CN115850418A

CN115850418A - Application of OsNPF7.3 protein in regulation and control of rice alkali resistance

Info

Publication number: CN115850418A
Application number: CN202211293154.5A
Authority: CN
Inventors: 张帆; 陆鲸冰; 徐建龙; 梅松; 赵秀琴; 王文生
Original assignee: Sanya National Academy Of Southern Propagation Chinese Academy Of Agricultural Sciences; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Sanya National Academy Of Southern Propagation Chinese Academy Of Agricultural Sciences; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2022-10-21
Filing date: 2022-10-21
Publication date: 2023-03-28

Abstract

The invention discloses application of OsNPF7.3 protein in regulation and control of rice alkali resistance, and belongs to the field of genetic engineering breeding. The technical problem to be solved by the invention is how to obtain the alkali-resistant rice. The invention provides a method for improving the stress resistance of plants, which comprises the steps of reducing or down-regulating the expression of an OsNPF7.3 protein coding gene in a receptor plant or reducing or down-regulating the activity or content of the OsNPF7.3 protein to obtain a target plant with higher stress resistance than the receptor plant. The invention also provides application of the OsNPF7.3 protein or a substance for regulating the expression of the coding gene of the OsNPF7.3 protein or a substance for regulating the activity or content of the OsNPF7.3 protein in regulating and controlling plant alkali resistance. The invention discloses the function of the OsNPF7.3 protein in the regulation of the alkali resistance of rice for the first time, and provides a foundation for the breeding of alkali-resistant rice.

Description

Application of OsNPF7.3 protein in regulation and control of rice alkali resistance

Technical Field

The invention belongs to the technical field of genetic engineering breeding, and particularly relates to application of OsNPF7.3 protein in regulation and control of rice alkali resistance.

Background

Rice is an important food crop in China, but rice production frequently suffers from various adversity stresses, wherein soil salinization is one of the most serious abiotic stresses which limit the growth and development of the rice and finally cause the yield reduction of the rice. Is limited by the influence of factors such as lack of saline-alkali tolerant germplasm resources, lag of resource identification work, low efficiency of conventional breeding technology, insufficient investment of saline-alkali tolerant breeding and the like, and has fewer new saline-alkali tolerant rice varieties with high quality and high yield, thereby being far incapable of meeting the requirements of current rice production. Therefore, further improving the saline-alkali tolerance level of the rice variety and further enlarging the planting area are important measures for increasing the total yield and ensuring the national food safety. The saline-alkali resistance of rice is a complex character controlled by a plurality of genes and is easily influenced by environmental factors. Under alkaline earth conditions, salt damage and alkali damage coexist, and the traditional saline-alkali tolerant breeding based on phenotype has low efficiency and slow progress, and can not meet the production requirement at the present stage. At present, the relatively consistent view is that a plurality of key genes are required to be polymerized for cultivating saline-alkali tolerant rice varieties. With the rapid development of biotechnology, molecular marker-assisted selection and gene editing technologies have gradually become common molecular breeding technologies for improving the saline-alkali tolerance breeding efficiency of rice. However, most of the rice alkali-resistant QTL (Quantitative trait loci) reported at present have small effect and are not finely positioned and cloned, and excellent alkali-resistant alleles are deficient, which all restrict the progress of alkali-resistant molecular breeding of rice. Therefore, the method can provide important theoretical guidance for reasonably establishing a rice alkali-resistant molecular breeding strategy by exploring a new rice alkali-resistant germplasm, identifying an excellent main effect QTL/gene of alkali resistance and analyzing an alkali-resistant genetic mechanism.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to obtain the rice with alkali resistance.

In order to solve the above technical problems, the present invention provides a method for improving plant stress resistance, comprising reducing or down-regulating the expression of a gene encoding an osnpf7.3 protein in a recipient plant or reducing or down-regulating the activity or content of the osnpf7.3 protein to obtain a target plant with higher stress resistance than the recipient plant,

the OsNPF7.3 protein can be A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is SEQ ID No. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding amino acid residues in an amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1) and is related to plant stress resistance;

a3 A) and a tag is linked to the N-terminus and/or C-terminus of A1) or A2).

In the present invention, SEQ ID No.1 consists of 593 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The protein-tag refers to a polypeptide or protein which is expressed by fusion with a target protein by using a DNA in vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and/or a SUMO protein tag and the like.

The stress resistance may be resistance of the plant to abiotic stress. Examples include low temperature, high temperature, drought, salt, alkali, flooding, excess light, ultraviolet radiation, mineral nutrient deficiency, oxygen deficiency, high winds, injuries, and air, soil or water contamination such as heavy metal, pesticide, ozone, and sulfur dioxide contamination.

Further, in the above method, the OsNPF7.3 protein may be derived from rice.

Further, in the above method, the stress resistance may be alkali resistance.

Further, in the above method, the recipient plant is rice, and the reducing or down-regulating the expression of the gene encoding the osnpf7.3 protein or the reducing or down-regulating the activity or content of the protein in the recipient plant is performed by subjecting a DNA molecule having a nucleotide sequence of SEQ ID No.3 in the recipient plant to at least one of the following mutations:

m1) deleting 13 nucleotides in total from 2070-2082 sites of a sequence 3 in a sequence table of a rice genome;

m2) deleting 2072 th nucleotide C in a sequence 3 in a sequence table of a rice genome;

m3), and inserting a nucleotide A between 2070 and 2071 of a sequence 3 in a sequence table of a rice genome.

