CN115044592A - Gene ZmADT2 for regulating and controlling corn plant type and resistance to smut, and coding protein and application thereof - Google Patents

Abstract

The invention discloses a gene ZmADT2 for regulating and controlling maize plant type and resistance to smut, and a coding protein and application thereof, belonging to the technical field of molecular genetics. The mutant strain of the gene ZmADT2 for regulating and controlling the corn strain type and the corn smut resistance is seriously dwarfed, and ears are susceptible to the corn smut. The invention identifies the gene sequence of ZmADT2 through map-based cloning, defines the encoding protein thereof, verifies that ZmADT2 gene regulates the corn plant type and the resistance of the tumor smut by CRISPR/Cas9 gene editing technology and genetic complementation. The genome sequence of the ZmADT2 gene is shown as SEQ ID NO.1, the CDS sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 3. The discovery and cloning of the gene provide a new gene resource for cultivating reasonable plant types and the resistance germplasm of the smut of corn by a biotechnology means.

Description

Gene ZmADT2 for regulating and controlling corn plant type and smut resistance, and encoding protein and application thereof

Technical Field

The invention relates to the technical field of molecular genetics, in particular to a gene ZmADT2 for regulating and controlling the resistance of a corn plant type and a corn smut disease, and a coding protein and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Crop pests pose a serious threat to crop yield and quality. Statistically, 20-40% of crops worldwide suffer from pest and disease damage. By 2050, the global demand for food is expected to increase gradually, causing more serious food safety problems. Fungal plant pathogens can threaten crop yield and may cause some diseases after ingestion, causing economic losses. The corn tumorous smut is a disease caused by corn infected by maize smut (Ustilago maydis), can infect all aboveground organs of corn, induces organs of corn such as leaves, stems, tassels and female ears to generate tumors after 4-7 days of infection, and forms mature winter spores after 14 days of infection.

In China, the corn tumor smut becomes one of the main corn diseases, and has serious influence on the corn yield. The smut disease of tumor can occur in each corn planting area in China, and the incidence of diseases in the planting area in the north is more extensive and serious compared with the planting area in the south, wherein the diseases in the northeast, the north China and the Huang-Huai-Hai area are more serious. The resistance of different corn varieties to the smut is different, and the yield statistics of some varieties shows that the infection of maize smut to stalks can cause 20 to 60 percent of yield reduction, while the infection to corn ears can cause nearly 80 percent of yield reduction, thus seriously threatening the yield of corn in China.

In the field, seedling infection symptoms are rare, and tumors are usually on the female and male ears. Researches show that the salicylic acid and the lignin play an important role in resisting smut infection of the corn. Screening corn materials with different disease resistance degrees, identifying resistance genes and researching the resistance molecular mechanism of corn to the smut disease are particularly important. However, the corn smut resistance gene cloned in corn is currently poorly understood.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a gene ZmADT2 for regulating and controlling the resistance of corn plant type and smut, and a coding protein and application thereof. The invention identifies a gene ZmADT2 which codes Arogenate dehydratase and prephenate dehydratase by a forward genetics method, and proves that the gene mutation causes the phenotype of corn development defect and weakened resistance to the smut.

The invention is realized by the following technical scheme:

in a first aspect of the invention, a gene ZmADT2 for regulating corn plant type and resistance to smut is provided, wherein the gene ZmADT2 is a nucleic acid molecule shown in the following i) or ii) or iii) or iv):

i) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

iii) a nucleic acid molecule which is 75% or more identical to the nucleotide sequence of i) or ii) and which expresses the same functional protein;

iv) a nucleic acid molecule other than ii) which encodes the amino acid sequence shown in SEQ ID NO. 3.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed using computer software, for example, as determined using the BLAST algorithm (Altschul et al 1990.journal of Molecular Biology 215: 403-.

In a second aspect of the present invention, there is provided a protein encoded by the above gene ZmADT2, wherein the protein is a protein represented by any one of the following (a1) to (A3):

(A1) a protein consisting of an amino acid sequence shown by SEQ ID NO.3 in a sequence table;

(A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.3 in the sequence table, is related to the corn plant type and the resistance to the smut and is derived from the SEQ ID NO. 3;

(A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1) or (A2).

Wherein, in order to facilitate purification of the protein of (A1) or (A2), a tag may be attached to the amino terminus or the carboxyl terminus of the protein of (A1) or (A2). The tag may be Poly-Arg (typically 6 RRRRR), Poly-His (typically 6 HHHHHHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

In a third aspect of the invention, a recombinant expression vector, a transgenic cell line or a genetically engineered bacterium containing the gene ZmADT2 is provided.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA3300, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant expression vectors. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CaMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when a recombinant expression vector is constructed using the gene of the present invention, an enhancer, including a translation enhancer or a transcription enhancer, may also be used.

In a fourth aspect of the present invention, there is provided an application of the gene ZmADT2, the protein encoded by the gene ZmADT2, the recombinant expression vector containing the gene ZmADT2, the transgenic cell line or the genetically engineered bacterium in at least one of the following (1) to (4):

(1) regulating and controlling the growth and development of plants;

(2) regulating and controlling the resistance of the plant to the smut disease;

(3) regulating and controlling the synthesis of plant salicylic acid, gibberellin and lignin;

(4) and (5) plant breeding.

In the above application, the plant is preferably corn.

In the above application, the plant breeding comprises: corn plant type breeding, and disease-resistant corn variety or strain breeding.

In a fifth aspect of the present invention, there is provided a method for adjusting the plant type of maize, comprising the steps of:

the protein coded by gene ZmADT2 or gene ZmADT2 is used as a target, and the expression level of gene ZmADT2 is regulated and controlled by a biotechnology means, or the activity of the protein coded by gene ZmADT2 is changed.

In the above method, the biotechnological means include, but are not limited to: gene editing, RNAi, T-DNA insertion, overexpression, etc.

In a sixth aspect of the invention, there is provided a method for restoring resistance to melanoma, comprising: a step of overexpressing a gene ZmADT2 in maize.

In the method, the gene ZmADT2 in the corn can be overexpressed by a method of transferring the gene ZmADT2 from an external source.

In a seventh aspect of the present invention, there is provided a cultivation method of corn with anti-tumor smut, the cultivation method comprising: transferring a gene ZmADT2 shown in SEQ ID NO.1 or SEQ ID NO.2 into corn to obtain corn with recovered resistance to the smut;

or regulating the expression of the gene ZmADT2 shown in SEQ ID NO.1 or SEQ ID NO.2 in the corn genome, and screening to obtain the corn plant with improved resistance to the smut.

In the breeding method, the method for transferring the gene ZmADT2 into the corn includes but is not limited to: polyethylene glycol method, Agrobacterium mediated method or gene gun bombardment method.

In the breeding method, the method for regulating the expression of the gene ZmADT2 in the maize genome comprises the following steps: introducing a DNA fragment capable of activating, increasing or inhibiting the transcriptional or translational level or protein activity of gene ZmADT 2; or controlling the synthesis of specific small RNA molecules and regulating the accumulation of mRNA of the gene ZmADT 2.

The specific small RNA molecule comprises: a microRNA (miRNA), an interfering small RNA (siRNA), or an artificial miRNA (artificial microRNA).

Although corn smut has become one of the major corn diseases with serious impact on corn yield, the corn smut causing smut (Ustilago maydis) is a fungus that induces the production of gall rich in amino acids and cholesterol-lowering β -glucose. The black truffle is also called black truffle in Mexico, can be used as food, and has unique flavor and high economic value.

