CN113969270A - Application of plant infection-related protein TaCIPK14 in regulation and control of stripe rust resistance of plants - Google Patents

Abstract

The invention discloses an application of a plant infection related protein TaCIPK14 in regulation and control of plant stripe rust resistance. The invention provides application of any one of substances a1) -a3) in regulation and control of stripe rust resistance of plants; a1) protein tacpk 14; a2) a nucleic acid molecule encoding the protein tacpk 14; a3) a recombinant vector, expression cassette, recombinant microorganism or recombinant transgenic plant cell line comprising a nucleic acid molecule encoding the protein tacpk 14; TaCIPK14 of the invention expresses a down-regulation expression trend in the interaction process of wheat and puccinia striiformis, and encoded proteins are distributed in cytoplasm, cell membrane and cell nucleus. The inhibition of the expression of the TaCIPK14 protein can reduce the stripe rust disease susceptibility of plants and enhance the disease resistance, and plays an important role in breeding plants with enhanced stripe rust resistance.

Description

Application of plant infection-related protein TaCIPK14 in regulation and control of stripe rust resistance of plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a plant infection-related protein TaCIPK14 in regulation and control of plant stripe rust resistance.

Background

Wheat stripe rust is one of the most important diseases of wheat in China, and has the advantages of strong popularity, wide distribution and serious harm. A large number of practices at home and abroad prove that the method for preventing and controlling the wheat rust by utilizing the disease resistance of the variety is the most economic and effective measure. However, since the toxicity of the stripe rust fungus is changed frequently and new small toxic species appear continuously, the rust resistance of wheat varieties is often lost, and since the 50 th century of the twentieth century, 7 batches of wheat improved rust resistant species lose the stripe rust resistance in China (plum shiver et al, 2002) successively exist in production. In recent years, with the appearance and development of small varieties such as No. 32, No. 33, V26 and the like in the wheat stripe rust fungus stripe, most main cultivars in wheat production in China lose the stripe rust resistance, so that the wheat in China is in the threat of the pandemic damage of stripe rust again. The research on the molecular mechanism of the interaction relationship between the wheat and the stripe rust is developed, and the method has an important theoretical guidance effect on the reasonable utilization of the stripe rust resistance of the wheat and the sustainable control of the stripe rust of the wheat.

Pathogenic bacteria infect plants to cause defense reaction of the plants, and meanwhile, some components of the hosts are utilized to adjust self growth, infection structure differentiation, negative regulation and control of plant immune reaction and absorption of nutrient substances from the hosts, so that host infection is caused (van Schie and Takken, 2014). All host genes that promote pathogen infestation and support affinity interactions are considered susceptible genes. In the early stage of pathogen infection, the disease-sensitive gene plays a role by promoting host recognition and pathogen invasion; in the invasion stage, the susceptibility gene increases susceptibility through negative regulation of host immune response; after successful invasion and establishment of parasitic relationships, pathogenic bacteria utilize host nutrients to meet their own metabolic and growth needs and promote susceptibility (van Schie and Takken, 2014). For example, sugar transporters (OsSWEET11 and OsSWEET13), homoserine kinase gene (HSK), aspartate kinase (AK2), and the like mediate host susceptibility to pathogenic bacteria (Lumbreras et al, 2010; vanDamme et al, 2009). The mutation or deletion of the susceptible gene can limit the pathogenic capability of pathogenic bacteria infection, thereby enhancing the disease resistance of the host. The genome editing technology can utilize artificially designed and modified nuclease to carry out precise site-directed modification on the genome, thereby realizing gene function knockout. The resistance of some important commercial crops to a plurality of main phytopathogens including plant viruses, bacteria and fungi can be improved by directly targeting the susceptible gene of a mutant host by using a gene editing technology (Zaidi et al, 2018). By using gene editing techniques, the mutant Arabidopsis and cucumber Eukaryotic translation initiation factor eIF4E (Eukaryotic translation initiation factor 4E), rice OsSWEET, citrus CsLOB1(Lateral organic bases 1), tomato Mlo and wheat Mlo and Raf-like mitogen-activated gene EDR1(enhanced disease resistance 1) confer resistance to potato Y virus, xanthomonas, citrus canker and powdery mildew, respectively, to the corresponding plants (Zaidi et al, 2018). Furthermore, the mutant DMR6(DOWNY MILDEW RESISTANCE 6) conferred plants with broad spectrum resistance to various pathogenic bacteria (de Toledo Thomazella et al, 2016). The disease resistance breeding is carried out by mutating host susceptible genes, so that crops obtain durable resistance to diseases, and part of bred varieties are commercialized (Parisi et al, 2016) at present. The strategy of improving disease resistance by mutating the susceptible gene greatly improves the efficiency of disease-resistant breeding and has wide application prospect.

Intracellular Ca after plants are stimulated by various biotic and abiotic stresses²⁺Short and complex changes occur. In order to properly perform the physiological responses, the calcium signals induced by each different environmental stimulus or different developmental information are specific, either in terms of different subcellular localization or in terms of the kinetics and magnitude of calcium oscillations. Calcium signals generated in response to stimuli are transmitted through the corresponding calcium sensor proteins to produce specific cellular functions. Plants contain 4 major classes of calcium sensing proteins, respectively: calmodulin (CaM), Calcineurin B-like proteins (CBL), Calcineurin Dependent Protein Kinase (CDPK) and Ca²⁺Or protein kinase CaM dependentEnzyme (CCaMK). The presence of multiple different calcium sensor proteins in a plant can decode specific calcium signals to trigger a specific set of cellular responses. Wherein, the CBL generates conformational change after being combined with calcium ions, interacts with downstream CBL-interacting protein kinases (CIPK) and regulates the kinase activity of the CBL-interacting protein kinases, and plays a role in signal transmission. Because a certain number of CBL and CIPK exist in the plant, different expression patterns and subcellular localization of the CBL and the CIPK are different, and different interaction of the CBL and the CIPK has preference, a plurality of CBL-CIPK combinations can be formed, and calcium signals generated after the plant is subjected to specific environmental stimuli or development signals can be transmitted by the specific CBL-CIPK combinations to carry out corresponding physiological responses.

CBL is a calcium sensing protein which is peculiar to plants and can sense calcium signals generated under various environmental stimuli. The CBL protein requires specific interaction with NAF/fish motifs in the C-terminal regulatory domain of CBL-interacting protein kinases (CIPKs) to modulate CIPK kinase activity and to exert a signaling effect. To date, CBLs and CIPKs have only been found in the plant kingdom (Pandey et al, 2004), bioinformatics analysis has identified 10 CBL family genes and 26 CIPK family genes in arabidopsis (kolukisaloglu et al, 2004), 10 CBL family genes and 33 CIPK family genes in rice (Kanwar et al, 2014), and 9 CBL family genes and 31 CIPK family genes in wheat (Sun et al, 2015). Because a certain number of CBL and CIPK exist in the plant, different expression patterns and subcellular localization of the CBL and the CIPK are different, and different interaction of the CBL and the CIPK has preference, a plurality of CBL-CIPK combinations can be formed, and calcium signals generated after the plant is subjected to specific environmental stimuli or development signals can be transmitted by the specific CBL-CIPK combinations to carry out corresponding physiological responses.

