (for example, the color of the grain is lightened, the endosperm of the grain is more shrunken, and the embryo of the grain is enlarged), thereby rapidly creating sweet corn germplasm.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

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.