Accordingly, in an eighth aspect of the present invention, there is provided a method of producing corn tumor melanoidin tumors comprising the steps of:

knocking out or silencing gene ZmADT2 in corn; or, the function of the protein coded by the gene ZmADT2 in the corn is lost, and corn material is obtained;

corn tumors, masa, were collected from the corn material obtained.

In the method, the gene ZmADT2 in the corn can be knocked out or silenced by using a CRISPR-Cas9 gene editing technology, a VIGS technology, a T-DNA insertion technology or an RNA interference technology.

The invention has the beneficial effects that:

the invention clones a gene ZmADT2 which can simultaneously regulate and control the corn development and the resistance of the smut, and a gene ZmADT2 which can regulate and control the synthesis of plant hormones, namely salicylic acid and gibberellin, the synthesis of lignin and the regulation of cell size. The invention provides an application of ZmADT2 in establishing a corn plant type and resisting a smut. Meanwhile, the plant type of the gene mutant is dwarfed, the ears are susceptible to the development of a large number of tumors, the tumors are edible and unique in flavor, and the gene mutant can be used as a material for producing and processing the corn tumor masa.

Drawings

FIG. 1 shows field characteristics of Wild Type (WT) and mutant (dsu).

FIG. 2 shows the developmental phenotypes of wild-type and mutant; in the figure, a hybrid plant selfed ears; B. wild Type (WT) and dsu mutant mature grain were cut longitudinally; seedlings of wild type (C) and dsu mutant (D) 3 weeks after planting; E. mature plants of wild type and dsu mutant; F. internodes from wild type and dsu mutant mature plants; G-H. wild type (G) and dsu mutant (H) mature leaves; I. pre-disease ears of wild type and dsu mutant; J. tassels of wild type and dsu mutant; K. roots of wild type and dsu mutant; plant height statistics for dsu mutants and wild type. Hundred grain weight statistics for dsu mutant and wild type; error bars are standard errors, x represents P < 0.001; represents P <0.05 (Student' st test). Panel A is 1cm, panel B is 2mm, panel C, D, F, G, H, I, J, K is 10cm and panel E is 50 cm.

FIG. 3 is a map-based cloning of a mutant gene; in the figure, (A) fine localization of the mutant gene; (B) the mutation site of the ZmADT2 gene has been marked for linkage analysis and primer position (F: dsu-jd-F; R: dsu-jd-R); (C) linkage analysis of mutant sites with phenotype.

FIG. 4 is a ZmADT2 gene complementation line phenotype; in the figure, F constructed by crossing A. pair dsu mutant with overexpression lines ₂ PCR identification of the generation plants; the primer pair of US5-1 and ZmADT2-R is used for distinguishing transgenic positive plants from transgenic negative plants, and the primer pair of F1 and R1 is used for distinguishing wild type genotypes from mutant genotypes; ' H ₂ O 'and P' are water as negative control and vector plasmid as positive control, respectively. B. The plant phenotypes of the four genotype materials are 50cm on a scale; C. the ear disease phenotype of the four genotype materials in the field is 10cm on a scale.

FIG. 5 is a ZmADT2 gene editing line phenotype; in the figure, a gene target sequence for CRISPR-Cas9 knockout system and the editing types of 2 transgenic lines were analyzed by nucleotide sequence alignment. B. The phenotype of homozygote mature plants (ZmADT2-KO1, ZmADT2-KO2) is knocked out by the wild type and transgenic CRISPR-Cas9 system, and the scale is 50 cm. C. The wild type and the transgenic CRISPR-Cas9 system knock out the ear field disease phenotype of homozygote mature plants (ZmADT2-KO1 and ZmADT2-KO2) with the scale of 10 cm.

FIG. 6 shows the measurement of salicylic acid content.

FIG. 7 shows gibberellin content determination.

FIG. 8 is lignin content determination.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As mentioned previously, corn tumor smut has become one of the major corn diseases, which has a serious impact on corn yield. Screening corn materials with different disease resistance degrees, identifying resistance genes and researching the resistance molecular mechanism of corn to the smut disease are particularly important. However, the corn smut resistance gene cloned in corn is currently poorly understood.

In view of the above, the inventor selects a maize mutant which is dwarf and highly susceptible to the tumor smut in the field, named as dsu, and identifies that the gene Zm00001d028712 on the first chromosome of maize has 72bp deletion through map-based cloning and candidate gene sequencing, and the deletion sequence is shown as SEQ ID No.4, and the details are as follows:

5'-GTGTGACTGACGACCAGGTGGGAGGGCCATAAGGCCTTTAGCGCGGCGGCGGCAATGGCGCCCACGGCCTGC-3', comprising a gene translation initiation codon.

It was further demonstrated by forward genetics that this gene (Zm00001d028712), which encodes Arogenate dehydratase and prephenate dehydratase, is a gene regulating the dsu phenotype and was designated ZmADT 2. The nucleotide sequence of the gene ZmADT2 is shown in SEQ ID NO.1, the coding region (CDS) sequence is shown in SEQ ID NO.2, and the amino acid sequence of the coding protein is shown in SEQ ID NO. 3.

The research of the invention finds that the mutation of the gene ZmADT2 causes the development defect of corn plants, the plant height is seriously dwarfed, and the corn tumor smut is very easy to infect in the field. The gene ZmADT2 can also regulate the synthesis of salicylic acid, gibberellin and lignin in plants. Therefore, the gene ZmADT2 can regulate and control the development of a corn plant type and the resistance to the smut, has the potential of cultivating corn varieties with resistance to the smut and an ideal plant type, can also construct a corn mutant strain which is easy to infect the smut, is used for the production and processing of the smut gall of the corn, and has high production and utilization values, thereby providing the invention.

It should be noted that, the nucleotide sequence at a non-critical position of the gene ZmADT2 of the present invention can be easily mutated by a person of ordinary skill in the art using a known method, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or higher identity with the nucleotide sequence of the gene ZmADT2 of the present invention, such as 75%, 80%, 85%, 90%, 95% or 99%, are derived from the nucleotide sequence of the present invention and are equivalent to the gene ZmADT2 of the present invention, as long as the encoded protein is functionally equivalent to the protein represented by SEQ ID NO.3, and shall fall within the scope of the present invention.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier. Wherein:

maize inbred lines W22, dsu mutant, B73 inbred line, maize a188 inbred line, publicly available from the applicant for use in the repeat of the present invention.

Example 1: phenotypic analysis and mutant gene cloning of corn developmental defect and tumor-susceptible smut mutant dsu

Phenotypic characterization of first, dsu maize mutants

A recessive mutant is separated from a background material of a corn inbred line W22 planted in a test field of Shandong agricultural university and is named dsu (dwarf and susceptible to Ustilago maydis). The phenotype of the mutant is plant height dwarfing, and a tumor grows on the ear, so that the ear is infected with corn smut and cannot normally fruit (figure 1). When Wild Type (WT) and dsu mutants with purified genetic background are planted in a field, the plant height of the dsu mutant is observed to be remarkably reduced relative to that of the wild type plant, and meanwhile, the plant height of the dsu mutant is observed to be remarkably reducedThe mutant was observed to have large numbers of tumors growing in petals on the upper part of the ear. dsu mutant and B73 inbred line are hybridized to obtain F ₁ Plants are substituted, and F ₁ Selfing and pollinating the plant to obtain F ₂ Instead, observation shows F ₂ The generation plants have the phenomenon of plant height separation, and the female ears of the dwarf plants are infected with corn smut and have tumor-shaped substances. Besides the dsu mutant has weak resistance to maize smut and is easy to infect maize tumor smut, the mutant has defects in growth and development compared with the wild type. The mutant seedlings show obvious difference from wild type seedlings in seedling period, plant height is shortened, leaves are shortened and narrowed, leaf color is lightened, roots, tassels and female ears are reduced, grains are reduced, and hundred grain weight is reduced (figure 2).