Early studies indicate that the CBL-CIPK signal pathway plays an important role in regulating high salt stress response, pH regulation, low potassium stress, nitrogen and phosphorus absorption, flooding stress, ABA signals, growth and development signal transduction and the like (Luan, 2009; Pandey et al, 2014). CBL-CIPK has less functional studies in biotic stress compared to abiotic stress.

Therefore, cloning the susceptible gene in the wheat and verifying the function of the susceptible gene in the interaction process of the wheat and the stripe rust has important significance, and can provide new gene resources and anti-source materials for cultivating the stripe rust resistant wheat variety.

Disclosure of Invention

An object of the present invention is to provide use of any one of the following substances a1) -a 3).

The invention provides application of any one of substances a1) -a3) in regulating and controlling plant disease resistance;

a1) protein TaCIPK14, derived from wheat variety water source 11(Triticum aestivum);

a2) a nucleic acid molecule encoding the protein tacpk 14;

a3) a recombinant vector, expression cassette, recombinant microorganism or recombinant transgenic cell line comprising a nucleic acid molecule encoding the protein tacpk 14;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

b2) a protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) subjecting the amino acid sequence of the protein according to b1) to a substitution of one or several amino acid residues and-

Or c1) -derived proteins which are deleted and/or added and have the same function.

In the above application, the nucleic acid molecule encoding the protein TaCIPK14 is a DNA molecule of any one of the following c1) -c 3):

c1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

c2) a DNA molecule which hybridizes with the DNA sequence defined in c1) under strict conditions and codes for a protein with the same function;

c3) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined under c1) and encoding a protein having the same function.

In order to facilitate purification of TaCIPK14 in (b1), the amino terminus or the carboxyl terminus of a protein consisting of the amino acid sequence shown in sequence No. 2 in the sequence listing is labeled as shown in Table 1.

Table 1 shows the sequence of the tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

TaCIPK14 of (b2) above may be synthesized artificially, or may be obtained by synthesizing a gene encoding the same and then performing biological expression. The coding gene of TaCIPK14 of (b2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 of the sequence table, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding tacpk 14 of the invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence of tacpk 14 isolated according to the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode tacpk 14 and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

The above expression cassette containing a nucleic acid molecule encoding tacpk 14 (tacpk 14 gene expression cassette) refers to a DNA capable of expressing tacpk 14 in a host cell, and the DNA may include not only a promoter that initiates transcription of tacpk 14 but also a terminator that terminates transcription of tacpk 14. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator and the nopaline and octopine synthase terminators.

The recombinant vector containing the TaCIPK14 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

The vector may be a plasmid, cosmid, phage or viral vector.

The microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

None of the above transgenic plant cell lines comprises propagation material.

The disease resistance is stripe rust resistance.

It is another object of the present invention to provide the use of a substance that reduces the activity or content of the protein TaCIPK 14.

The invention provides the application of substances for reducing the activity or content of the protein TaCIPK14 in d1 or d 2;

or, the invention also provides the use of a substance inhibiting the expression of a nucleic acid molecule encoding the protein TaCIPK14 in d1 or d 2as follows;

d1, improving plant disease resistance;

d2, cultivating disease-resistant plants;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) protein composed of amino acid sequence shown in sequence 2 in sequence table

b2) A protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) subjecting the amino acid sequence of the protein according to b1) to a substitution of one or several amino acid residues and-

Or c1) -derived proteins which are deleted and/or added and have the same function.

In the above application, the nucleic acid molecule encoding the protein TaCIPK14 is a DNA molecule of any one of the following c1) -c 3):

c1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

c2) a DNA molecule which hybridizes with the DNA sequence defined in c1) under strict conditions and codes for a protein with the same function;

c3) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined under c1) and encoding a protein having the same function.

In the above application, the substance is 1) or 2) or 3):

1) a silencing sequence;

the silencing sequence is 94 th-387 th nucleotides of the sequence 1 or 971 th-1254 th nucleotides of the sequence 1;

2) a recombinant vector comprising the silencing sequence;

the recombinant vector is a viral vector containing the silencing sequence;

3) a system comprising said recombinant vector.

In the above application, the disease resistance is stripe rust resistance.

In the above application, the stripe rust resistance is embodied as any one of the following (1) to (5): (1) under the condition of stripe rust infection, the stripe rust severity of the wheat plant with TaCIPK14 protein expression inhibition is weaker than that of a control plant, and the generation of summer sporophyte on the wheat leaf is obviously reduced; (2) under the condition of rust stripe fungus infection, the number of rust stripe fungus haustorium at the wheat plant infection point with the inhibited TaCIPK14 protein expression is obviously less than that of a control plant; (3) under the condition of rust infection, the number of the mother cells of the rust sucker of the wheat plant infected point with the inhibition of TaCIPK14 protein expression is obviously less than that of a control plant; (4) under the condition of rust stripe fungus infection, the length of the rust stripe fungus hypha of the wheat plant infection point with the inhibited TaCIPK14 protein expression is obviously smaller than that of a control plant; (5) under the condition of rust stripe, the colony area of the rust stripe of the wheat plant infection point with the inhibited expression of the TaCIPK14 protein is obviously smaller than that of a control plant. The results show that the susceptibility of the wheat plants with the expression of the TaCIPK14 protein inhibited is reduced, and the TaCIPK14 gene has the stripe rust susceptibility function.

In the above application, the plant is a monocotyledon. The monocotyledon can be wheat; the wheat may specifically be a water source 11.

The 3 rd object of the present invention is to provide a method for breeding a disease-resistant transgenic plant.

The method provided by the invention is 1) or 2):

1) the method comprises the following steps: inhibiting or reducing the content and/or activity of protein TaCIPK14 in a target plant to obtain a transgenic plant;

2) the method comprises the following steps: inhibiting or reducing the expression of a nucleic acid molecule encoding a protein TaCIPK14 in a target plant to obtain a transgenic plant;

the disease resistance of the transgenic plant is higher than that of the target plant;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) protein composed of amino acid sequence shown in sequence 2 in sequence table

b2) A protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) subjecting the amino acid sequence of the protein according to b1) to a substitution of one or several amino acid residues and-

Or c1) -derived proteins which are deleted and/or added and have the same function.

The above-mentioned disease resistance higher than that of the target plant is embodied in any of the following 1) to 3):

1) under the condition of rust stripe infection, the number of infection suckers of the transgenic plants is less than that of target plants;

2) under the condition of rust stripe infection, the colony area of the transgenic plant is smaller than that of the target plant;

3) under the condition of rust stripe infection, the length of hypha of the transgenic plant is smaller than that of the target plant.

