CN118147176A - Application of maize gene ZmGPAT in salt tolerance and cereal Gu Tanju disease resistance - Google Patents

Abstract

The invention relates to the field of genetic engineering breeding, and in particular discloses application of a gene ZmGPAT of maize 'one-factor multiple-effect' in salt tolerance and cereal Gu Tanju disease resistance. The invention discovers that the corn mutant with ZmGPAT knocked out gene simultaneously shows the phenotype of salt resistance and cereal Gu Tanju resistance, and the plant with ZmGPAT gene over-expression simultaneously shows the phenotype of salt sensitivity and cereal Gu Tanju resistance, thereby providing the application of the corn ZmGPAT6 gene or the biological material containing the coding gene thereof in regulating and controlling the salt resistance and cereal Gu Tanju resistance of corn. The invention lays a theoretical foundation for researching the mechanism of salt tolerance and cereal Gu Tanju disease resistance of corn response, and simultaneously provides a new gene resource for cultivating a new variety of salt tolerance and cereal Gu Tanju disease resistance 'one-factor multiple-effect' corn.

Description

Application of maize gene ZmGPAT in salt tolerance and cereal Gu Tanju disease resistance

Technical Field

The invention belongs to the field of genetic engineering breeding, and particularly relates to application of ZmGPAT gene in salt tolerance and cereal Gu Tanju disease resistance.

Background

With the increasing rise of global environmental problems, crops are subjected to a wide variety of abiotic and biotic stresses during growth and development. Corn is one of three major food crops, and salt stress and infection of grass Gu Tanju pathogenic bacteria cause significant yield reduction in corn. Grain safety issues are impacted (Liang et al, 2023; guo et al, 2017; park et al, 2016); the pathogenic bacteria of corn stalk Gu Tanju disease is stalk Gu Tanju bacteria, which only infects corn, and has the advantages of rapid onset, easy transmission, large hazard area, great challenges for prevention and control of corn, and great losses to corn planting areas (Torres et al, 2014). By utilizing the modern molecular biology technical means, salt-tolerant and grass Gu Tanju-resistant mutant materials can be obtained through transgenic technology, the molecular mechanism of corn salt tolerance and grass Gu Tanju-resistant stress is further explored, and the cultivation of new corn salt-tolerant grass Gu Tanju-resistant varieties plays an important role in future corn molecular breeding.

In recent years, researches on the infection mechanism of salt and alkali tolerance of corn or corn stalk Gu Tanju diseases are frequent, but the excavation and the researches on key genes of salt tolerance and stalk Gu Tanju disease resistance are not reported. Therefore, the research of the gene ZmGPAT for resisting salt tolerance and grass Gu Tanju disease of corn is of great significance to the improvement of corn germplasm resources and agricultural production.

Disclosure of Invention

The invention aims to provide an application of a gene 'one-cause multiple effect' ZmGPAT in salt tolerance and cereal Gu Tanju disease resistance.

ZmGPAT6, one of the members of the glycerol-3-phosphate acyltransferase (GPAT) family, is a key rate-limiting enzyme involved in plant lipid biosynthesis and is responsible for catalyzing the first step of de novo synthesis of membrane lipids and storage lipids (Ohlrogge et al, 1995). GPAT has been found to be involved in a variety of plant stress responses, and AtGPAT and AtGPAT have phosphatase activity in addition to sn-2 acyltransferase activity in Arabidopsis thaliana, so that the conversion of the phosphate group of the intermediate sn-2 PA to sn-2 Monoacylglycerol (MAG) can be directly carried out to participate in the synthesis of cutin (Yang et al, 2010), affecting the normal development of leaf stomata and thus responding to a variety of biotic and abiotic stresses (Hsu et al, 2010). Through protein sequence alignment and key functional domain analysis, zmGPAT6 has a domain of sn-2 acylase and a domain of phosphatase at the same time. The research finds that ZmGPAT gene plays a negative regulation role in salt tolerance and cereal Gu Tanju disease resistance of corn.

The cDNA sequence of ZmGPAT gene is shown as SEQ ID NO.1, and the amino acid sequence of ZmGPAT gene coded protein is shown as SEQ ID NO. 2.