M1) mutation causes the 123 rd to 135 th nucleotides of the coding gene of the OsNPF7.3 protein, namely, the sequence 2 in the sequence table to be deleted, thereby generating frame shift mutation and causing translation to be terminated early, and further knocking out the OsNPF7.3 protein.

M2) mutation causes deletion of the 125 th nucleotide of the coding gene of the OsNPF7.3 protein, namely, the sequence 2, thereby generating frame shift mutation and causing premature termination of translation, and then knocking out the OsNPF7.3 protein.

M3) mutation to cause the insertion of nucleotide A between 123 th and 124 th positions of the coding gene of the OsNPF7.3 protein, namely a coding sequence, in a sequence 2 in a sequence table, thereby generating a frame shift mutation to cause premature termination of translation, and further knocking out the OsNPF7.3 protein.

In order to solve the above technical problems, the present invention provides in a second aspect the use of an osnpf7.3 protein, or a substance that regulates the expression of a gene encoding said osnpf7.3 protein, or a substance that regulates the activity or content of said osnpf7.3 protein, wherein said use may be any one of the following:

d1 Application of OsNPF7.3 protein or an expression substance for regulating and controlling the coding gene of the OsNPF7.3 protein or a substance for regulating and controlling the activity or the content of the OsNPF7.3 protein in regulating and controlling the stress resistance of plants;

d2 Application of OsNPF7.3 protein or an expression substance for regulating and controlling the coding gene of the OsNPF7.3 protein or a substance for regulating and controlling the activity or the content of the OsNPF7.3 protein in preparing a product for regulating and controlling the stress resistance of plants;

d3 Application of OsNPF7.3 protein or an expression substance for regulating and controlling the coding gene of the OsNPF7.3 protein or a substance for regulating and controlling the activity or the content of the OsNPF7.3 protein in preparing a product for cultivating stress-resistant plants;

d4 Application of OsNPF7.3 protein or substance for regulating and controlling expression of OsNPF7.3 protein coding gene or substance for regulating and controlling activity or content of OsNPF7.3 protein in plant breeding;

the OsNPF7.3 protein can be A1), A2) or A3) as follows:

a1 Protein with an amino acid sequence of SEQ ID No. 1;

a3 A) and a tag is attached to the N-terminus and/or C-terminus of A1) or A2).

Further, in the above application, the plant may be any one of:

p1) a monocotyledonous plant,

p2) a plant of the order Hedera,

p3) a plant of the family Poaceae,

p4) plants of the genus Oryza,

p5) Rice.

Further, in the above application, the protein may be derived from rice.

Further, in the above application, the purpose of the plant breeding may be to cultivate stress-resistant plants.

Further, in the above application, the plant breeding purpose may be to cultivate stress-resistant rice.

Further, in the above application, the plant breeding purpose may be to cultivate alkali-resistant rice.

Further, in the above-mentioned use, the substance regulating the osnpf7.3 protein or the substance regulating the expression of the gene encoding the osnpf7.3 protein or the substance regulating the activity or content of the osnpf7.3 protein is a biomaterial, and the biomaterial may be any one of the following B1) to B9):

b1 Nucleic acid molecules encoding an OsNPF7.3 protein;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A transgenic plant cell line containing the nucleic acid molecule according to B1), or a transgenic plant cell line containing the expression cassette according to B2), or a transgenic plant cell line containing the recombinant vector according to B3);

b6 A transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2), or a transgenic plant tissue containing the recombinant vector of B3);

b7 A transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2), or a transgenic plant organ containing the recombinant vector of B3);

b8 A nucleic acid molecule that inhibits or reduces the expression of a gene encoding an osnpf7.3 protein or a nucleic acid molecule that inhibits or reduces the activity of an osnpf7.3 protein;

b9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule according to B8).

In the above-mentioned related biological materials, the expression cassette described in B2) means DNA capable of expressing the above-mentioned protein in a host cell, and the DNA may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence.

The plant expression vector carrying the protein-encoding gene of the present invention can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant cells or tissues into plants.

In the related biological material, the recombinant microorganism in B4) can be yeast, bacteria, algae and fungi.

In the above related biological materials, the plant tissue of B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above related biological materials, the transgenic plant organ of B7) may be root, stem, leaf, flower, fruit and seed of the transgenic plant.

In the related biological materials, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may or may not include propagation material.

Further, in the above application, the nucleic acid molecule of B1) may be a DNA molecule represented by any one of the following B1) to B3):

b1 The coding sequence of the coding chain is a DNA molecule shown in SEQ ID No. 2;

b2 The nucleotide sequence of the coding chain is a DNA molecule shown in SEQ ID No. 3;

b3 A DNA molecule which has 80 percent or more than 80 percent of identity with the nucleotide sequence defined by b 1) or b 2) and codes the OsNPF7.3 protein.

In the present invention, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

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

Further, in the above-mentioned applications, the nucleic acid molecule of B8) may be a DNA molecule expressing a gRNA of the coding gene of the osnpf7.3 protein or a gRNA targeting the coding gene of the osnpf7.3 protein.