Second, map-based cloning of mutant genes

Using the above F ₂ Mutant phenotypic material isolated from the population is subjected to map-based cloning of the mutated gene. F was selected by developing molecular markers (primers 1-20, 1-28, 1-47, 2-6, 2-8, 2-20, 4-2, 4-10, 4-16, 4-23, Z8, Z9, Z13, Z14, 2-54, 1-55, 1-59, 1-67, 1-70 for map-based cloning) ₂ The crossover individual in (1) is combined with the linkage relation between the genotype and the phenotype to further narrow the candidate interval.

The results showed that the mutant gene was located on chromosome 1, and the interval was finally narrowed to an interval of about 120Kb for two molecular markers, Z8 and Z13, containing 4 genes in total (FIG. 3A). The full lengths of the wild type and mutant 4 genes were amplified separately (primer Zm028709 for Zm00001D 028709; primer Zm028710 for Zm00001D 028710; primer Zm028711 for Zm00001D 028711; Zm028712D1, Zm028712D2, and Zm028712D3 for Zm00001D028712 in three segments), and sequencing comparison revealed that one of the genes (Zm00001D028712) had a 72-nucleotide deletion (FIG. 3B) with the deletion sequence:

5'-GTGTGACTGACGACCAGGTGGGAGGGCCATAAGGCCTTTAGCGCGGCGGCGGCAATGGCGCCCACGGCCTGC-3', comprising a gene translation initiation codon. The initiation codons of a plurality of transcripts are positioned in a deletion region, and primers (dsu-jd) are designed on both sides of the deletion site to homozygously identify the genotypes of the wild type and the mutant, so that the genotype of the wild type plant is a normal genotype, and the genotype of the mutant is a mutant genotype (figure 3C).

Therefore, the gene (Zm00001d028712) is named as ZmADT2, the nucleotide sequence of the gene ZmADT2 is shown as SEQ ID NO.1, the coding region (CDS) sequence is shown as SEQ ID NO.2, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 3.

The map cloning method is as follows:

(1) extraction of corn DNA

Firstly, taking about 200mg of fresh corn material, putting the fresh corn material into a 1.5mL centrifuge tube filled with steel balls, freezing the fresh corn material for 1min by using liquid nitrogen, and then grinding the fresh corn material for 1min by using a tissue grinder 55 Hz;

② adding 500 mul PL buffer after grinding fully, mixing fully, incubating for 25min at 65 ℃, shaking up for several times every 5 min;

③ after cooling, adding 500 mu L of chloroform, placing on a horizontal shaking bed, and fully shaking at 110 rpm;

fourthly, centrifuging at 11000rpm for 10min, sucking 400 mu L of upper-layer water phase to a new centrifugal tube of 1.5mL, adding 1mL of absolute ethyl alcohol (precooling at minus 20 ℃), slightly reversing and mixing evenly, wherein flocculent precipitate can be seen;

fifthly, centrifuging at 8000rpm for 5min, discarding the supernatant, adding 750 mu L of 75% ethanol, washing the precipitate;

sixthly, centrifuging for 3min at 8000rpm, fully removing the supernatant, uncovering the cover and drying at room temperature for 4 h;

seventhly, after drying, 200 to 250 microliter ddH is added ₂ O, incubating at 37 ℃ for 30min, shaking and mixing uniformly, and storing at-20 ℃ for later use.

(2) PCR reaction system and reaction conditions

PCR amplification was performed using molecular marker primers, and a 20. mu.L volume PCR reaction was prepared as follows:

the PCR amplification reaction was performed as follows:

(3) PCR product band detection

Simultaneously with PCR amplification, 4% agarose gel was prepared. The size of the bands was checked by 4% agarose gel with dsu mutant, B73 inbred line and F ₁ PCR products amplified by taking the material DNA as a template are used as a control, and 10 mu L of standard DNA Marker is added into each row of reserved glue holes for comparing the DNA size of the amplified products.

(4) Amplification and sequencing of candidate genes

Designing a specific primer sequence according to a candidate gene DNA sequence, taking wild type and mutant DNA as templates, and carrying out PCR amplification by adopting Phanta EVO ultrahigh fidelity DNA polymerase, wherein the specific reaction system is as follows:

the PCR amplification reaction was performed as follows:

after the PCR product was detected by 1% agarose gel, DNA was recovered and used

Construction of Cloning vector by Blunt3 Cloning Kit, transformation of vector into E.coli TOP10 E.coli rod competent cells, screening positive clones for sequencing.

Primer sequences involved in the above experiments:

example 2: obtaining and identifying transgenic plants

Identification and genetic complementation of over-expressed plants

Using specific primersThe protein coding region (CDS) of the ZmADT2 gene is amplified and is connected into a vector after the pCAMBIA3300 is modified, and the maize A188 inbred line immature embryo is transformed by an agrobacterium-mediated genetic transformation technology. Positive plants, T, were screened by resistance screening and PCR (primer set: US5-1/ZmADT2-R) identification ₀ The expression level of the ZmADT2 gene in the generation transgenic plant is obviously improved compared with the gene expression level of an A188 inbred line. The foreign DNA sequence was introduced into the dsu mutant by crossing the positive transgenic plant with the dsu mutant and identified by PCR (primer set: US5-1/ZmADT 2-R; F1/R1) (FIG. 4A). Phenotypic analysis showed that the dsu mutant phenotype was completely restored by transferring CDS into ZmADT2 gene (FIG. 4B-C), which demonstrates that ZmADT2 gene is the gene regulating the phenotype.

Second, knockout strain phenotype identification

Meanwhile, a CRISPR-Cas9 knockout vector is constructed, guide sequences respectively targeting two targets of a ZmADT2 gene are introduced into a pBUE411-2gR expression vector, and an A188 inbred line immature embryo is transformed by an agrobacterium-mediated genetic transformation technology. Through PCR identification and amplification sequencing, 2 strains of different edit types (ZmADT2-KO1, ZmADT2-KO2) were screened (FIG. 5A). Homozygotes of both lines had similar dwarfing and other organ development-deficient phenotypes as the dsu mutant compared to the wild-type, while the ears grew tumors similar to those grown on the dsu mutant ear during later development, indicating that ZmADT2 regulates maize development and resistance to smut (fig. 5B-C).

The above experimental method is as follows:

(1) construction of recombinant expression vectors

1) Overexpression vector construction

Firstly, primer design: downloading a CDS sequence of the gene from an http:// ensemble.gramene.org/Zea _ mays/Info/Index website, and selecting a proper enzyme cutting site from expression vector plasmid multiple cloning sites according to the enzyme cutting sites in the CDS sequence to be added at the 5' end of the forward and reverse amplification primers. In the experiment, two enzyme cutting sites of BamHI and ScaI are selected, and a primer pair is adopted: OE-ZmADT 2-F; OE-ZmADT 2-R.

Performing PCR amplification by adopting Phanta EVO ultrahigh fidelity DNA polymerase and using cDNA of wild corn as a template, performing PCR electrophoresis detection and product recovery after the amplification is finished, connecting with a cloning vector, and sequentially performing escherichia coli competent cell transformation, escherichia coli colony PCR screening of positive colonies, bacteria sample sequencing and plasmid extraction.