In the above, the plant is a dicotyledonous plant or a monocotyledonous plant.

It is a further object of the present invention to provide a substance.

The substance provided by the invention is 1) or 2) or 3):

1) a silencing sequence;

the silencing sequence is 94 th-387 th nucleotides of the sequence 1 or 971 th-1254 th nucleotides of the sequence 1;

2) a recombinant vector comprising the silencing sequence;

the recombinant vector is a viral vector containing the silencing sequence; the recombinant vector is the following BSMV viral vector gamma containing the DNA molecule shown in the 94 th-387 th site from the 5 'end of the sequence 1 or the BSMV viral vector gamma containing the DNA molecule shown in the 971 th-1254 th site from the 5' end of the sequence 1;

3) the system containing the recombinant vector is A) or B) as follows: A) BSMV viral vector alpha, BSMV viral vector beta and BSMV viral vector gamma containing DNA molecules shown in 94-387 bit from the 5' end of a sequence 1; B) BSMV viral vector alpha, BSMV viral vector beta and BSMV viral vector gamma containing a DNA molecule shown in the 971-1254 th site of the 5' end of the sequence 1.

The above substances have the following functions: reduce the activity or content of the protein TaCIPK14, or inhibit the expression of a nucleic acid molecule encoding the protein TaCIPK 14.

The pathogenic bacteria of the rust disease are puccinia striiformis physiorace CYR31 in the embodiment of the invention.

Experiments prove that TaCIPK14 obtained by VIGS is used for instantly silencing a plant to have lower susceptibility to stripe rust than a control plant, and the TaCIPK14 protein has stripe rust susceptibility function. The protein and the gene provided by the invention provide a basis for controlling stripe rust for people, and play an important role in cultivating the plants with enhanced stripe rust disease resistance.

Drawings

FIG. 1 shows the expression profile of TaCIPK14 in a combination of wheat and stripe rust interaction.

FIG. 2 shows the results of the localization of TaCIPK14 in wheat protoplasts.

FIG. 3 is a schematic structural diagram of a recombinant vector of the TaCIPK14 gene and barley mosaic virus (BSMV).

FIG. 4 is a phenotypic analysis of the inoculation of Puccinia striiformis after silencing of TaCIPK14 by the VIGS method.

FIG. 5 is a histological microscopic observation analysis of the effect of transient silencing of TaCIPK14 on the growth and development of Puccinia striiformis.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

In the following examples,% is by mass unless otherwise specified.

The reagent formulations used in the following examples are as follows:

table 2 shows the formula of the cellulose hydrolysate

Table 3 shows PEG4000 solutions (five days in storage in one preparation, but preferably in use, 100. mu.l of PEG4000 solution per sample, the total amount of the solution preparation being adjusted according to the amount of the sample to be tested)

Table 4 shows W5 solution

Table 5 shows MMG solution

Table 6 is the WI solution

Cellulase R10(YaKult Honsha) Cellulase (Yakult, C6270-1g)

Mecerozyme R10(YaKult Honsha) pectinase (Rongxing Bio, RX-L0042-100mg)

mannitol (Beijing Mengyi Mei Shang center, M0122-500g)

KOH (Beijing xi Yang Hui Zhi science and technology Co., Ltd., XYHZ-2017-

KCl (Beijing Baoruijie science and technology Co., Ltd., 7447-40-7)

MES (Beijing Bylendi Biotechnology Co., Ltd., DE-E169-100g)

CaCl2 (Beijing Bylendi biotechnology, Ltd., 031-

NaCl (Beijing Bylendi biotechnology Co., Ltd., 7647-14-5)

MgCl2 (Beijing Bylendi Biotechnology Co., Ltd., DE-0288-

Glucose (Beijing Byleddi Biotech Co., Ltd., 049-

PEG4000 (Beijing Byleddi Biotech Co., Ltd., BR-0084)

BSA bovine serum albumin (Beijing Zeping science, Inc., 0219989980.)

beta-Mercaptoethanol (0482-100 ML Biotech, Inc., Redbou Biotech, Beijing).

The 16318hGFP (Green fluorescent protein) vector in the following examples is described in the literature "molecular characterization and functional characterization of the millet WRKY36 transcription factor [ J ]. Chinese agricultural science 2015,48(5): 851-.

BSMV viral vectors (including α, β and γ plasmids) in the following examples are disclosed in the literature "Hein I, Barciszewska-Pacak M, Hrubikova K, et al. Virus-induced gene sizing-based functional characterization of genes associated with a pore and tissue restriction in barrel [ J ]. Plant Physiology,2005.138,2155-2164 ]. Publicly available from the applicant.

The P.cereus physiognomonas tritici strain CYR23 in the examples described below is disclosed in "Liu P, Guo J, Zhang R, et al. TaCIPK10 organisms with and phosphorylates TaNH2 to activate woat damage responses to stripe run [ J ]. Plant biotechnology journel, 2019,17(5): 956-. Publicly available from the applicant.

The physiological race of puccinia striiformis CYR31 in the following examples was disclosed in the study of "royal phoenix, wulian, xueshuan, jin jun, jia qin, yuanwen, yangjiaxiu, study of nos. 30 and 31 in new race of puccinia striiformis [ J ] plant protection article, 1996(01):39-44. Publicly available from the applicant.

The wheat variety water source 11 in the following examples is disclosed in the documents "Cao Zhang Jun, Jingjin, Wangmen, et al, domestic important anti-source variety water source 11, Water Source 92 and Hybrid46 anti-stripe rust Gene relation analysis [ J ]. Western North plant Proc., 2003,23(1): 64-68". Publicly available from the applicant.

The 1 XFES Buffer in the following examples contains 100mM glycine (glycine), 60mM K2HPO4(pH9.2), 1% (mass%) sodium pyrophosphate (sodium pyrophosphate), 1% (mass%) bentonite (bentonite) and 1% (mass%) diatomaceous earth (celite), with the balance being water.

Example 1 acquisition of TaCIPK14 protein and Gene encoding the same

First, mRNA isolation and TaCIPK14 amplification

Taking 11 normal-growing wheat seedlings growing for 7 days, quickly freezing the seedlings by using liquid nitrogen, and storing the seedlings at minus 80 ℃ for later use. Total RNA from wheat leaves was extracted by Trizol method (Tianggen) and first strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized by SMART method and the PCR product was detected by 1.0% agarose gel electrophoresis. The amplification primers are as follows:

TaCIPK14-F:5’-ATGGCAAACAGAGGGAAGATTCT-3’

TaCIPK14-R:5’-CTACTCTAGCTGCTGCTGGTGGT-3’。

a1335 bp PCR product was obtained.