The invention carries out salt stress treatment on a corn mutant library created by a gene editing method in earlier work, and screens and obtains Zmgpat mutant materials with salt-tolerant phenotype by taking plant height, biomass and sodium content of plants as indexes.

The invention further carries out over-expression on ZmGPAT genes, carries out salt stress verification on ZmGPAT-OE over-expression materials, and discovers that transgenic plants over-expressing the genes have obvious salt-sensitive phenotype.

DAB and NBT staining and ROS scavenging enzyme activity measurement are carried out on Zmgpat mutant and ZmGPAT-OE over-expression materials under salt stress, and the results show that: compared with the wild type, the hydrogen peroxide and superoxide anion content of Zmgpat mutant plants is obviously reduced, and the ROS scavenging enzyme activity is obviously increased; compared with the wild type, the hydrogen peroxide and superoxide anion content of ZmGPAT-OE over-expression plants is obviously increased, and the ROS scavenging enzyme activity is obviously reduced. The above results indicate that: under salt stress, zmgpat mutant can timely remove the ROS content in the body, so that plants are prevented from being subjected to oxidative stress, and a salt-tolerant phenotype is shown; and ZmGPAT-OE over-expression material can not timely remove ROS content under salt stress, so that plants are subjected to oxidative stress, and a salt-sensitive phenotype is shown.

According to the invention, the Zmgpat mutant material and the ZmGPAT-OE overexpression material are subjected to cereal anthracnose pathogen infection analysis, and the Zmgpat mutant material is determined to be in a disease-resistant phenotype, and the ZmGPAT6-OE overexpression material is determined to be in a disease-resistant phenotype by counting the disease spot area and cereal Gu Tanju pathogen biomass serving as indexes. Further analysis of the Zmgpat mutant material and ZmGPAT-OE overexpression material for chitin-induced ROS bursts was performed as follows: compared with the wild type, the Zmgpat mutant has obviously increased ROS burst induced by the chitin, so that the infection of the grass Gu Tanju bacteria is inhibited to show an anti-disease phenotype; compared with the wild type, the ROS burst induced by the chitin in ZmGPAT-OE over-expression material is obviously reduced, so that the infection of the grass Gu Tanju bacteria is promoted to show an infectious phenotype.

The invention determines ZmGPAT genes which are possible 'one-cause multiple-effect' key genes for salt tolerance and cereal Gu Tanju disease resistance of corn.

The invention provides application of a corn ZmGPAT gene or a biological material containing the coding gene thereof in regulating and controlling salt tolerance and cereal Gu Tanju disease of corn, which comprises the following aspects:

(1) Improving plant salt tolerance and cereal anthracnose resistance germplasm resources;

(2) Selecting transgenic plants with salt tolerance and grass Gu Tanju disease resistance;

(3) The survival rate of the plants in a high-salt environment is improved;

(4) The survival rate of the plant cereal anthracnose in the environment is improved;

(5) The survival rate of the plants in the high-salt and cereal anthracnose environment is improved.

The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

The invention also provides a preparation method of the transgenic plant, and the method is used for respectively obtaining the knock-out mutant or the super-expression material of ZmGPAT by knocking out the gene or the super-expression gene, so that the plant which has salt tolerance and is resistant to the cereal Gu Tanju disease or salt sensitivity and is sensitive to the cereal Gu Tanju disease is respectively obtained.

The invention provides new gene resources for cultivating new varieties of salt-tolerant and grass Gu Tanju-disease-resistant plants, and lays a theoretical foundation for researching mechanisms of salt tolerance and disease stress resistance of corn response.

Drawings

FIG. 1Zmgpat6 mutant Material salt stress phenotype and physiological analysis

Phenotype analysis of Zmgpat mutants under 100mM NaCl salt stress; and B, performing salt stress on the aerial parts of the Zmgpat mutant materials for biomass analysis. And C, carrying out salt stress aerial part Na ⁺ content analysis on Zmgpat mutant materials. D: salt stress analysis of Zmgpat mutant materials on the lower Na ⁺ content. The data are obtained from three independent replicates.

FIG. 2ZmGPAT6 Gene expression analysis of overexpressed Material

Analysis of expression level of ZmGPAT-OE 1-ZmGPAT-OE 5 strain ZmGPAT 6. The data are obtained from three independent replicates.