After the plasmid extraction is finished, the restriction enzyme of the selected enzyme cutting site is used for respectively carrying out enzyme cutting on the expression vector plasmid and the cloning vector plasmid, and then electrophoresis detection and product recovery are carried out.

And fifthly, connecting the target DNA fragment into an expression vector, and sequentially carrying out escherichia coli competent cell transformation, escherichia coli colony PCR screening of positive colonies, bacteria sample sequencing and plasmid extraction.

2) CRISPR/Cas9 gene editing vector construction

Firstly, primer design: the CDS sequence and gDNA sequence of the gene are downloaded from http:// ensemble.gramene.org/Zea _ mays/Info/Index website, 19bp base at the 5 ' end of 5 ' -NGG-3 ' PAM motif is selected as a guide sequence in the exon region, the specificity of the sequence is detected by BLAST tool, and a high specificity sequence is selected for primer synthesis.

② 4 primers (MT1T2-BsF, MT1T2-F0 and MT1T 2-R0) taking pCBC-MT1T2 as a template

MT1T2-BsR) PCR amplification method, which integrates a guide sequence into a vector fragment for PCR electrophoresis detection and product recovery. Carrying out enzyme digestion connection on the recovered PCR product and pBUE411-2gR vector plasmid, and carrying out reaction in a PCR instrument under the reaction conditions of 37 ℃ and 5 hours; 50 ℃ for 5 min; 80 ℃ for 10 min.

The system is as follows:

and thirdly, after the enzyme digestion ligation reaction is finished, carrying out escherichia coli competent cell transformation, escherichia coli colony PCR screening of positive colonies, bacterial sample sequencing and plasmid extraction in sequence.

(2) Obtaining of recombinant Agrobacterium

The EHA105 Agrobacterium competent cells (50. mu.L) were thawed on ice, 5. mu.L of the above recombinant vector was added after thawing completely, and the mixture was flicked and mixed. After ice-bath for 5min, liquid nitrogen is frozen for 8min, and then quickly transferred to a 37 ℃ water bath and placed for 5 min. After ice-cooling for 2min, 1mL of YEP liquid medium was added and the mixture was incubated at 28 ℃ and 200rpm for 3 hours. 200. mu.L of the bacterial suspension was spread evenly on YEP plates containing kanamycin (50mg/L) and rifampicin antibiotic (50mg/L), and cultured in an inverted state at 28 ℃ for 2.5 days. And (5) screening positive strains by colony PCR.

(3) Obtaining and identifying transgenic lines

Activating and culturing the recombinant Agrobacterium in YEP liquid culture medium containing kanamycin and rifampicin antibiotics, collecting appropriate amount of thallus in AB induction culture medium (Glucose 20 g/L; MES 19.52 g/L; NH) containing kanamycin (50mg/L), rifampicin antibiotics (50mg/L) and acetosyringone (100mg/L) ₄ Cl 1g/L；MgSO ₄ ·7H ₂ O 0.3g/L；FeSO ₄ ·7H ₂ O 2.5mg/L；KCl 150mg/mL；CaCl ₂ 10mg/mL；NaH ₂ PO ₃ 0.156g/L) in the culture medium, OD ₆₆₀ Adjusting to 0.8-1.0, infecting corn embryo for 9-11 days after pollination, spreading the infected embryo with its bow facing upwards on co-culture medium (N6 salt; N6 Vitamins; Thiamin HC10.5 mg/L; Sucross 30 g/L; L-proline 1.38 g/L; Myo-insulin 0.1 g/L; Casein hydrosate 0.1 g/L; 2, 4-D0.5 mg/L; MES0.1g/L; 6-BA 0.01 mg/L; Cys 100 mg/L; Agarose 6.5g/L), and dark-culturing at 22 deg.C for 20-24 h. Transferred to a recovery medium (MS salt; MS Vitamins; Cross 30 g/L; L-proline 0.7 g/L; Myo-Inositol 0.1 g/L; Casein hydrosate 0.1 g/L; 2, 4-D0.5 mg/L; MES 0.5 g/L; Dicamba 0.5 mg/L; phytagel 3g/L) and dark-cultured at 28 ℃ for 7 days. Further subcultured on a new recovery medium, followed by transfer to a differentiation medium (MS salt; MS Vitamins; Sucross 60 g/L; Myo-Inositol 100 mg/L; Agarose 3g/L) for differentiation culture until emergence of seedlings.

Extracting the above obtained T ₀ Generating transgenic line DNA, identifying positive plants by specific primer sequence (CAS9-2 sequencing F, CAS9-2 sequencing R), simultaneously amplifying DNA fragments of knockout lines containing two target points, and analyzing gene editing situation by sequencingThe method is described.

Primer sequences involved in the above experiments:

example 3: determination of salicylic acid, gibberellin and lignin content

The wild type planted in the field and the young ear of the dsu mutant are taken for salicylic acid content detection, and the salicylic acid content of the dsu mutant is obviously lower than that of the wild type (figure 6). Analysis of gibberellin content in wild-type and mutant caulicles showed that the gibberellin content of dsu mutant was significantly lower than that of wild-type (fig. 7). Meanwhile, the dsu mutant had lower lignin levels than the wild type (FIG. 8). The ZmADT2 gene is shown to regulate the synthesis of salicylic acid, gibberellin and lignin.

The experimental procedure described above is as follows:

(1) determination of salicylic acid and gibberellin

Taking the young ears of wild type and mutant, grinding in liquid nitrogen thoroughly in a mortar, taking about 200mg of the ground powder containing ² H ₄ ]-overnight extraction in methanol with standard SA internal standard, and purification of crude extract by Oasis MAX SPE, for assay after further pretreatment.

Taking wild type and mutant caulicles, freezing the wild type and mutant caulicles in a mortar by using liquid nitrogen, fully grinding, taking about 200mg of ground powder, extracting the powder by using 90% methanol, further purifying a crude extract by using a gibberellin standard product to be detected as a reference, and then carrying out quantitative detection by UPLC MS/MS.

(2) Tissue staining method lignin content

The wild type and mutant were taken from the same internode and the material was cut into 150 μm thick slices by using a shaking microtome. Staining with 2% (w/v) phloroglucinol solution in 95% ethanol for 2min, followed by treatment with 50% HCl for 2min, coverslipping, and observation under an optical microscope.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> gene ZmADT2 for regulating and controlling maize plant type and resistance to smut, and coding protein and application thereof

<130> 2022

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 7570

<212> DNA

<213> corn (Zea mays)