The target sequence of the gRNA is GTTCGCAGATTCGCAGCCCG (SEQ ID No.3, 2054-2073)

In order to solve the technical problems, the invention provides the OsNPF7.3 protein and the biological material in the application in a third aspect.

In order to solve the above technical problems, the present invention provides a method for producing an alkali-tolerant plant, comprising reducing or down-regulating the expression of the gene encoding the osnpf7.3 protein in a recipient plant or reducing or down-regulating the activity or content of the osnpf7.3 protein, thereby obtaining a plant of interest having higher alkali tolerance than the recipient plant.

Further, in the above method, the OsNPF7.3 protein may be derived from rice.

Further, in the above method, the stress resistance may be alkali resistance.

Further, in the above method, the recipient plant may be rice, and the reducing or down-regulating the expression of the gene encoding the osnpf7.3 protein or the reducing or down-regulating the activity or content of the protein in the recipient plant is carried out by mutating a DNA molecule having a nucleotide sequence of SEQ ID No.3 in the recipient plant by any one of the following mutations:

M1) mutation causes the coding gene of the OsNPF7.3 protein, namely 123 th to 135 th nucleotides in a sequence 2 in a sequence table to be deleted, thereby generating frame shift mutation and causing translation to be terminated early, and further knocking out the OsNPF7.3 protein.

M2) mutation results in the deletion of the 125 th nucleotide of the coding gene of the OsNPF7.3 protein, namely the sequence 2, the frame shift mutation is generated, the translation is terminated early, and the OsNPF7.3 protein is knocked out.

The beneficial technical effects obtained by the invention are as follows:

1) The invention discloses the correlation between rice OsNPF7.3 protein and rice alkali resistance for the first time, and provides the application of OsNPF7.3 protein in the regulation of rice alkali resistance;

2) Compared with wild rice, the alkali-resistant rice obtained by the method can obviously reduce physiological damage to the rice caused by alkali stress in a water culture experiment and obviously improve the alkali resistance of the rice.

Drawings

FIG. 1 is a phenotypic analysis of alkali resistance-related traits in rice seedling stage.

Figure 2 is the GWAS results for alkali-resistance related traits in the total population at seedling stage.

Figure 3 shows the GWAS results of alkali-resistance related traits in indica subpopulation at seedling stage.

FIG. 4 shows the GWAS results of the seedling-stage alkali-resistant related traits in japonica rice subpopulations.

FIG. 5 is a Wein chart showing the GWAS correlation results among the total population, indica rice and japonica rice.

FIG. 6 is an analysis of the candidate gene in locus 19.

FIG. 7 shows the detection of OsNPF7.3 target site mutation types.

FIG. 8 shows the seedling alkali tolerance identification of OsNPF7.3 mutant.

FIG. 9 shows the result of the alkali-resistant survival rate of OsNPF7.3 mutant in seedling stage.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples were processed using SPSS11.5 statistical software and the results were expressed as mean ± standard deviation using One-way ANOVA test with significant difference (P < 0.05), with very significant difference (P < 0.01), and with very significant difference (P < 0.001).

Example 1 identification of alkali resistance of Rice at seedling stage

1.1 Experimental methods

306 parts of materials from 3K rice germplasm resources are selected for identifying the alkali resistance of rice at the seedling stage. These materials include 137 parts of indica (Xian/indica), 166 parts of japonica (Geng/japonica), 1 part of Aus,1 part of Basmati and 1 part of Admix (Table 1).

TABLE 1.306 names and types of rice seedling-stage germplasm resources

TABLE 1 (continuation)

TABLE 1 (continuation)

TABLE 1 (continuation)

TABLE 1 (continuation)

And (3) placing 306 parts of material seeds in a 55 ℃ oven for drying for 3 days, breaking dormancy, soaking the seeds in 3% sodium hypochlorite solution for disinfection, and repeatedly washing the seeds with distilled water. After the seeds are disinfected, water culture is carried out for germination acceleration for 2 days, 36 uniform seeds are selected for each material after the seeds are exposed, and the test is repeated for 3 times, wherein 12 seeds are repeated. The seeds are sown in a 96-hole PCR plate with holes at the bottom, then the PCR plate with the exposed white seeds is placed in a plastic box with clear water (pH 5.5) and cultured in an artificial climate chamber under the conditions of 14h of illumination/10 h of darkness, the culture temperature is 28 ℃/26 ℃ and the humidity is 70 percent. Culturing for 7 days, changing clear water to Yoshida nutrient solution (pH 5.5) (Yoshida et al, 1976), and culturing under alkali stress until the seedling grows to two-leaf one-heart stage, wherein the alkali solution contains 0.15% Na ₂ CO ₃ The Yoshida nutrient solution has a pH of about 9.5, and the control contains no 0.15% Na ₂ CO ₃ The Yoshida nutrient solution has a pH of about 5.5. The solution was changed every 3 days.

On day 23 of alkali treatment, the alkali damage grade (Score of alkali damage severity, SAT) of each strain was evaluated with reference to the rice Standard evaluation System (Chaudhary, 1996), and was classified into 1-9 different grades in the order of the degree of alkali damage to seedlings from light to heavy (Table 2). Materials with the alkali damage level not more than 3 are divided into alkali-resistant materials, materials with the alkali damage level not less than 7 are divided into alkali-sensitive materials, and the rest materials are divided into alkali-resistant intermediate materials. Recording was started when the first dead Seedling appeared within the line, and the number of days to live of the line was calculated using a weighted average (SSD) until all seedlings died. The ratio of survival days to alkali damage grade, namely the growth index (VGI), is used for comprehensively evaluating the alkali resistance of the rice in the seedling stage.