FIG. 3ZmGPAT6 overexpressing material salt stress phenotype and physiological analysis

A ZmGPAT A6-OE phenotyping at 100mM NaCl salt stress; and B, carrying out salt stress on the aerial parts of the ZmGPAT-OE material for biomass analysis. Salt stress aerial parts of the C ZmGPAT-OE material were analysed for Na ⁺ content. And D, zmGPAT-OE material salt stress underground Na ⁺ content analysis. The data are obtained from three independent replicates.

FIG. 4Zmgpat6 mutant and DAB, NBT staining analysis under salt stress of ZmGPAT6 overexpressed material

Blade DAB staining phenotype analysis under salt stress of Zmgpat mutant and ZmGPAT-OE over-expression material; b, carrying out phenotype analysis on NBT staining results of leaves under salt stress of Zmgpat mutant and ZmGPAT-OE over-expressed material;

FIG. 5 determination of ROS enzyme activity under salt stress of Zmgpat6 mutant and ZmGPAT over-expressed material

Zmgpat6 mutant material salt stress POD, CAT, SOD enzyme activity detection analysis; zmGPAT6-OE over-expression material salt stress POD, CAT, SOD enzyme activity detection analysis;

FIG. 6 phenotype and physiological analysis of Zmgpat6 mutant and ZmGPAT over-expressed Material infection of Po Gu Tanju

Phenotype analysis of Zmgpat mutant materials four days after infection of Gu Tanju bacteria; and B, carrying out statistical analysis on the area of the lesions after the Zmgpat mutant material infects the Gu Tanju bacteria for four days. Biomass analysis four days after C Zmgpat mutant material infects Gu Tanju bacteria. D: phenotype analysis of ZmGPAT-OE four days after infection with Gu Tanju bacteria; statistical analysis of the lesion area four days after infection of the grass Gu Tanju with E ZmGPAT-OE material. Biomass analysis four days after F: zmGPAT-OE material infection with Gu Tanju bacteria. The data are obtained from three independent replicates.

FIG. 7 analysis of ROS burst induced by chitin in Zmgpat6 mutant and ZmGPAT over-expressed material

Zmgpat6 mutant material was analyzed for chitin-induced ROS bursts; b ZmGPAT A6-OE material was analyzed for chitin-induced ROS bursts.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 21), or the conditions recommended by the manufacturer's instructions. The main reagents in the following examples were: various restriction enzymes, taq DNA polymerase, T4 ligase, pyrobest Taq enzyme, KOD from NEB, toyobo and other biological companies; dNTPs are available from Genestar; the plasmid miniprep kit and the agarose gel recovery kit are purchased from Shanghai Jierui bioengineering company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), and the like, and companies such as Glucose, BSA, and LB Medium are available from Sigma, bio-Rad, and the like; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and the various other chemical reagents used in the examples were all imported or custom analytical pure reagents. The primers used in the examples were synthesized by Hexakuda and subjected to related sequencing.

Example one salt stress phenotype identification of maize Zmgpat6 mutant

Salt stress screening is carried out by using the corn mutant library material edited by the genes, and Zmgpat mutant material with potential salt tolerance is screened by taking the salinization degree of the first leaf of the plant and the height of the plant as observation indexes. Further, the salt-tolerant phenotype of Zmgpat mutant is further identified by taking plant height, biomass and sodium content as indexes.

Seeds of Wild Type (WT) and Zmgpat mutant were sown to vermiculite and nutrient soil (1000 g) 1:1, adding water (CK) and 100mM NaCl saline to Zmgpat mutant and WT respectively, and culturing in a greenhouse at 28 deg.C until soil is fully saturated with water. Maize plants were grown to three weeks, WT and Zmgpat mutant phenotypes were observed, and biomass (fresh weight of aerial parts) in g (g) under water and 100mM NaCl treatment was recorded. The aerial parts of WT and Zmgpat mutant corn materials were sampled and filled into dried cowhide bags, the sample names and sampling dates were marked, dried in an oven at 80℃to constant weight, and the dry weight of the samples was recorded.