After sequencing, the PCR product has the nucleotide shown as a sequence 1 in a sequence table, the gene of the nucleotide is named as a TaCIPK14 gene, the amino acid sequence of the protein coded by the gene is shown as a sequence 2 in the sequence table, and the protein is named as a TaCIPK14 protein.

Secondly, RT-PCR detection of TaCIPK14 induced expression condition by rust

1. Preparation of test materials

The rust fungus is inoculated in the first leaf and first heart stage of wheat, and the inoculation method is described in the literature, "Kangsheng, Li Shuangqi. Loffian 10 discovery of new pathogenic bacteria at normal temperature [ J ]. proceedings of university of agriculture and forestry in northwest (Nature science edition), 1984(04):18-28. The 11 leaves of the wheat water source are respectively inoculated with a non-affinity combination microspecies CYR23 and an affinity combination microspecies CYR31, and sterile water is inoculated as a control.

Sampling is carried out at 0h, 6h, 12h, 24h, 48h, 72h and 120h after inoculation respectively, and the comparison sampling time point is consistent with the treatment. When sampling, fresh leaves are cut, wrapped with tin-platinum paper, put into liquid nitrogen for quick freezing, and then stored at-80 ℃ for later use. Total RNA from wheat leaves was extracted by Trizol method (Tianggen) and first strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized by SMART method.

2. RT-PCR detection of TaCIPK14 expression level

Specific quantitative PCR primers are designed according to the sequences (GenBank accession number: U76744) of the TaCIPK14 and the TaEF-1 alpha of the elongation factor gene of wheat.

The RT-PCR primer sequence is as follows:

Qcipk14-F：5’-AAGCACCAGGGCATCTATCCA-3’

Qcipk14-R：5’-TCAAAGGCATTTAAGTTTGTCACC-3’。

QTaEF-F：5’-TGGTGTCATCAAGCCTGGTATGGT-3’

QTaEF-R：5’-ACTCATGGTGCATCTCAACGGACT-3’

RT-PCR was performed using the above cDNA as a template and RT-PCR primers.

Before the quantitative PCR primer is used, the specificity and the amplification efficiency (more than or equal to 90%) of an amplification product of the quantitative PCR primer need to be detected, and TaEF-1 alpha is used as an internal reference gene in Real-time PCR analysis. Real-time quantitative PCR amplification was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and a Bio-Rad CFX Manager quantitative PCR instrument (Bio-Rad, Hercules, California) using cDNAs of each processing sample point as a template, respectively, according to the instructions. At least 3 replicates were made per reaction, and the Ct values for each replicate, as well as their mean and standard deviation, were generated by a quantitative PCR instrument by manually adjusting the baseline. Each reaction is repeated for 3 times, Ct values are averaged, and experimental data are analyzed by adopting a Delta Delta Delta Ct method to determine the relative expression quantity of the genes.

The qRT-PCR result is shown in figure 1, TaCIPK14 has expression patterns of 0h, 6h, 12h, 24h, 48h, 72h and 120h after respectively inoculating a non-compatible race CYR23 and a compatible race CYR31 of the stripe rust to a wheat water source 11, wherein TaCIPK14 shows a down-regulation expression trend in the early stage (6-24h) and the later stage (72-120h) of stripe rust infection in a non-compatible and compatible combination, wherein the expression level in 6h after inoculation in the non-compatible combination is the lowest and is 0.027 times of a control, and the expression level in 24h after inoculation in the compatible combination is the lowest and is 0.0017 times of the control. Where ". x" indicates significance at a level of p <0.05 compared to 0h, respectively.

The above results indicate that TaCIPK14 is induced to express by Puccinia striiformis.

Subcellular localization analysis of TaCIPK14

1. Vector construction

And (3) performing PCR amplification by using the PCR product TaCIPK14 gene amplified from the previous step as a template and using a primer with a BamHI enzyme cutting site, wherein the amplified product is subjected to BamHI enzyme cutting and then is connected with a 16318hGFP (green fluorescent protein) vector subjected to the same enzyme cutting to obtain a recombinant vector GFP-TaCIPK14, and expressing the fusion protein.

The amplification primers used for construction of the tacpk 14 subcellular localization vector are: (underlined indicates the cleavage site)

TaCIPK14-GFP-F:5’-TATCTCTAGAGGATCCATGGCAAACAGAGGGAAGATTCT-3’

TaCIPK14-GFP-R:5’-TGCTCACCATGGATCCCTCTAGCTGCTGCTGGTGGT-3’。

2. Protoplast preparation

1) Wheat protoplast preparing and transforming method

First, a wheat variety water source 11 for sowing and planting in a soil culture chamber is provided.

And under the condition of good growth, taking leaves before blooming to prepare the protoplast.

And thirdly, cutting the well-growing leaves in the middle part, and cutting the leaves into leaves with the width of 0.5-1mm by a blade.

The cut leaf strips were put into the previously prepared cellulose enzymatic hydrolysate shown in table 2 (about 10 to 20 leaves were required per 5 to 10ml of the enzymatic hydrolysate). The leaves were completely immersed in the enzymatic hydrolysate with tweezers.

And fifthly, vacuumizing for 30 minutes in the dark (wrapped by the tin foil paper) by using a vacuum pump. (in this case, PEG4000 solution shown in Table 3, 200ul and 1000ul tips were removed to make the suction and the beating gentle in the operation)

Sixthly, performing enzymolysis for at least 3h under the dark condition without shaking at room temperature (the wheat is shaken at 50rpm and 28 ℃). When the enzymolysis liquid turns green, the culture dish is slightly shaken to promote the release of the protoplast. (at this point a pre-cooled amount of W5 solution)

The protoplasts in solution were examined under a microscope, wheat mesophyll protoplasts approximately 30-50um in size.

And the enzyme solution containing protoplasts was diluted with an equal amount of W5 solution shown in Table 4 before removing undissolved leaves by filtration.

The self-skin is prepared by wetting 35-75um nylon membrane or 60-100 mesh sieve with W5 solution, and filtering the enzymolysis solution containing protoplast with the above solution.

A30 ml round-bottom centrifuge tube is used for 1-2min with 100g, and is centrifuged at 4 ℃ to precipitate protoplast, and the supernatant is removed as much as possible. The protoplasts were then gently resuspended in 10ml of W5 solution pre-cooled on ice.

The sample was allowed to stand on ice for 30 minutes until protoplast formation.

The following operations were carried out at room temperature 23 ℃

Centrifuging for 8-10min with 100g water to precipitate protoplast. The W5 solution was removed as much as possible without touching the protoplast pellet. The protoplasts were then resuspended in the appropriate amount of MMG solution (1m, Table 5) to a final concentration of 2X10⁵One per ml.

10ul or 20ul DNA (10-20. mu.g of about 5-10kb recombinant vector 16318hGFP-TaCIPK14) was added to the 2ml EP tube for selection.

To this was added 100ul protoplasts (2X 10)⁴One), gently mix.