<400> 1

ctgggctaga cagaccggct catgttggca aaatattctg ggccgtatca tgttggcaaa 60

aatatactga ctccggtccg caactccgga tgtgtcggcg accaggcaaa gctccgtgtc 120

ctctcgtctc gtgtgactga cgaccaggtg ggagggccat aaggccttta gcgcggcggc 180

ggcaatggcg cccacggcct gcctccggac ccccttcttc cccgcccgtg cccgatccac 240

caacgccacc ccgttctcgc cacaccgccg cttttctctc aaatgcagcg cctccaacgg 300

tgacaactcc aaccccaact ccatctccat ctctcccacc tcgccgcctc ggctaacggt 360

gtcggacggc gtcgagtctg tggacgttaa cgggttgcgt cgcccgccag cgcccgcctc 420

cgtccccgcc gcccgcgatc cccactggct gcctcgtaag tcgcgcggac gcgacggtcc 480

ttgcgtttgc taatctcggg tttgattaat caaacggtgc taatctgtga tctgcgtgtt 540

ttttttttgg ctgtgtttag gtccgctcac aagtgcggac gtcatggagg tggacgggaa 600

ggggctcaag gttgcttacc aggtaggtgc tcgacgaaac gcgtctcacg ggtgctgttt 660

caatttcctt aggactgtgc ttgtgtgttt cagataattt aagggagttt ggttatggtg 720

ttaaacttag ggttgtgctg gagcgtacag cgaagcagca gcaaagaagg cgtacccgaa 780

ttgtgagact gtgccctgtg agcacttcga cactgcattt caggtaacaa agggtgttgc 840

ctgttcagca catcctagtt actttacttg actcatgttg cgattttggt aggataccag 900

attgcaagtg gttaaatgtt gtcttacgaa acctattgag aagtaccaag gctgaattgc 960

agtggcgtac ctaggatttt agtgacgggt ggtctaacta gatttttttg actatataaa 1020

tgcaacacat cggtttctaa ataaaaaaat taccaaatac aatataatca aataaaaagg 1080

tcttataatc atctaacata ttaaaaataa aagtataaca attatacctt acatactaat 1140

gatgatagtt taagaaaaaa tataattcta ctttttttgt tggacctaca cctcttaata 1200

gccatgaaag tatttatgat atcatcttca cccacttata agaaaatata tcgttcgaca 1260

aaaaatgacg agacaatcat caaaagacta tctccatctt atttcttaat ttattcttga 1320

caagtattat tgcagaaaat actctatcaa cacgtgttgt tgcgacgggt acaaacaata 1380

ctcgtttgac ggggatagtg acaacaggct aattagcttt gtcggttgga gcctttacaa 1440

acgtgaaacg tcaagtcgta cttgtacctg ctgcctgcta gactgcagct tgccgccagg 1500

gttgctcaca gttgcgtcac agagatgcct acagcctatg gccacgtccg ctgccccgtc 1560

tgcagtaaat ttgttgaagt gtcgctccat ccgattgtcc caattgtaga cttgtatgga 1620

ctttggttgt actgagcgca tggtggtccg tggcccacaa ggaccaccat gtagctccgc 1680

cactgctgaa ttggactggc tccatattgc ttttgtttaa cacactaatt ggtagaaata 1740

atcatcacta acacataata tgcattaaat gcttcccatg ctgtgttaga aaaaacaagc 1800

tctagccagc aatgttgttc ataccatttt ttgggggggc tattgctcat acgaatttat 1860

cgtgcttgcg gaatgtaaaa aatgccacat atctgctctt gtgtagttgt gttctttgaa 1920

aattgagata gatgaggcta aatttactta tcatgtcaat ttcctgaaat agactataag 1980

ggaagcacat ttttgtgact tggtagcagt agggtaatat ttttttctag cacaggagag 2040

atttgtacca ttgcactaaa agtaggttaa catgtaacat aatttttttt cttctaaaac 2100

tatttttcag agaggtatat gtgttttgtc ttaaagatac acttctctag ttagtgtatt 2160

gatgtgactt ttgttttatc tcatgttctc ctttgacagg ctgttcaaaa ctgggtagct 2220

gatagggccg tactgccact tgagaattca ttgggaggta gcatacatag gaattatgat 2280

cttttgcttc ggcatagttt gcacattgta ggagaagttc gccttgcggt tcgccattgc 2340

ttattagcca atcctggcgt gaagattgaa aacctgaaaa gtgctatgag tcatccccag 2400

gtgaactgct tggttgatct gtctttacta ctctcattac tgcttgtttt catcggaggt 2460

aatcttgcaa ccatatcatt tattttaatt gataggctct tgcgcaatgt gagcacacat 2520

taacatcact aggaattgag catagagaag ctgttgacga cactgctggt gcagcaaagg 2580

tagattgagg ggttttgttc acctaaaaat caccttttaa ttcagatatc ccttttttag 2640

caactctttt tttattcttt tatagattgt gatttcagtt acccattatg ttgaactctc 2700

tgtttttaat catagcaata gcattctttc ctagattgtt gcagaacata tgctccagga 2760

cactggtgct attgctagtt cgttggcggc taaactgtat ggattggatg ttcttgctga 2820

gaatattcag gtacagcgag ttcatgctaa aatatttaaa taagatacac attctgatct 2880

ttgttctgaa aattaacctg tcaatttatt taggatgaca aagataatgt cacccgtttt 2940

atgatgctag ctcgagaacc catcatacct cgcactgata aaccatttaa ggtacccata 3000

tttgtgcaag ctcttttcca ttatcactgc ttatcctcat tgtcaccaca gctgatttgt 3060

tgtatgacca ttttttagac aagcatagtc ttttcactag aagaaggacc tggacagctc 3120

tttaaggcac ttgctgtatt tgcactgagg gacatcaacc tcacaaaggt ctaccaaaca 3180

actttgtctt gtcagttcca tgcattttgt acaaatgatt cactatctgt gctctgtttt 3240

tgaatggatg tgcagattga aagtcgtcca cacaaggaaa ggcctcttcg ggtatctgat 3300

gactgttcct cactcttaaa gttagatgct aaacctgtgg ccttctaaat cattcaattt 3360

cactacctgc agttttgcat gaatatgagc attacttgtg ctgggcaagt gtttatatct 3420

ttctaaccca tccaatcctg gattgcacca ggaactttga ttaccttttt tatgttgacc 3480

ttgaagcatc aatggctgat ccaaagactc agaatgctct tggaaatctg aaggtgctct 3540

ttctctgtta gtacaatctg gtgaatgtaa actgtgctca tgttgcttta tttcaggagt 3600

ttgcaacatt tttaagagtt cttggaagct atcctacaga tgtgaatgaa ccttagattg 3660

aaagtactgc agattacata ctggtgagta atgaataaca ttgtctactt tttgtgcttt 3720

gttactatag tatggtgaac gaaagaacat tcttttattt tcagtttcaa cgacataata 3780

tgttggagtt gggcaagtca cttgagtgac atgcttcttt tataaaatgt aatgcatggt 3840

ttgacttggt gcaccttaaa acgaatgggg agactactac tagacatttg tgggtgaata 3900

tccgttcctt agtttgcttg ggttgagtag atgtagaaca actttttggt atatgatcag 3960

aagtattcta atcagcaaaa aaacacacat taaaaaggag aattgttcca cataattcaa 4020

ttcttccaac caacaaaaac attgttctgt tagattggac gcactaacca gttcaaccca 4080

aaagtttaag ctgatgggag aaggtagtca atccacttat acatttcaac accccactca 4140

cgtgcagaca gagagaaaag tgcaaacgtg gaaataaatg ggtgaaggca taaataaagc 4200

ctctgtcatg attcgaactc gagacctcta gctccgatac catgttagag tggatgcact 4260

aactagttca acccaaaagc ttaaactgat aaaagaagat aaccaatcca cttatacact 4320

tcaacatgtt tcacattgaa aaggtactgt tctagaatta aattattctg aactataaaa 4380

ttgttaggca ttgaaaagaa atttgttcca gactttcagg cataaaatca aattgttttt 4440