Grinding the dry sample into powder by using a mortar, weighing 0.1 g into a 10 mL tube, adding 10 mL of 1M HCl, placing the mixture in a shaking table at 28 ℃ for overnight soaking, and filtering the overnight soaked sample into a new 10 mL tube by using filter paper to prepare mother liquor; samples were diluted to the appropriate range (sample measurement concentration at the middle of standard curve) with HCl 1M, and Na ⁺ content in mg g ^-1 (mg/g) was measured on an ion-body atomic emission spectrometer 4100MP-AE, depending on the sample.

The WT and Zmgpat mutant were analyzed for biomass, aboveground and underground sodium ion content, and other data under CK and 100mM NaCl treatment, and the results are shown in fig. 1: 100 Under the treatment of mM NaCl salt stress, the Zmgpat mutant exhibited a salt-resistant phenotype (FIG. 1A), with aerial biomass (FIG. 1B) significantly higher than wild type, and aerial (FIG. 1C) and subsurface (FIG. 1D) Na ⁺ levels significantly lower than wild type. All data were obtained as three independent replicates, plotted using GRAPHPAD PRISM software, and error analyzed using SPSS software.

EXAMPLE two ZmGPAT creation, screening and salt stress verification analysis of transgenic plants overexpressing the 6-OE

1. ZmGPAT6 construction of an overexpression vector: collecting samples of different tissue parts (young leaves, roots, stems, filaments and anthers) of the corn B73, extracting total RNA of the samples by using a Trizol method, obtaining cDNA templates of the different tissue parts by using 5 x All-in-One RT Master Mix (ABM, canada) reverse transcription, and placing the templates at the temperature of-20 ℃ for standby. Downloading ZmGPAT gene cDNA sequence in MaizeGDB, searching specific Primer sequence by using Primer 5.0 on-line Primer design website, using cDNA at different tissue positions of B73 as template of Primer 1 (SEQ ID NO. 3: CTCACCAACTTGTGACTACAC) and Primer 2 (SEQ ID NO.4: GCACCGCAGAGATACAATAAAG), specifically amplifying CDS sequence with KOD FX DNA Polymerase, detecting PCR amplified product by agarose gel electrophoresis, and sequencing the PCR amplified product to Hexahua corporation. The CDS sequence of ZmGPAT gene with correct sequence is inserted into pCAMBIA1300 vector by CE Mix recombinase after XbaI digestion, 5 mu L of digestion-connection system product is taken to transform E.coli competence, screening is carried out on LB plate containing 50 mu g/mu L kanamycin, monoclonal colony sequencing is selected, and the recombinant expression vector after correct sequencing is named ZmGPAT-OE.

2. ZmGPAT6 transformation of the overexpression vector: the ZmGPAT-OE vector constructed was transferred into Agrobacterium EHA105 by heat shock and PCR identified. The positive colonies obtained were isolated at 1: mixing agrobacterium with concentration of 1, respectively containing two knockdown vectors, adding glycerol, and preserving at-80deg.C.

The vectors constructed in the embodiment all carry out agrobacterium-mediated transformation of B73-329 immature embryos on a plant genetic transformation platform of Beijing university biological agriculture institute. Taking young embryo of freshly stripped corn B73-329 with the thickness of about 1.5mm as a receptor material, placing the stripped corn embryo into 2mL plastic centrifuge tubes containing 1.8mL suspension for not more than 1 hour, and placing about 100 young embryos into each centrifuge tube; the suspension was aspirated and the young embryos were rinsed 2 times with fresh suspension, the bottom of the tube remained a small amount of suspension that could have passed through the young embryos, then heat shock was applied for 2 minutes at 43℃followed by an additional ice bath for 1 minute, the residual wash solution at the bottom was aspirated with a pipette, and 1.0 mL of Agrobacterium infestation was added, gently shaken for 30 seconds, and then allowed to stand in the dark for 8 minutes. Pouring the young embryo and the infection liquid in the centrifuge tube into a co-culture medium, shaking uniformly, sucking out excessive infection liquid by using a pipetting gun, and co-culturing in darkness at 23 ℃ for 3 days with scutellum of all young embryos facing upwards. After the co-cultivation is finished, the young embryo is transferred to a recovery culture medium by sterile forceps, and is cultivated for 7 days at 28 ℃, and the young embryo growing on the young embryo needs to be removed at any time in the middle process. After the recovery culture, the young embryo is placed on 1.5 mg/L biamap screening medium for screening and culturing for 3 rounds, each round of screening and culturing for 2 weeks, and then 2 rounds of screening and culturing for 2 weeks on 2 mg/L biamap screening medium are carried out. The resistant calli were transferred to expansion medium and dark cultured for 2 weeks at 28 ℃. The propagated calli were then transferred to induction medium and incubated for 2 weeks at 28℃in the dark. Then transferring to a differentiation medium, culturing at 25 ℃ under 5000 lx under light for 2 weeks. After the cultivation is finished, single seedlings are separated from the differentiated seedling clusters and placed in a rooting medium, and the temperature is 25 ℃,5000 lx and the seedlings are subjected to illumination cultivation until rooting; transferring the young seedling into a small nutrition pot for growth, and transplanting the young seedling into a greenhouse after the young seedling survives growth.