Add 110ul of PEG solution and mix thoroughly by gently tapping the centrifuge tube (approximately 6-10 samples can be converted each time).

The transformation mixture was then subjected to induction for 20-30min (depending on the experimental conditions, higher expression levels may require longer transformation times).

⒄ the conversion reaction was terminated by diluting the conversion mixture with 400-440ul of W5 solution at room temperature and gently shaking the tube upside down to mix well.

Centrifugation at 100g for 2min at a medium temperature and then removal of the supernatant. Then 1ml of W5 solution was added to the suspension and washed once, and the supernatant was centrifuged at 100g for 2 min.

⒆ protoplasts were gently resuspended in a multi-well tissue culture dish with 1ml of WI solution (Table 6).

⒇ protoplasts were induced at room temperature (20-25 ℃) for more than 18 hours.

The 16318hGFP vector was used as a control.

GFP tag expression was then observed under a confocal laser microscope.

3. Wheat protoplast microscopic examination:

protoplasts after dark culture for 18h were pelleted, and then GFP (green fluorescent protein) fluorescence was observed in a Laser scanning confocal microscope (Bio-rad micro surgery, LSMC) and subjected to scanning photography. The operating parameters of the LSCM are: ex 488nm, Em 525 ± 15nm, Power 10%, Zoom7, Frame512 × 512 by medium speed scan. The software is TIME-COARSE and PHOTOSHOP 5.0.

The results are shown in FIG. 2, which is a control of protoplasts transferred into empty vector 16318 hGFP; the following is a map of the localization of TaCIPK14 in protoplasts transfected with a recombinant vector GFP-TaCIPK14(GFP-TaCIPK14), and it is known that TaCIPK14 is distributed in cell membranes, cytoplasm and nuclei.

Example 2 application of inhibiting TaCIPK14 gene expression in regulation and control of stripe rust resistance of plants

Firstly, obtaining TaCIPK14 gene silencing plant

1. Construction of TaCIPK14 gene BSMV-VIGS vector system

(1) Obtaining silencing sequences

1) Obtaining of silencing sequence TaCIPK14-S1

PCR amplification was carried out using the 1335bp TaCIPK14 fragment (SEQ ID NO: 1) amplified in example 1as a template and using primer pairs TaCIPK14-S1F and TaCIPK14-S1R to obtain a PCR amplification product (corresponding to nucleotide sequences 94 to 387 from the 5' end of the sequence 1) of 294bp in size, which was designated as a silencing sequence TaCIPK 14-S1.

The nucleotide sequences of the above primer pair TaCIPK14-S1F and TaCIPK14-S1R are as follows (the underlined sequences are restriction enzyme digestion recognition sites for PacI enzyme and NotI enzyme):

TaCIPK14-S1F:5’-TTAATTAACTAGAGTCGAACCGAAGCGTC-3’；

TaCIPK14-S1R:5’-GCGGCCGCTTGGCTGTGGCAGTAATCCAC-3’。

2) obtaining of silencing sequence TaCIPK14-S2

A1335 bp TaCIPK14 fragment amplified in example 1 was used as a template, and TaCIPK14-S2F and TaCIPK14-S2R were subjected to PCR amplification by using primers to obtain a PCR amplification product of 284bp (a sequence of nucleotides 971-1254 from the 5' end of the sequence 1), which was designated as a silent sequence TaCIPK 14-S2.

The nucleotide sequences of the primer pair TaCIPK14-S2F and TaCIPK14-S2R are as follows (the sequences shown by underlining are restriction enzyme cutting recognition sites of PacI enzyme and NotI enzyme):

TaCIPK14-S2F:5’-TTAATTAACGGGGTTTGACCTGTCCG-3’；

TaCIPK14-S2R:5’-GCGGCCGCCTCCCGGTACTCCAGCGGAT-3’。

(2) construction of silencing vectors

1) Construction of gamma-TaCIPK 14-S1 silencing vector

And (2) respectively carrying out enzyme digestion on the silencing sequence TaCIPK14-S1 and the BSMV-VIGS virus vector gamma obtained in the step (1) by using PacI and NotI, and then connecting the enzyme-digested silencing sequence TaCIPK14-S1 with the enzyme-digested BSMV-VIGS virus vector gamma vector framework to obtain a recombinant vector gamma-TaCIPK 14-S1.

The recombinant vector gamma-TaCIPK 14-S1 is DNA molecules between PacI and NotI enzyme cutting sites of the BSMV-VIGS viral vector gamma which are replaced by the silencing sequence TaCIPK14-S1 obtained in the step (1), other sequences of the BSMV-VIGS viral vector gamma are kept unchanged, and the recombinant vector gamma-TaCIPK 14-S1 is obtained, wherein TaCIPK14-S1 is reversely inserted into the BSMV-VIGS viral vector gamma.

Carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaCIPK 14-S1 by using a primer pair gamma-F and gamma-R, wherein positive cloning is a vector obtained by inserting TaCIPK14-S1 (a sequence of 94-387 nucleotides from the 5' end of a sequence 1 in a sequence table) between PacI and NotI enzyme cutting sites of a gamma chain of a BSMV-VIGS virus vector in the opposite direction of gene expression and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged.

The gamma-F and gamma-R primer sequences are as follows:

γ-F:5’-AAAGTGAGGTTAACGCAATACG-3’；

γ-R:5’-TCAGGCATCGTTTTCAAGTT-3’。

2) construction of gamma-TaCIPK 14-S2 silencing vector

And (2) respectively carrying out enzyme digestion on the silencing sequence TaCIPK14-S2 and the BSMV-VIGS virus vector gamma obtained in the step (1) by using PacI and NotI, and then connecting the enzyme-digested silencing sequence TaCIPK14-S2 with the enzyme-digested BSMV-VIGS virus vector gamma vector framework to obtain a recombinant vector gamma-TaCIPK 14-S2.

The recombinant vector gamma-TaCIPK 14-S2 is DNA molecules between PacI and NotI enzyme cutting sites of the BSMV-VIGS viral vector gamma which are replaced by the silencing sequence TaCIPK14-S2 obtained in the step (1), other sequences of the BSMV-VIGS viral vector gamma are kept unchanged, and the recombinant vector gamma-TaCIPK 14-S2 is obtained, wherein TaCIPK14-S2 is reversely inserted into the BSMV-VIGS viral vector gamma.

Carrying out PCR amplification and sequencing identification on the recombinant vector gamma-TaCIPK 14-S2 by using a primer pair gamma-F and gamma-R, wherein the positive clone is the vector obtained by inserting TaCIPK14-S2 (the sequence of nucleotide 971-1254 from the 5' end of the sequence 1 in the sequence table) between PacI and NotI enzyme cutting sites of the gamma chain of the BSMV-VIGS virus vector in the opposite direction of gene expression and keeping other sequences of the BSMV-VIGS virus vector gamma unchanged.