TABLE 2 evaluation criteria for alkali damage rating

1.2, results of the experiment

The results of the evaluation of the alkali damage level, the survival days and the growth index of 306 materials are shown in Table 3 and FIG. 1. Table 3 shows statistics of expression data of alkali-resistant related traits at seedling stage, fig. 1 shows phenotype analysis of alkali-resistant related traits at seedling stage of rice, wherein (a) in fig. 1 shows alkali damage level, survival days and growth index of rice under alkali stress, (b) in fig. 1 shows distribution of alkali-resistant related traits of indica rice and japonica rice subgroups, (c) in fig. 1 shows a diagram of analysis of alkali-resistant related traits at seedling stage of total population, and represents significance of P <0.01 and P <0.001, respectively, and (d) in fig. 1 shows a diagram of analysis of alkali-resistant related traits at seedling stage of indica rice (upper) and japonica rice (lower) subgroups.

Overall, each trait exhibited wide variation (fig. 1 (a)). The average alkali damage rating of the total population was 5.65, the average survival days was 26.16 days, and the average growth index was 5.83. Judging the alkali-resistant material, the alkali-sensitive material and the alkali-resistant intermediate material according to the evaluation standards of Table 2 and the evaluation method of 1.1, and identifying 51 parts of the alkali-resistant material, 109 parts of the alkali-sensitive material and 146 parts of the alkali-resistant intermediate material. Wherein the alkali damage level of "NONA BOKRA" is grade 1 at 23 days of alkali stress, and the survival time under alkali stress of "KHAO KAI" is longest and is 41.47 days.

Phenotypic analysis of variance among indica-japonica populations shows that the alkali damage level of japonica rice is significantly lower than that of indica rice, the survival days and the growth index are significantly higher than those of indica rice, and japonica rice at the seedling stage is more alkali-resistant than indica rice (fig. 1 (b)). These 3 traits showed significant correlation between each other, both in the total population and in the indica subpopulation (fig. 1 (c), (d)), alkali grade showed very significant negative correlation with survival days and growth index, and very significant positive correlation with survival days and growth index.

TABLE 3 expression of alkali-resistant related traits in seedling stage

Note: SAT: alkali hazard level, SSD: days to live, VGI: growth index (SSD/SAT).

Example 2 Whole genome Association analysis

2.1 Experimental methods

Downloading a 4.8M high density SNP dataset from Rice SNP Seek database (http:// SNP-Seek. Irri. Org /) (ALEXANDROV et al, 2015) SNP genotype information was extracted for 306 materials using the Plink software (PURCELL et al, 2007), with SNP markers with deletion rate < 20% and minimum allele frequency > 5% retained. After genotype filtration, 2652345, 1946491 and 1130390 SNPs were finally obtained for genome-wide association analysis of the total population, indica and japonica rice populations. The association between SNPs and alkali resistance-related traits was detected using EMMAX (KANG et al, 2010) based on Mixed linear model analysis (MLM). Calculation of the affinity matrix in the filtration of linked SNPs first using Plink (parameter indep-pair 50 0.1) and then using EMMAX to calculate the affinity matrix (parameter EMMAX-kin-v-h-d 10). A make-GRM module based on GCTA software (YANG et al, 2011) generates a GRM matrix, performs principal component analysis, and extracts the first 3 principal components for use as covariates to control population structure. The number of effective independent SNPs, N, was calculated using GEC software (LI et al, 2012), and significance thresholds (1/N) of survivive P-value were calculated using Bonferroni correction method, setting P =2.51E-06, 2.85E-06, 8.73E-06 as the whole genome significance thresholds for the total population, indica and japonica populations, respectively. Linkage disequilibrium distance (WANG et al, 2018), classifying significant SNPs within 300kb as one locus, and defining the SNP with the smallest P value in one locus as lead SNP.

2.2 Experimental results

The experimental results are shown in table 4 and fig. 2 to 5, and table 4 shows the results of the alkali-resistant related traits GWAS at the rice seedling stage. Figure 2 is the GWAS results for alkali-resistance related traits in the total population at seedling stage. Figure 3 shows the GWAS results of alkali-resistance related traits in indica subpopulation at seedling stage. FIG. 4 shows the GWAS results of the seedling-stage alkali-resistant related traits in japonica rice subpopulations. Fig. 5 is a wien diagram showing GWAS correlation results among the total population, indica type rice and japonica rice, wherein (a) in fig. 5 is a wien diagram showing significant SNPs among the total population, indica type rice and japonica rice, and (b) in fig. 5 is a wien diagram showing genes corresponding to the significant SNPs among the total population, indica type rice and japonica rice.