ttctcgaaag cgcaggagaa ctacgcaata atgtattaaa gaagaggtac atatacaaga 4500

acaaactagg ggcccctacg gcagccaata acaggcatac agaaacccat ccatgactaa 4560

aaaactaaca agaactacca caatgctagc taactagaag ggaggcccgg aatgggggcg 4620

gtaagcaaag acaacttctt tgcaccagcc atctcccaaa ggtcaacctc caacccaact 4680

cttctaatac catcagtaat actggggctc ttgttgtaaa aaacacaacc attgtgctgc 4740

ttccataagg tccaaactcc caggattact agagaattga ggccatccct tgcgattcca 4800

gagaccttag tacataacat tctccaccat tcaaggaagg cactatcctc ggattggggg 4860

caagaagctg aaggttcact tgatgaagta gtttgaacca aaattctcga gcaaaaacac 4920

atccaatcaa catatgatcc aaggtttctt gatcctgatc acataatgga catctctccg 4980

gatggtccgt gccttttttc tgtagcctat ctgatgttca aaccttcctg tgtgcgacca 5040

gccacataaa aaacttagtt tttggaggag cccaagtttt ccatattcta tgaaaaggct 5100

caaactcaga agatccaata aaggaacccc ctatatgctt cctttgagga gaactttccg 5160

tttgcagcta ggcgataaaa atgcttgtct tcaacttgag gtcttagaac aattgagtca 5220

actaaatccc acaagatgag aaactcattg ataattccaa cagaaagagc tccctgaatg 5280

ccagaaagcc agctctgatt agagatagca tccaccactg ttctcttgtt aactcttttc 5340

ttaggaatga tcccaaataa tcgaggggct aagtctacaa ttctgtgacc caatagccac 5400

ctatcggtcc agaaaaaaaa tcaaattgtt ctaacccaca aaaagttgtt ccatgcaaaa 5460

gataaattgt tctaggacat taaattgaat tattctaatc aacaaccaaa aaaatcaaat 5520

tgttttaacc agcaaaaacg tgttccacac aaaagatgaa ttgttctagg gtattaaatt 5580

agattgtttc taatcaccaa ccaaaaaacc gttccatgtt aaaaaatggc ttgttccagg 5640

tataaaaccg aatgttgtaa aaatgaagaa ttgcatagag aagatgagtg ggtggtgtca 5700

agcagtggag cttgaagctt tggtaattga aggatggagg ctcaagcaac gtgaggatga 5760

aaaaggagca ggcaggctat catggctgac attgagtgaa ttgggctgac gtgatggtcc 5820

tcggttctgt ggatggactg cgtggggaat aaaaagaaga ttgtcacgtg gatgactttt 5880

agttgccgca gccaaattca cgccttagag gacttgtcat ctttgctttc ttatacatag 5940

tcattttgag tttactgaaa gaatcctata aaacttctaa taaactaggc aactaagtag 6000

cagaagttga tgaccatggt tattaaagca ctaaagcact actgagatgt taaagcgctg 6060

caaagcttca cgaattgacg taaagcgccc gttttaagcg ctaaagcgcc actttttagc 6120

gttaaaacac tttgggccgt aaaaaagcca cattttgtcg ctttttcaga tttccaaccc 6180

agcacaggcc tcctaaccta aaacaggggg catcattcct ttgtcacgcc tgcagctagc 6240

cgtcacggac tcacgctacg cgttcgtccg acatccctag gcttcaccgc tggccggtaa 6300

gaaccttgct tgagctcgct gcctccctcc cctcctccct gagttgctcc ctgctcgcgc 6360

cacgctccct ctcccccgcg gccgcgctcg gcgctcgttg ctccttggtc cctgctcgcg 6420

ctcggcgccg ccgctgcctc cctgtgatgc actccctgct ctcgttcggt cagctcgccg 6480

gtcctcgctt agttgcctcc ctgctctgcc tctgttagct tgccaagtcg cctctctgct 6540

ctgcctttgt tagctgcccc tgtccctgct ctcatcctcc tccccttccc tgctcacatt 6600

ctcctctgat tttgattatt tgcagactga aaatggcaac cagtagtgat gctaatgcat 6660

ctgtggtcgt ggacattgca ttccacttta gttgcttttg tgctatgttt atgattgtgg 6720

actactgcac atttcattcc actttgtttg tggttgtcga cttgctagtt gctattagtt 6780

gaacacttga acttgtgtta ttgtggattt gtggtagagt ggttgtggac atgttataat 6840

tttacgtgaa attatgttat attcgtattt tgtccatgtt ctttaaaaag acgttttaaa 6900

gctcgcttaa acgttttaaa gtcaaaaagt tctcatgagc gcttcaacgt tttatcgctt 6960

caataacctt gttgatgact actatttgta ggagaaaata ccagaaaata atagatatgg 7020

aaaggtgtat tgcatgtaat ttcccttcaa gagcaacaga agttactata ttatttgttt 7080

attctctcaa ggatctaatt tgttctcaaa cgttttctga tccaggatta gcaggaatag 7140

gcaaaatggt tagagttggt ttcctgagca agttaaaatg aggaactcta ttaaggtgat 7200

gctaactttt agtcttttac agagtaaaag gatctactct agaccagtag ggaccggtgc 7260

attcgaggct cacatttggt tgtgatattg agaggaaagt ctgatgtttt gctggtgatt 7320

tttctgatga tttaagtctt ctccaatctc ctatggatac caaagggttg aatattcgga 7380

aatacttcaa tgtacaaggt ttatatgttt catgttggag aatctgtgtc atggtagcat 7440

gcaggttgct aaagcacagt tacagtgaaa taatgggaca atgtctaaga acaccgccct 7500

ttttttgtga caatataata agaactgaat aactgatgtt tccatgtctg ggcgattggg 7560

ccggcgttcc 7570

<210> 2

<211> 1182

<212> DNA

<213> corn (Zea mays)

<400> 2

atggcgccca cggcctgcct ccggaccccc ttcttccccg cccgtgcccg atccaccaac 60

gccaccccgt tctcgccaca ccgccgcttt tctctcaaat gcagcgcctc caacggtgac 120

aactccaacc ccaactccat ctccatctct cccacctcgc cgcctcggct aacggtgtcg 180

gacggcgtcg agtctgtgga cgttaacggg ttgcgtcgcc cgccagcgcc cgcctccgtc 240

cccgccgccc gcgatcccca ctggctgcct cgtccgctca caagtgcgga cgtcatggag 300

gtggacggga aggggctcaa ggttgcttac cagggttgtg ctggagcgta cagcgaagca 360

gcagcaaaga aggcgtaccc gaattgtgag actgtgccct gtgagcactt cgacactgca 420

tttcaggctg ttcaaaactg ggtagctgat agggccgtac tgccacttga gaattcattg 480

ggaggtagca tacataggaa ttatgatctt ttgcttcggc atagtttgca cattgtagga 540

gaagttcgcc ttgcggttcg ccattgctta ttagccaatc ctggcgtgaa gattgaaaac 600

ctgaaaagtg ctatgagtca tccccaggct cttgcgcaat gtgagcacac attaacatca 660

ctaggaattg agcatagaga agctgttgac gacactgctg gtgcagcaaa gattgttgca 720

gaacatatgc tccaggacac tggtgctatt gctagttcgt tggcggctaa actgtatgga 780

ttggatgttc ttgctgagaa tattcaggat gacaaagata atgtcacccg ttttatgatg 840

ctagctcgag aacccatcat acctcgcact gataaaccat ttaagacaag catagtcttt 900

tcactagaag aaggacctgg acagctcttt aaggcacttg ctgtatttgc actgagggac 960

atcaacctca caaagattga aagtcgtcca cacaaggaaa ggcctcttcg ggtatctgat 1020

gactgttcct cactcttaaa gaactttgat tacctttttt atgttgacct tgaagcatca 1080

atggctgatc caaagactca gaatgctctt ggaaatctga aggagtttgc aacattttta 1140

agagttcttg gaagctatcc tacagatgtg aatgaacctt ag 1182

<210> 3

<211> 393

<212> PRT

<213> corn (Zea mays)