(3) BSMV-VIGS vector system

The BSMV-VIGS viral vectors alpha, beta and gamma vectors together constitute the viral vector system BSMV gamma.

The BSMV-VIGS viral vectors alpha and beta and the recombinant vector gamma-TaCIPK 14-S1 jointly form a viral silencing vector system BSMV capable of silencing TaCIPK14 genes, namely TaCIPK 14-S1.

The BSMV-VIGS system vectors alpha and beta and the recombinant vector gamma-TaCIPK 14-S2 jointly form a virus silencing vector system BSMV capable of silencing TaCIPK14 genes, namely TaCIPK 14-S2.

The structure of the constructed barley mosaic virus-induced gene silencing (BSMV-VIGS) vector system is shown in FIG. 3, and is the structure of alpha, beta and gamma chains in the vector system respectively; the silent fragments TaCIPK14-S1 and TaCIPK14-S2 of the TaCIPK14 gene are reversely inserted into restriction enzyme NotI and PacI enzyme cutting sites of a gamma chain respectively, and two BSMV-VIGS vector systems BSMV, TaCIPK14-S1as and BSMV, TaCIPK14-S2as of the TaCIPK14 gene are constructed respectively.

2. BSMV in vitro transcription

(1) Linearization of vectors

Respectively carrying out enzyme digestion on the alpha vector and the gamma vector of the BSMV virus vector by MluI, carrying out enzyme digestion on the gamma-TaCIPK 14-S1 and the gamma-TaCIPK 14-S2 by BssHII, carrying out enzyme digestion on the beta chain of the BSMV virus vector by SpeI, and respectively obtaining linearized plasmids.

(2) And (2) carrying out in vitro transcription by using the linearized plasmid obtained in the step (1) as a template to respectively obtain in vitro transcribed BSMV viral vectors of alpha, beta, gamma-TaCIPK 14-S1 and gamma-TaCIPK 14-S2.

The in vitro transcription reaction was performed according to the instructions of RiboMAXtMLage Scale RNA production System-T7 (product of Promega corporation, cat. No.: P1300).

The transcription reaction system and conditions are respectively as follows: the total reaction volume is 20.0 μ L, including 6.5 μ L of linearized plasmid, 4.0 μ L of 5 × Transcription Buffer, 1.5 μ L of Cap (product of Promega corporation, Cat: P1718), rNTP PreMix6.0 μ L, 2.0 μ L of Enzyme Mix, reaction at 37 ℃ for 2h, taking 1 μ L to determine the concentration, requiring the concentration of in vitro Transcription product to be not less than 0.125 μ g/μ L to ensure successful inoculation of wheat leaf virus, and storing the rest Transcription products at-70 ℃ for later use.

3. Inoculation of BSMV

Preparing a BSMV gamma recombinant virus vector solution, a BSMV TaCIPK14-S1 recombinant virus vector solution and a BSMV TaCIPK14-S2 recombinant virus vector solution.

The BSMV-TaCIPK 14-S1 recombinant virus vector solution is a solution obtained by diluting in vitro transcription products of BSMV-VIGS vectors alpha, beta and gamma-TaCIPK 14-S1 to 0.125 mu g/mu L by DEPC water, sucking 10 mu L of each transcription product, mixing the diluted products, and adding 170 mu L of 1 XFES Buffer. Wherein, the concentrations of alpha, beta and gamma-TaCIPK 14-S1 in vitro transcription products in the BSMV TaCIPK14-S1 recombinant virus vector solution are all 6.25 ng/mu L.

The BSMV-TaCIPK 14-S2 recombinant virus vector solution is a solution obtained by diluting in vitro transcription products of BSMV-VIGS vectors alpha, beta and gamma-TaCIPK 14-S2 to 0.125 mu g/mu L by DEPC water, sucking 10 mu L of each transcription product, mixing the diluted products, and adding 170 mu L of 1 XFES Buffer. Wherein, the concentrations of alpha, beta and gamma-TaCIPK 14-S2 in vitro transcription products in the BSMV TaCIPK14-S2 recombinant virus vector solution are all 6.25 ng/mu L.

The above-mentioned BSMV-gamma recombinant virus vector solution is a solution obtained by diluting the in vitro transcription products of BSMV-VIGS vectors alpha, beta and gamma to 0.125. mu.g/. mu.L with DEPC water, mixing 10. mu.L of each of them, and adding 170. mu.L of 1 XFES Buffer. Wherein, the concentrations of alpha, beta and gamma in-vitro transcription products in the BSMV-gamma recombinant virus vector solution are all 6.25 ng/mu L.

Sowing wheat water source 11 in nutrient soil, growing to two-leaf stage, respectively taking 10 μ L BSMV: gamma recombinant virus vector solution, BSMV: TaCIPK14-S1 recombinant virus vector solution, BSMV: TaCIPK14-S2 recombinant virus vector solution and 1 xFES Buffer, coating and inoculating on the second flat leaf of wheat, and using DEPC ddH after 10min₂Spraying O, and adjusting the temperature to 25 ℃ for moisture preservation for 24 h. Then, the strain is transformed to normal condition culture at 25 ℃ to obtain transformed BSMV gamma plants, transformed BSMV TaCIPK14-S1as plants, transformed BSMV TaCIPK14-S2as plants and simulated inoculated plants respectively.

The plants of the transformed BSMV TaCIPK14-S1as and the transformed BSMV TaCIPK14-S2as are wheat plants which silence TaCIPK14 genes, the plants of the transformed BSMV Gamma are negative control plants, and the simulated inoculated plants coated with 1 XFES Buffer are blank control plants.

4. qRT-PCR verification of wheat for silencing TaCIPK14 gene

After culturing the transformed BSMV plant TaCIPK14-S1as and the transformed BSMV plant TaCIPK14-S2as, the transformed BSMV plant Gamma and the simulated inoculation plant (MOCK) obtained in the step 3 for 10 days under normal conditions, the puccinia striiformis physiological race CYR31 is inoculated, and the inoculation method is described in the literature, "Kangsheng, Lishuangqi, Loff forest 10 normal temperature pathogenic new bacterial system discovery [ J ]. Notice of northwest university of agriculture and forestry science and technology (Nature science edition), 1984(04):18-28. And (3) respectively shearing the inoculated strain leaves at 24h, 48h and 120h after inoculation, and using the inoculated strain leaves for extracting RNA and synthesizing cDNA through reverse transcription. RT-PCR was performed using the synthesized cDNA as a template and the method of example 1 to detect the relative expression level of TaCIPK 14.