Genome-wide association analysis of 3 traits, scale of Alkali Toxicity (SAT), days of survival (SSD), and growth index (VGI), of 306 material based on Mixed Linear Model (MLM) correlated 90, 514, and 4 significant SNPs in the total population (fig. 2), indica population (fig. 3), and japonica population (fig. 4), respectively, with 32 SNPs being commonly associated in the total population and indica subpopulations and no significant SNPs between indica subpopulations being commonly associated (fig. 5 (a)). Further analysis shows that there is no overlap between genes of significant SNPs detected in indica rice population and japonica rice population (fig. 5 (b)), and it is presumed that there may be different alkali-resistant genetic mechanisms between indica rice and japonica rice. In order to reduce the redundancy of the same locus caused by repeated positioning in different traits, adjacent SNPs within 300kb are defined as a locus, and as a result, 44 loci (loci 1-loci 44) are detected in 3 traits, and the loci with significant association are detected in the rest chromosomes except the 9 th chromosome (Table 4).

TABLE 4 Rice seedling stage alkali-resistant related traits GWAS results

/>

Table 4 (continuation)

Table 4 (continuation)

The genes of the significant SNPs detected by each population are annotated with functions, and reported genes related to alkali resistance are not found, but genes related to abiotic stress are detected in the total population and the indica rice subpopulation, namely OsRACK1A, osNPF7.3, osNPF7.4, osZIP6, osGSTU4, osBBX6, osMGD and OVP3. In addition, several new sites were found that were very significantly associated with alkali resistance during the seedling stage, such as: SNP rs1_28342694 (P = 2.34E-07) with a very significant association with the alkali injury level on chromosome 1 of rice; SNP rs11_17463475 (P = 7.65E-09) with a very significant association with days of survival on chromosome 11 of rice.

Among the 44 sites detected, there were some sites associated with multiple traits at the same time. For example, locus3 on chromosome 1 was significantly associated with the alkali injury level, survival days, and growth index simultaneously in the total population; loci locus 19 and locus 40, which affect both survival days and growth index, were detected on

chromosome

4 and 11, respectively, in the total population and indica subpopulation.

Example 3 analysis of candidate genes

3.1 Experimental methods

Taking a gene at least meeting one of the following conditions as a seedling-stage alkali-resistant candidate gene: (1) Functional annotation of genes associated with salt and alkali stress based on nippon reference genome IRGSP 1.0 (KAWAHARA et al, 2013) and funriceigenes database (YAO et al, 2018); (2) The gene corresponding to the SNP associated in 3 traits at the same time; (3) Genes corresponding to SNPs with a very significant association (P-value < 0.05/N) with each trait. If the SNP is located between two genes, the downstream gene is selected.

Local linkage disequilibrium analysis was calculated for each candidate gene using LDBlockshow (DONG et al, 2021). Haplotyping of non-synonymous mutant SNPs on the coding region of candidate genes was performed using Nipponbare as reference genome (ZHANG et al, 2021), and Duncan multiple range test was performed on the differences between different haplotypes (containing at least 10 materials) using the "agricolae" software package in R (DE MENDIBURU, 2017).

3.2 results of the experiment

According to the method described in 3.1, 7 candidate genes were predicted (Table 5). Through haplotype analysis of the 7 candidate genes, the significant difference of alkali-resistant related trait phenotypes among different haplotypes of 4 genes (LOC _ Os01g06740, LOC _ Os01g49310, LOC _ Os02g55910 and LOC _ Os04g 50950) is found, and the genes are screened as important seedling-stage alkali-resistant candidate genes.

The specific experimental results are shown in table 5 and fig. 6, and table 5 shows the information of 7 seedling-stage alkali-resistant candidate genes. FIG. 6 is an analysis of the candidate gene in locus 19. Wherein (a) in FIG. 6 is local Manhattan plot and LD analysis of VGI of locus 19 in the total population, red dots represent the position of lead SNP rs4_30165974 (candidate gene LOC _ Os04g 50950), FIG. 6 (b) is the gene structure plot of LOC _ Os04g50950 and the major haplotype in the CDS region, FIG. 6 (c) is the haplotype analysis result of LOC _ Os04g50950 in the total population, letters on the box plot represent multiple comparison results at a significance level of 0.05, FIG. 6 (d) is the subgroup distribution of LOC _ Os04g50950 haplotype.

TABLE 5.7 information of alkali-resistant candidate genes in seedling stage

LOC _ Os04g50950 was detected in both the total population and indica subpopulation, affecting both survival days and growth index, with an LD interval of about 34.99kb, containing 139 SNPs (FIG. 6 (a)). A total of 2 major haplotypes were found in the total population (fig. 6 (b)), with hap1 having a significantly higher number of days to live and growth index than hap2 (fig. 6 (c)). Most of hap1 is japonica rice material, and most of hap2 is indica rice material (fig. 6 (d)), which shows significant indica differentiation.

The amino acid sequence of LOC _ Os04g50950 is SEQ ID No.1. The coding sequence is SEQ ID No.2. The genome sequence is SEQ ID No.3, wherein the 230 th to 280 th sites of the SEQ ID No.3 are first exons, the 452 th to 514 th sites are second exons, the 2062 th to 2370 th sites are third exons, and the 2461 th to 3819 th exons are 4 th exons.