<400> 3

Met Ala Pro Thr Ala Cys Leu Arg Thr Pro Phe Phe Pro Ala Arg Ala

1 5 10 15

Arg Ser Thr Asn Ala Thr Pro Phe Ser Pro His Arg Arg Phe Ser Leu

20 25 30

Lys Cys Ser Ala Ser Asn Gly Asp Asn Ser Asn Pro Asn Ser Ile Ser

35 40 45

Ile Ser Pro Thr Ser Pro Pro Arg Leu Thr Val Ser Asp Gly Val Glu

50 55 60

Ser Val Asp Val Asn Gly Leu Arg Arg Pro Pro Ala Pro Ala Ser Val

65 70 75 80

Pro Ala Ala Arg Asp Pro His Trp Leu Pro Arg Pro Leu Thr Ser Ala

85 90 95

Asp Val Met Glu Val Asp Gly Lys Gly Leu Lys Val Ala Tyr Gln Gly

100 105 110

Cys Ala Gly Ala Tyr Ser Glu Ala Ala Ala Lys Lys Ala Tyr Pro Asn

115 120 125

Cys Glu Thr Val Pro Cys Glu His Phe Asp Thr Ala Phe Gln Ala Val

130 135 140

Gln Asn Trp Val Ala Asp Arg Ala Val Leu Pro Leu Glu Asn Ser Leu

145 150 155 160

Gly Gly Ser Ile His Arg Asn Tyr Asp Leu Leu Leu Arg His Ser Leu

165 170 175

His Ile Val Gly Glu Val Arg Leu Ala Val Arg His Cys Leu Leu Ala

180 185 190

Asn Pro Gly Val Lys Ile Glu Asn Leu Lys Ser Ala Met Ser His Pro

195 200 205

Gln Ala Leu Ala Gln Cys Glu His Thr Leu Thr Ser Leu Gly Ile Glu

210 215 220

His Arg Glu Ala Val Asp Asp Thr Ala Gly Ala Ala Lys Ile Val Ala

225 230 235 240

Glu His Met Leu Gln Asp Thr Gly Ala Ile Ala Ser Ser Leu Ala Ala

245 250 255

Lys Leu Tyr Gly Leu Asp Val Leu Ala Glu Asn Ile Gln Asp Asp Lys

260 265 270

Asp Asn Val Thr Arg Phe Met Met Leu Ala Arg Glu Pro Ile Ile Pro

275 280 285

Arg Thr Asp Lys Pro Phe Lys Thr Ser Ile Val Phe Ser Leu Glu Glu

290 295 300

Gly Pro Gly Gln Leu Phe Lys Ala Leu Ala Val Phe Ala Leu Arg Asp

305 310 315 320

Ile Asn Leu Thr Lys Ile Glu Ser Arg Pro His Lys Glu Arg Pro Leu

325 330 335

Arg Val Ser Asp Asp Cys Ser Ser Leu Leu Lys Asn Phe Asp Tyr Leu

340 345 350

Phe Tyr Val Asp Leu Glu Ala Ser Met Ala Asp Pro Lys Thr Gln Asn

355 360 365

Ala Leu Gly Asn Leu Lys Glu Phe Ala Thr Phe Leu Arg Val Leu Gly

370 375 380

Ser Tyr Pro Thr Asp Val Asn Glu Pro

385 390

<210> 4

<211> 72

<212> DNA

<213> corn (Zea mays)