The results of measuring the relative expression level of tacpk 14 gene are shown in fig. 4A, in which MOCK: simulating inoculated blank control plants; gamma in BSMV: inoculating a negative control plant of BSMV gamma; TaCIPK14-1as and TaCIPK14-2as represent plants transformed with TaCIPK14 gene expression down-regulated by silent segments S1 and S2 to BSMV TaCIPK14-S1as and TaCIPK14-S2as, respectively; as can be seen from FIG. 4A, after 0h, 48h, 72h and 120h of transformation of BSMV: TaCIPK14-S1as and transformation of BSMV: TaCIPK14-S2as plants after the rust streak fungus is inoculated, the expression level of the TaCIPK14 gene in the plants is 36% -66% of that of control plants which synchronously transform BSMV: gamma, which indicates that the expression level of TaCIPK14 is successfully inhibited in silent plants, two silent sequences selected in the experiment, namely TaCIPK14-S1 and TaCIPK14-S2, are effective, and both silent segments S1 and S2 can significantly reduce the expression of the TaCIPK 14-gene (the expression of p is represented by 'x' to be less than 0.05).

Secondly, analyzing the stripe rust resistance of TaCIPK14 gene-silenced plants

Effectively silenced transgenic BSMV: TaCIPK14-S1as plants, transgenic BSMV: TaCIPK14-S2as plants, negative control plants (BSMV: gamma) and blank control plants (MOCK) were inoculated with puccinia striiformis physiological race CYR31 after the fourth leaf development. Sampling is carried out 48h and 120h after inoculation, and WGA staining is carried out for observing the development condition of the rust stripe after the rust stripe is infected. The onset of the disease was observed at 14d after inoculation.

The results are shown in FIG. 4B, MOCK: simulating inoculated blank control plants; gamma in BSMV: inoculating a negative control plant of BSMV gamma; TaCIPK14-S1as as BSMV and TaCIPK14-S2as as BSMV represent plants transformed with TaCIPK14-S1as and plants transformed with TaCIPK14-S2as respectively; under the condition of rust infection, visible prunella was observed on all treated leaves, and the severity of rust of the tacpk 14 gene-silenced plants was weaker than that of the control plants.

The statistical analysis of the number of leaf sporocysts is shown in FIG. 4c, TaCIPK14-S1as and TaCIPK14-S2as are plants transformed with BSMV: TaCIPK14-S1as and plants transformed with BSMV: TaCIPK14-S2as respectively, gamma is a negative control plant inoculated with BSMV: gamma, and TaCIPK14 gene-silenced plants TaCIPK14-S1as and TaCIPK14-S2as are inoculated per 0.5cm²The number of sporozoites was significantly reduced (FIG. 4C).

The wheat leaves with the expression of the TaCIPK14 down-regulated are shown to reduce the susceptibility to the puccinia striiformis physiological race CYR 31.

Histological observation shows that TaCIPK14-S1as and TaCIPK14-S2as are respectively a plant transformed with BSMV, TaCIPK14-S1as and a plant transformed with BSMV, TaCIPK14-S2as, gamma is a negative control plant inoculated with BSMV, as shown in figure 5A, and the number of haustorium cells infecting the rust stripe is counted by microscopic observation 48h after the rust stripe is inoculated; FIG. 5B, microscopic observation and statistics of the number of haustoria of the infected rust stripe at 48h after the rust stripe is inoculated; FIG. 5C, microscopic observation measured the length of hyphae infecting the rust streak fungus 120h after inoculation of the rust streak fungus; FIG. 5D, microscopic observation 120h after inoculation of Puccinia striiformis, measures the colony area of infecting Puccinia striiformis. 50 infection points were counted for each biological experiment and 3 biological replicates were performed. Error bars are shown in the figures as standard deviations, where "+" indicates significance at p <0.05 levels compared to 0h, respectively. From the figure, it can be seen that under the condition of rust, the number of infected suckers, the colony area and the hypha length of the TaCIPK14 silent plant are lower than those of the control plant, which shows that the reduction of the expression of TaCIPK14 reduces the susceptibility of wheat.

Therefore, the TaCIPK14 is an important stripe rust disease related important gene, and the inhibition of the expression of the gene can improve the stripe rust disease resistance of plants.

SEQUENCE LISTING

<110> northwest agriculture and forestry science and technology university

<120> application of plant infection-related protein TaCIPK14 in regulation and control of plant stripe rust resistance