Example 4 obtaining of candidate knockout plants

4.1 Experimental methods

In order to verify the effect of the candidate gene OsNPF7.3 on the alkali resistance of rice in the seedling stage, the T0 plant of the mutant is obtained by knocking out OsNPF7.3 by using a CRISPR/Cas9 technology on japonica rice variety 'Zhonghua 11' (ZH 11) as target rice (only the OsNPF7.3 is knocked out, and other gene editing is not carried out). T is ₀ The plants were provided by Baige Gene science and technology, inc. (Jiangsu), and the preparation method was as follows:

4.1.1, osNPF7.3 gene editing and knocking-out target primer design and CRISPR/Cas9 vector construction

According to the gene editing principle, a specific segment with the length of 20bp in the OsNPF7.3 full-length cDNA sequence is screened as a target sequence for OsNPF7.3 gene editing and knockout through online Blast homology analysis, and the nucleotide sequence of the target sequence is (5 '-3'): GTTCGCAGATTCGCAGCCCG (SEQ ID No.3, positions 2054-2073, located in the second intron and the third exon of the OsNPF7.3 gene). And designing an annealing primer according to the target sequence to anneal and synthesize a DNA molecule containing the target sequence, and constructing to BGK032 to obtain a BGK032-SG recombinant vector, wherein the BGK032-SG recombinant vector expresses Cas9 protein and sgRNA targeting the target sequence. The nucleotide sequence of the BGK032-SG recombinant vector is SEQ ID No.4. Wherein, the No.4 No. 519-538 bits of SEQ ID are spacer region of sgRNA, namely target sequence of the sgRNA, and the No. 539-614 bits are framework region of the sgRNA. 2815-6915 shows a Cas9 coding sequence.

4.1.2 obtaining of OsNPF7.3-KO transgenic Rice

The recombinant BGK032-SG vector is transformed into Agrobacterium EHA105 by a freeze-thaw method. The method comprises the steps of transforming japonica rice variety medium flower 11 (generally represented by ZH 11) by an agrobacterium-mediated method, taking mature seeds of the medium flower 11, mechanically shelling, selecting plump, smooth and non-plaque seeds, disinfecting, and inoculating to an induction culture medium for induction culture. Selecting rice callus with good appearance and good growth capacity as a receptor material, transferring a BGK032-SG recombinant vector into the rice callus by adopting an agrobacterium-mediated method, transforming the rice callus by using a culture solution containing 100 mu M acetosyringone and agrobacterium with an OD value of 0.3-0.5, placing the callus soaked by a transformation solution (infection culture medium) on a co-culture medium for co-culture, and co-culturing for 50-55 h at 28 ℃ under a dark condition. The callus without obvious agrobacterium on the surface is selected and transferred to an N6 bacteriostatic culture medium containing 2.0mg/L of 2,4-D and 500mg/L of cefuroxime and cultured in dark for 3-4 days at 28 ℃. The calli were transferred to selection medium for 30d, subcultured every 10 d. Taking a fresh hygromycin-resistant callus, inoculating the callus into a pre-regeneration culture medium, culturing for 7 days at 28 ℃ in the dark, then placing the callus in a light culture room (12 h light/12 h dark) for continuous culture for 7 days, transferring the callus onto a regeneration culture medium (250 mL tissue culture bottle), and culturing continuously in the light until a regeneration plant grows out.

The medium formulation referred to in this example is as follows:

TABLE 6 culture media and formulations for genetic transformation

/>

4.1.3 sequencing of mutations in regenerated plants

T to be obtained ₀ Transplanting the seedlings to a field, taking leaves to extract DNA, and designing specific primers on a Primer3.0 website according to the target point information of OsNPF7.3, wherein the specific primers are named as OsNPF7.3-F/R. Sequencing analysis was performed on all sampled plants and the sequencing results were analyzed using DNAMAN and Chromas software. Screening out homozygous mutant plants, harvesting, and separating T ₁ Planting seed into plant line, randomly selecting 7 plants for each plant line, sampling, extracting whole genome DNA, amplifying and sequencing, selecting homozygous mutant plants, and selfing and reproducing to T ₂ 。

Primers (5 '-3') used for sequencing mutant plants

OsNPF7.3-F：AGGCCTTATTTCGTTGCAAACT；

OsNPF7.3-R：TGGCAATGCCGTTAAATGCT。

Wherein, the TPS method is adopted for extracting the DNA of the rice, and the steps are as follows:

(1) Placing a small amount of leaves in a 2mL centrifuge tube, adding 2 steel balls, covering, placing in liquid nitrogen for freezing for 30s, and pulverizing with a pulverizer.

(2) Adding 1000 μ L of TPS extractive solution, standing in 65 deg.C oven for 30min, and shaking vigorously every 10min.

(3) Standing for 10min, and centrifuging at 12000rpm for 10min.

(4) Taking 500 mu L of supernatant fluid to a new 1.5ml centrifuge tube, adding equal volume of precooled isopropanol, shaking slightly and shaking uniformly, standing at-20 ℃ for about 1h, and centrifuging at 12000rpm for 10min.

(5) Pouring out the supernatant, adding 75% ethanol, washing the precipitate, drying in a fume hood, and oven dryingAfter drying, 200. Mu.L of ddH was added ₂ O。

(6) The DNA quality was verified using the software Nano Drop and stored at4 ℃ until use.

4.2, results of the experiment

Will T ₀ After sequencing the PCR amplification product of the plant, performing sequence comparison by using DNAMAN software, and respectively comparing the OsNPF7.3 sequence of the wild type Zhonghua 11 (ZH 11) with the mutant sequence. And checking the sequencing result by using Chromas software, and carrying out statistical analysis on the mutation types at the positions of the gene targets.