<400> 4

gtgtgactga cgaccaggtg ggagggccat aaggccttta gcgcggcggc ggcaatggcg 60

cccacggcct gc 72

<210> 5

<211> 20

<212> DNA

<213> 1-20

<400> 5

tgctgatcaa ccgagtatgc 20

<210> 6

<211> 20

<212> DNA

<213> 1-20

<400> 6

ctctcctcgt tcgttcttcg 20

<210> 7

<211> 24

<212> DNA

<213> 1-28

<400> 7

gtacaacgga ggcattctca agtt 24

<210> 8

<211> 24

<212> DNA

<213> 1-28

<400> 8

tgtacatggt ggtcttgttg aggt 24

<210> 9

<211> 20

<212> DNA

<213> 1-47

<400> 9

aggaagctgt atcccacacg 20

<210> 10

<211> 20

<212> DNA

<213> 1-47

<400> 10

tcatgggttt cttctttgcg 20

<210> 11

<211> 19

<212> DNA

<213> 2-6

<400> 11

ggacctccag gcaaggaac 19

<210> 12

<211> 20

<212> DNA

<213> 2-6

<400> 12

cagtagtagc cagttcccgc 20

<210> 13

<211> 20

<212> DNA

<213> 2-8

<400> 13

cacaaacgtc caagcgtcag 20

<210> 14

<211> 20

<212> DNA

<213> 2-8

<400> 14

gcagatctac tcttcggccc 20

<210> 15

<211> 20

<212> DNA

<213> 2-20

<400> 15

gctctggctt gccattctca 20

<210> 16

<211> 20

<212> DNA

<213> 2-20

<400> 16

tgtctggcat ggtttccgtt 20

<210> 17

<211> 20

<212> DNA

<213> 4-2

<400> 17

gccagaggtc tattcggtct 20

<210> 18

<211> 19

<212> DNA

<213> 4-2

<400> 18

ggaagcccct acagcagaa 19

<210> 19

<211> 22

<212> DNA

<213> 4-10

<400> 19

cgagctggat tatgcaacaa gt 22

<210> 20

<211> 20

<212> DNA

<213> 4-10

<400> 20

tcgcagaaac cccgaatgtt 20

<210> 21

<211> 20

<212> DNA

<213> 4-16

<400> 21

tctcgcctct ccattgatgc 20

<210> 22

<211> 21

<212> DNA

<213> 4-16

<400> 22

tgctttactt gcttctcctc a 21

<210> 23

<211> 20

<212> DNA

<213> 4-23

<400> 23

ttttgtgtca ttgcggcctt 20

<210> 24

<211> 19

<212> DNA

<213> 4-23

<400> 24

gggaaatacg gggttctgg 19

<210> 25

<211> 18

<212> DNA

<213> Z8

<400> 25

gtcgtaactg ggtacgca 18

<210> 26

<211> 19

<212> DNA

<213> Z8

<400> 26

gaagcggatc ataaaggac 19

<210> 27

<211> 19

<212> DNA

<213> Z9

<400> 27

gcatacagca tcagagtct 19

<210> 28

<211> 20

<212> DNA

<213> Z9

<400> 28

ggaatgcagg tttgctgatg 20

<210> 29

<211> 20

<212> DNA

<213> Z13

<400> 29

cctttgcatt tccgagaacc 20

<210> 30

<211> 19

<212> DNA

<213> Z13

<400> 30

tactttgctg tcctgcgag 19

<210> 31

<211> 18

<212> DNA

<213> Z14

<400> 31

gctacggctt cgtcagtc 18

<210> 32

<211> 19

<212> DNA

<213> Z14

<400> 32

gcgtccgcct ccttgaacg 19

<210> 33

<211> 20

<212> DNA

<213> 2-54

<400> 33

ggtgagttct ccagtccctg 20

<210> 34

<211> 20

<212> DNA

<213> 2-54

<400> 34

gaacaaggtc ttaagtggcc 20

<210> 35

<211> 20

<212> DNA

<213> 1-55

<400> 35

ggtgatgacc acgtacaagc 20

<210> 36

<211> 20

<212> DNA

<213> 1-55

<400> 36

ctgtgacatg tttgatgccc 20

<210> 37

<211> 20

<212> DNA

<213> 1-59

<400> 37

accctcatct tcagcagtcg 20

<210> 38

<211> 20

<212> DNA

<213> 1-59

<400> 38

ggtgaaacgg cagtacaagg 20

<210> 39

<211> 23

<212> DNA

<213> 1-67

<400> 39

agaccatatt ccaggcttta cag 23

<210> 40

<211> 21

<212> DNA

<213> 1-67

<400> 40

acaactagca gcagcacaag g 21

<210> 41

<211> 19

<212> DNA

<213> 1-70

<400> 41

cggttcatgc tagctctgc 19

<210> 42

<211> 18

<212> DNA

<213> 1-70

<400> 42

gttgtggctg tggtggtg 18

<210> 43

<211> 20

<212> DNA

<213> Zm028709

<400> 43

aattctgagt gcccacctga 20

<210> 44

<211> 20

<212> DNA

<213> Zm028709

<400> 44

agactcggac tcaagttgca 20

<210> 45

<211> 20

<212> DNA

<213> Zm028710

<400> 45

cccatcttcg ctgcatttca 20

<210> 46

<211> 20

<212> DNA

<213> Zm028710

<400> 46

caacctccac ctcgatgaga 20

<210> 47

<211> 20

<212> DNA

<213> Zm028711

<400> 47

ctggtccgtt caaactccct 20

<210> 48

<211> 20

<212> DNA

<213> Zm028711

<400> 48

cgccttttga tccgaacctt 20

<210> 49

<211> 25

<212> DNA

<213> Zm028712D1

<400> 49

ctgggctaga cagaccggct catgt 25

<210> 50

<211> 19

<212> DNA

<213> Zm028712D1

<400> 50

taagagtgag gaacagtca 19

<210> 51

<211> 19

<212> DNA

<213> Zm028712D2

<400> 51

tacctcgcac tgataaacc 19

<210> 52

<211> 18

<212> DNA

<213> Zm028712D2

<400> 52

agcagggagc aactcagg 18

<210> 53

<211> 19

<212> DNA

<213> Zm028712D3

<400> 53

ggcaactaag tagcagaag 19

<210> 54

<211> 20

<212> DNA

<213> Zm028712D3

<400> 54

caaaccgact aacctgctcg 20

<210> 55

<211> 20

<212> DNA

<213> dsu-jd

<400> 55

ccacgacatc ctcaagcaag 20

<210> 56

<211> 21

<212> DNA

<213> dsu-jd

<400> 56

acagattagc accgtttgat t 21

<210> 57

<211> 23

<212> DNA

<213> OE-ZmADT2-F

<400> 57

ggatccatgg cgcccacggc ctg 23

<210> 58

<211> 25

<212> DNA

<213> OE-ZmADT2-R

<400> 58

gagctcctaa ggttcattca catct 25

<210> 59

<211> 36

<212> DNA

<213> MT1T2-BsF

<400> 59

aataatggtc tcaggcgagt catcagatac ccgaag 36

<210> 60

<211> 40

<212> DNA

<213> MT1T2-F0

<400> 60

gagtcatcag atacccgaag gttttagagc tagaaatagc 40

<210> 61

<211> 33

<212> DNA

<213> MT1T2-R0

<400> 61

ttgcttatta gccaatcctc gcttcttggt gcc 33

<210> 62

<211> 36

<212> DNA

<213> MT1T2-BsR

<400> 62

attattggtc tctaaacttg cttattagcc aatcct 36

<210> 63

<211> 19

<212> DNA

<213> CAS9-2 sequencing F

<400> 63

gttagtgtat tgatgtgac 19

<210> 64

<211> 19

<212> DNA

<213> CAS9-2 sequencing R

<400> 64

gatcagccat tgatgcttc 19

<210> 65

<211> 19

<212> DNA

<213> ZmADT2-R

<400> 65

ctaaggttca ttcacatct 19

<210> 66

<211> 21

<212> DNA

<213> US5-1

<400> 66

ttttagccct gccttcatac g 21

<210> 67

<211> 20

<212> DNA

<213> F1

<400> 67

ctagacagac cggctcatgt 20

<210> 68

<211> 20

<212> DNA

<213> R1

<400> 68

ccgttaacgt ccacagactc 20

Claims (10)

1. A gene ZmADT2 for regulating and controlling corn plant type and smut resistance, wherein the gene ZmADT2 is a nucleic acid molecule shown in the following i) or ii) or iii) or iv):

i) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

iii) a nucleic acid molecule which is 75% or more identical to the nucleotide sequence of i) or ii) and which expresses the same functional protein;

iv) a nucleic acid molecule other than ii) which encodes the amino acid sequence shown in SEQ ID NO. 3.

2. The protein encoded by the gene ZmADT2 of claim 1, which is a protein represented by any one of (a1) to (A3) below:

(A1) a protein consisting of an amino acid sequence shown by SEQ ID NO.3 in a sequence table;

(A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.3 in the sequence table, is related to the corn plant type and the resistance to the smut and is derived from the SEQ ID NO. 3;

(A3) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1) or (A2).

3. A recombinant expression vector, transgenic cell line or genetically engineered bacterium comprising the gene ZmADT2 of claim 1.

4. Use of the gene ZmADT2 of claim 1, the protein encoded by the gene ZmADT2 of claim 2, or the recombinant expression vector, transgenic cell line or genetically engineered bacterium comprising the gene ZmADT2 of claim 3 in at least one of the following (1) to (4):

(1) regulating and controlling the growth and development of plants;

(2) regulating and controlling the resistance of the plant to the smut disease;

(3) regulating and controlling the synthesis of plant salicylic acid, gibberellin and lignin;

(4) plant breeding;

preferably, the plant is maize.

5. The use of claim 4, wherein the plant breeding comprises: corn plant type breeding, and disease-resistant corn variety or strain breeding.

6. A method for adjusting the plant type of corn is characterized by comprising the following steps:

the gene ZmADT2 or the protein coded by the gene ZmADT2 is taken as a target, the expression level of the gene ZmADT2 is regulated and controlled through a biotechnology means, or the activity of the protein coded by the gene ZmADT2 is changed;

preferably, the biotechnological means comprises: gene editing, RNAi, T-DNA insertion, or overexpression.

7. The cultivation method of the corn with the anti-tumor smut disease is characterized by comprising the following steps: transferring a gene ZmADT2 shown in SEQ ID NO.1 or SEQ ID NO.2 into corn to obtain corn with anti-tumor powdery mildew;

or regulating the expression of the gene ZmADT2 shown in SEQ ID NO.1 or SEQ ID NO.2 in the corn genome, and screening to obtain the corn plant with improved resistance to the smut.

8. The breeding method of claim 7, wherein the method of modulating the expression of gene ZmADT2 in the maize genome comprises: introducing a DNA fragment capable of activating, increasing or inhibiting the transcriptional or translational level or protein activity of gene ZmADT 2; or controlling the synthesis of specific small RNA molecules and regulating the accumulation of mRNA of the gene ZmADT 2.

9. A method for producing corn tumor masa, comprising the steps of:

knocking out or silencing gene ZmADT2 in corn; or, the function of the protein coded by the gene ZmADT2 in the corn is lost, and corn material is obtained;

corn tumors, masa, were collected from the corn material obtained.

10. The method of claim 9, wherein the gene ZmADT2 in maize is knocked out or silenced using CRISPR-Cas9 gene editing technology, VIGS technology, T-DNA insertion, or RNA interference technology.

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