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1335

<212> DNA

<213> Artificial sequence

<400> 1

atggcaaaca gagggaagat tctaatggag cggtacgagc tgggaagatt gttggggaaa 60

ggaacattcg gcaaggtaca ctatgcaagg agcctagagt cgaaccgaag cgtcgccata 120

aagatgctgg acaaggagaa ggtgctcaag gttgggctct cggagcaaat caggcgtgag 180

gtcacaacca tgcggttggt ggcacacaag aacattgttc agcttcatga ggtcatggcg 240

acacgaaaca aaatatactt tgtcatggag tatgtgaaag gcggtgagct ctttgacaag 300

gttgcaaaga gtggcaagct cacagagggt gctgcacata agtatttcca gcagctcatc 360

agtgcagtgg attactgcca cagccaaggc gtgtatcacc gggatctcaa gctggagaac 420

ctgctcctgg atgagaatga gaaccttaag gtctcggatt ttggattgag cgcactttca 480

gagtcaaaga ggcaagatgg cttgctgcac accacctgcg gaacacccgc atatgtagct 540

ccggaggtca tcagcaagac aggttatgat ggtgcgaaat cagatatctg gtcttgtggt 600

gttatccttt ttgttcttgt tgctggttat ctccctttcc atggttccaa cttgatggac 660

atgtaccgga agattgagca aggagatttc aggtgcccca gctggttctc acacaaactc 720

cagaagctct tgctcaagat cctggacccc aatccaagca ccagggcatc tatccagaag 780

ataaaagagt ctacctggtt ccggaaaggt ccaaggggca cccttgcagt gaaggagaga 840

actcccagtg agaatgtcat cacaaatgct cctcctacag ctggtgtgag gccaaggaag 900

aacactcatg aagatgtgaa gcccctaatg gtgacaaact taaatgcctt tgagatcatc 960

tccttctcca cggggtttga cctgtccggc ctattcatcc aagaggactg cagaaaggag 1020

acaaggttca cttcagacaa gcctgcttca accatcatct cgaagctgga atatgttgcg 1080

aaggcgctga atctcagggt aaggaagaag gacaatggtg tggtgaagat gcaagcaagg 1140

aaggagggaa ggaatggtgc tgttcagtta gacatggaga tcttcgagat cacaccttcc 1200

caccacctca ttgagatgaa acaaacaagt ggtgatccgc tggagtaccg ggagctattg 1260

gaggacatcc ggccagcgct gaaggacata gtctgggcct ggcacggaga tgaccaccag 1320

cagcagctag agtag 1335

<210> 2

<211> 444

<212> PRT

<213> Artificial sequence

<400> 2

Met Ala Asn Arg Gly Lys Ile Leu Met Glu Arg Tyr Glu Leu Gly Arg

1 5 10 15

Leu Leu Gly Lys Gly Thr Phe Gly Lys Val His Tyr Ala Arg Ser Leu

20 25 30

Glu Ser Asn Arg Ser Val Ala Ile Lys Met Leu Asp Lys Glu Lys Val

35 40 45

Leu Lys Val Gly Leu Ser Glu Gln Ile Arg Arg Glu Val Thr Thr Met

50 55 60

Arg Leu Val Ala His Lys Asn Ile Val Gln Leu His Glu Val Met Ala

65 70 75 80

Thr Arg Asn Lys Ile Tyr Phe Val Met Glu Tyr Val Lys Gly Gly Glu

85 90 95

Leu Phe Asp Lys Val Ala Lys Ser Gly Lys Leu Thr Glu Gly Ala Ala

100 105 110

His Lys Tyr Phe Gln Gln Leu Ile Ser Ala Val Asp Tyr Cys His Ser

115 120 125

Gln Gly Val Tyr His Arg Asp Leu Lys Leu Glu Asn Leu Leu Leu Asp

130 135 140

Glu Asn Glu Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Leu Ser

145 150 155 160

Glu Ser Lys Arg Gln Asp Gly Leu Leu His Thr Thr Cys Gly Thr Pro

165 170 175

Ala Tyr Val Ala Pro Glu Val Ile Ser Lys Thr Gly Tyr Asp Gly Ala

180 185 190

Lys Ser Asp Ile Trp Ser Cys Gly Val Ile Leu Phe Val Leu Val Ala

195 200 205

Gly Tyr Leu Pro Phe His Gly Ser Asn Leu Met Asp Met Tyr Arg Lys

210 215 220

Ile Glu Gln Gly Asp Phe Arg Cys Pro Ser Trp Phe Ser His Lys Leu

225 230 235 240

Gln Lys Leu Leu Leu Lys Ile Leu Asp Pro Asn Pro Ser Thr Arg Ala

245 250 255

Ser Ile Gln Lys Ile Lys Glu Ser Thr Trp Phe Arg Lys Gly Pro Arg

260 265 270

Gly Thr Leu Ala Val Lys Glu Arg Thr Pro Ser Glu Asn Val Ile Thr

275 280 285

Asn Ala Pro Pro Thr Ala Gly Val Arg Pro Arg Lys Asn Thr His Glu

290 295 300

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

305 310 315 320

Ser Phe Ser Thr Gly Phe Asp Leu Ser Gly Leu Phe Ile Gln Glu Asp

325 330 335

Cys Arg Lys Glu Thr Arg Phe Thr Ser Asp Lys Pro Ala Ser Thr Ile

340 345 350

Ile Ser Lys Leu Glu Tyr Val Ala Lys Ala Leu Asn Leu Arg Val Arg

355 360 365

Lys Lys Asp Asn Gly Val Val Lys Met Gln Ala Arg Lys Glu Gly Arg

370 375 380

Asn Gly Ala Val Gln Leu Asp Met Glu Ile Phe Glu Ile Thr Pro Ser

385 390 395 400

His His Leu Ile Glu Met Lys Gln Thr Ser Gly Asp Pro Leu Glu Tyr

405 410 415

Arg Glu Leu Leu Glu Asp Ile Arg Pro Ala Leu Lys Asp Ile Val Trp

420 425 430

Ala Trp His Gly Asp Asp His Gln Gln Gln Leu Glu

435 440

Claims (10)

1. The application of any one of the following substances a1) -a3) in regulating and controlling plant disease resistance;

a1) protein tacpk 14;

a2) a nucleic acid molecule encoding the protein tacpk 14;

a3) a recombinant vector, expression cassette or recombinant bacterium comprising a nucleic acid molecule encoding protein TaCIPK 14;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

b2) a protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) c1) derived protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein of b1) and having the same function.

2. Use according to claim 1, characterized in that:

the nucleic acid molecule encoding the protein TaCIPK14 is a DNA molecule of any one of the following c1) -c 3):

c1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

c2) a DNA molecule which hybridizes with the DNA sequence defined in c1) under strict conditions and codes for a protein with the same function;

c3) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined under c1) and encoding a protein having the same function.

3. Use according to claim 1 or 2, characterized in that: the disease resistance is stripe rust resistance.

4. The use of a substance which reduces the activity or content of the protein TaCIPK14 in d1 or d 2as follows;

or, the use of a substance inhibiting the expression of a nucleic acid molecule encoding the protein TaCIPK14 in d1 or d 2;

d1, improving plant disease resistance;

d2, cultivating disease-resistant plants;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) protein composed of amino acid sequence shown in sequence 2 in sequence table

b2) A protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) c1) derived protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein of b1) and having the same function.

5. Use according to claim 4, characterized in that:

the nucleic acid molecule encoding the protein TaCIPK14 is a DNA molecule of any one of the following c1) -c 3):

c1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

c2) a DNA molecule which hybridizes with the DNA sequence defined in c1) under strict conditions and codes for a protein with the same function;

c3) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined under c1) and encoding a protein having the same function.

6. Use according to claim 4 or 5, characterized in that:

the substances are 1) or 2) or 3) as follows:

1) a silencing sequence;

the silencing sequence is 94 th-387 th nucleotides of the sequence 1 or 971 th-1254 th nucleotides of the sequence 1;

2) a recombinant vector comprising the silencing sequence;

the recombinant vector is a viral vector containing the silencing sequence;

3) a system comprising said recombinant vector.

7. Use according to any one of claims 4 to 6, characterized in that:

the disease resistance is stripe rust resistance.

8. A method for breeding disease-resistant transgenic plants comprises the following steps 1) or 2):

1) the method comprises the following steps: inhibiting or reducing the content and/or activity of protein TaCIPK14 in a target plant to obtain a transgenic plant;

2) the method comprises the following steps: inhibiting or reducing the expression of a nucleic acid molecule encoding a protein TaCIPK14 in a target plant to obtain a transgenic plant;

the disease resistance of the transgenic plant is higher than that of the target plant;

the protein TaCIPK14 is any one of the following (b1) - (b 3):

b1) protein composed of amino acid sequence shown in sequence 2 in sequence table

b2) A protein formed by adding a tag sequence to the end of the amino acid sequence of the protein shown in b 1);

b3) c1) derived protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein of b1) and having the same function.

9. The method of claim 8, wherein:

the disease resistance is stripe rust resistance.

10. A substance which is 1) or 2) or 3) below:

1) a silencing sequence;

the silencing sequence is 94 th-387 th nucleotides of the sequence 1 or 971 th-1254 th nucleotides of the sequence 1;

2) a recombinant vector comprising the silencing sequence;

the recombinant vector is a viral vector containing the silencing sequence;

3) a system comprising the recombinant vector;

the substance has the following functions: reduce the activity or content of the protein TaCIPK14, or inhibit the expression of a nucleic acid molecule encoding the protein TaCIPK 14.

Images