Statistical analysis is performed on the mutation types generated at the target position of the OsNPF7.3 gene, and two homozygous mutation types are found, wherein one mutation type is single base insertion, and the other mutation type is 13bp deletion near the target position (figure 7). And (4) harvesting the seeds of the homozygous mutant single plant and continuing to breed.

For OsNPF7.3 mutant T ₁ The whole genome DNA is extracted from the plant, 7 homozygous mutant strains (shown in table 6) are screened out by using OsNPF7.3-F/R, the mutation types are divided into 3 types, and the mutation types are respectively 13bp deletion (3 strains) at a target point, one base C deletion (3 strains) at the target point and one base A insertion (1 strain) at the target point. Selfing and generation adding to obtain T ₂ And the mutant strains are respectively named as qat4-1, qat4-2 and qat4-3 and are used for verifying the alkali resistance phenotype at the subsequent seedling stage.

TABLE 6 mutation detection of T1 transgenic plants

The mutation types of the homozygous mutant lines qat4-1, qat4-2 and qat4-3 are specifically as follows:

compared with the wild type middle flower 11, the OsNPF7.3 in the mutant plant qat4-1 is changed as follows: the OsNPF7.3 gene in the two homologous chromosomes is mutated into Osnpf7.3-1, osnpf7.3-1 is the 2070-2082 nucleotide deletion of a sequence 3 in a sequence table, the coding sequence of Osnpf7.3-1 is the 123-135 nucleotide deletion of a sequence 2 in the sequence table, a frame shift mutation is generated, translation is terminated early, and the OsNPF7.3 protein is knocked out.

Compared with the wild type middle flower 11, the OsNPF7.3 in the mutant plant qat4-2 is changed as follows: the OsNPF7.3 gene in the two homologous chromosomes is mutated into Osnpf7.3-2, osnpf7.3-2 is the 2072 nucleotide deletion of a sequence 3 in a sequence table, the coding sequence of Osnpf7.3-1 is the 125 nucleotide deletion of the sequence 2 in the sequence table, a frame shift mutation is generated, translation is terminated early, and the OsNPF7.3 protein is knocked out.

Compared with the wild type middle flower 11, the OsNPF7.3 in the mutant plant qat4-3 is changed as follows: the OsNPF7.3 gene in the two homologous chromosomes is mutated into Osnpf7.3-3, the Osnpf7.3-3 is inserted between 2070 and 2071 of a sequence 3 in a sequence table, the coding sequence of Osnpf7.3-1 is inserted between 123 and 124 of a sequence 2 in the sequence table, a frame shift mutation is generated, translation is terminated early, and the OsNPF7.3 protein is knocked out.

Example 5 functional verification of alkali-resistant candidate genes

5.1 test methods

Wild type and T ₂ The seedling culture mode of the homozygous mutant plant is shown in example 1, after the seeds are exposed to white, 36 full and consistent seeds are selected from each material, the seeds are averagely divided into 2 parts and are respectively used for control and alkali stress treatment, and 3 times of biological repetition is set in the test. On the 17 th day of alkali stress, 6 seedlings of each material under alkali stress and normal conditions are randomly selected to measure the height and the length of the seedlings, the roots are simply washed by distilled water, residual nutrient solution on the surfaces of the seedlings is washed away, water is sucked by filter paper, the overground part and the roots of each material are respectively weighed to measure the fresh weight of the seedlings, then the overground part and the root samples of each material are respectively put into different envelope bags, the temperature is adjusted to 80 ℃ for drying for several days after the water is removed at 105 ℃ for 30min, and the dry weights of the overground part and the roots are weighed. Transferring the dried samples under alkali stress and contrast conditions into test tubes, adding acetic acid solution (20 mL of overground part and 10mL of root part) into the test tubes, placing the test tubes in a 90 ℃ constant-temperature oscillation water bath kettle for oscillation for 3h, taking and diluting supernate after the solution is cooled, and respectively measuring Na (sodium) of the overground part and the root part of each material by using a flame photometer ⁺ And (4) concentration. The calculation formula is as follows:

na in the sample ⁺ Mass fraction (mg/g) = C × V × N/(M × 1000) [ C: on-machine testSample element concentration (mg/L), V: volume of acetic acid in sample extract (mL), N: dilution factor, M: sample quality (g) formula (3-1).

The seedling stage culture mode of the wild type and T2 homozygous mutant plants is shown in example 1, after the seeds are exposed to white, 40 full and consistent seeds are selected from each mutant material and used for measuring the survival rate of the mutants after alkali stress treatment, and 3 biological repetitions are set in the test. After the seeds appeared white, 40 full and consistent seeds are selected from the wild type and used for measuring the survival rate of the wild type after alkali stress treatment, and 9 biological repetitions are set in the test. At day 36 of alkaline stress, the number of survivors was counted for all remaining material and survival was calculated.

5.2 and OsNPF7.3 seedling stage alkali resistance function identification experiment result

Experimental results As shown in FIGS. 8 and 9, in FIG. 8, (a) shows phenotypes before treatment (left) and under alkali stress of OsNPF7.3 mutant and wild-type ZH11 (right), and in FIG. 8, (b-i) shows shoot heights (b), root lengths (c), fresh weight of aerial parts (d), fresh weight of roots (e), dry weight of aerial parts (f), dry weight of roots (g), na of aerial parts (g) of control and alkali stress of 17d wild-type ZH11 and OsNPF7.3 mutant ⁺ Content (h), root Na ⁺ Content (i), scale 3cm. * And each represents P<0.05 and P<Significance of 0.01. Data represent mean ± sd of 3 replicates. FIG. 9 shows the survival rate of mutant and wild type at day 36 of alkaline stress. The length of the scale in FIG. 9 represents 5cm. Wild type data represent the mean ± sd of 9 replicates and mutant data represent the mean ± sd of 3 replicates.

The 3 mutants of osnpf7.3 showed stronger alkali resistance than the wild type (fig. 8 (a)). The alkali stress inhibited the growth of the aerial parts, resulting in a decrease in fresh and dry weight of the aerial parts, but there was no significant difference between the mutant and the wild type (fig. 8 (b), (d), (f)). For roots, the qat4-2 mutant root length under alkaline stress was significantly lower than the wild type, but there was no significant difference in root fresh and dry weights (c), (e), (g) in fig. 8). Measurement of Na in aerial parts and roots ⁺ Content, found that alkali stress causes Na in aerial parts and roots ⁺ And (4) increasing. But there was no significant difference between the wild type and the mutant ((h), (i) in fig. 8). OsNPF7.3-KO transgenic lineqat4-1, qat4-2 and qat4-3 are more alkali-resistant than wild type middle flower 11 at seedling stage. The results in FIG. 9 show that: after 36 days of alkali stress, the average survival rate of nine repeated 360 strains of 11 flowers in the wild type is 10.0 percent, the average survival rate of three repeated 120 strains of qat4-1 is 36.7 percent, the average survival rate of three repeated 120 strains of qat4-2 is 47.5 percent, the average survival rate of three repeated 120 strains of qat4-3 is 68.3 percent, and the survival rate of 3 OsNPF7.3-KO transgenic strains is obviously higher than that of the wild type (figure 9).

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

Claims

1. A method for improving the stress resistance of a plant comprises the steps of reducing or down-regulating the expression of an OsNPF7.3 protein coding gene in a receptor plant or reducing or down-regulating the activity or content of the OsNPF7.3 protein to obtain a target plant with the stress resistance higher than that of the receptor plant,

the OsNPF7.3 protein is A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is SEQ ID No. 1;

a3 A) and a tag is attached to the N-terminus and/or C-terminus of A1) or A2).

2. The method of claim 1, wherein: the OsNPF7.3 protein is derived from rice.

3. The method according to claim 1 or 2, characterized in that: the stress resistance is alkali resistance.

4. The method according to any one of claims 1-3, wherein: the receptor plant is rice, and the reduction or down-regulation of the expression of the OsNPF7.3 protein coding gene or the reduction or down-regulation of the activity or the content of the protein in the receptor plant is realized by performing at least one mutation on a DNA molecule with a nucleotide sequence of SEQ ID No.3 in the receptor plant:

the receptor plant is rice, and the reduction or down-regulation of the expression of the OsNPF7.3 protein coding gene or the reduction or down-regulation of the activity or the content of the protein in the receptor plant is realized by performing at least one mutation on a DNA molecule with a nucleotide sequence of SEQ ID No.3 in the receptor plant:

m2) and deleting 2072 th nucleotide C in a sequence 3 in a sequence table of a rice genome;

Use of the OsNPF7.3 protein or a substance regulating the expression of a gene encoding said OsNPF7.3 protein or a substance regulating the activity or content of said OsNPF7.3 protein, characterized in that: the application is any one of the following:

the OsNPF7.3 protein is A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is SEQ ID No. 1;

a3 A) and a tag is attached to the N-terminus and/or C-terminus of A1) or A2).

6. Use according to claim 5, characterized in that: the plant is any one of the following plants:

p1) monocotyledonous plants,

p2) plants of the order Hematoda,

p3) a plant of the family Poaceae,

p4) plants of the genus Oryza,

p5) Rice.

7. Use according to claim 5 or 6, characterized in that: the substance regulating the OsNPF7.3 protein or the substance regulating the expression of the gene encoding the OsNPF7.3 protein or the substance regulating the activity or content of the OsNPF7.3 protein is a biological material, and the biological material is any one of the following B1) to B9):

b1 Nucleic acid molecules encoding an OsNPF7.3 protein;

b6 A transgenic plant tissue containing the nucleic acid molecule according to B1), or a transgenic plant tissue containing the expression cassette according to B2), or a transgenic plant tissue containing the recombinant vector according to B3);

8. Use according to any one of claims 5 to 7, characterized in that:

b1 The nucleic acid molecule is a DNA molecule shown in any one of the following b 1) to b 3):

b3 A DNA molecule having 80% or more 80% identity to the nucleotide sequence defined in b 1) or b 2) and encoding the protein of claim 1;

b8 The nucleic acid molecule is a DNA molecule expressing a gRNA targeted to the gene encoding the protein of claim 1 or is a gRNA targeted to the gene encoding the protein of claim 1.

9. An osnpf7.3 protein for use according to claim 1 and a biological material for use according to any one of claims 5 to 8.

10. A method for producing an alkali-tolerant plant, comprising reducing or down-regulating expression of a gene encoding said osnpf7.3 protein or reducing or down-regulating activity or content of said osnpf7.3 protein in a recipient plant, resulting in a plant of interest having an alkali tolerance higher than that of said recipient plant.