Transferring the T0 generation transgenic seedling to nutrient soil for growing for about 7d, taking transgenic seedling leaves in a 2.0mL centrifuge tube, extracting leaf genome DNA by adopting a CTAB method, amplifying and screening ZmGPAT-OE transgenic strains by using a primer 3 (SEQ ID NO.5: ATGGTGAGCAAGGGCGA) and a primer 4 (SEQ ID NO.6: TTATCGGACGATGCCGTCG), and sequencing and identifying PCR products. The obtained transgenic positive plants are subjected to selfing or backcross pollination, and stable genetic materials named ZmGPAT-OE-1, zmGPAT-OE-2, zmGPAT-OE-3, zmGPAT-OE-4 and ZmGPAT-OE-5 are screened.

3. ZmGPAT6 qRT-PCR analysis of transgenic plants overexpressed by ZmGPAT

In this example, over-expressed strains ZmGPAT-OE-1, zmGPAT-OE-2, zmGPAT-OE-3, zmGPAT-OE-4 and ZmGPAT-OE-5 with higher expression levels were isolated, and the expression of ZmGPAT genes in the over-expressed strains ZmGPAT-OE-1, zmGPAT-OE-2, zmGPAT-OE-3, zmGPAT6-OE-4 and ZmGPAT-OE-5 was detected by real-time quantitative PCR. The specific method comprises the following steps: the total RNA of leaf blade of the above-mentioned over-expression strain was extracted, and cDNA was obtained by reverse transcription using 5×all-in-One RT Master Mix (ABM, canada). After 5-fold dilution of cDNA obtained by reverse transcription, quantitative reverse transcription polymerase chain reaction detection was performed on QuantStudio5 QuantStudio Real-TIME PCR SYSTEM (ABI, U.S.) using TB Green ™ Premix Ex Taq ™ (TaKaRa, japan), with OE amplification primer of F (SEQ ID NO.7: GCAGAGATGGTGAAGAAGGC), R (SEQ ID NO.8: ACACCATCGGCTTTGGGTA), UBI as a reference gene, and with amplification primer of: f (SEQ ID NO.9: CGACAACGTGAAGGCGAAGA) and R (SEQ ID NO.10: ACGCAGATACCCAGGTACAGC).

After the PCR reaction is completed, the relative expression quantity between the wild type (B73-329) and the over-expression strain (OE) is calculated according to the principle of 2 ^-Δ(ΔCt), and is plotted and analyzed, and three biological repetitions are carried out, wherein the three trends are similar. Simultaneously with the amplification of the identified genes, each sample was amplified simultaneously with the UBI gene as an internal reference. From the results, fig. 2, it can be seen that: the ZmGPAT expression level of ZmGPAT-OE-1 and ZmGPAT-OE-4 plants is obviously higher than that of other strains, so ZmGPAT-OE-1 and ZmGPAT-OE-4 are selected as ZmGPAT over-expression materials for experimental verification.

4. Salt stress identification of maize ZmGPAT-OE overexpression material

Salt stress screening is carried out by taking the ZmGPAT-OE over-expression materials ZmGPAT-OE-1 and ZmGPAT-OE-4 as research materials; data collection and analysis were performed for phenotyping, biomass assays, and Na ⁺ assays as in example one salt stress identification method, and the results are shown in fig. 3: zmGPAT6-OE-1 and ZmGPAT6-OE-4 materials exhibited salt-sensitive phenotypes (FIG. 3A), and ZmGPAT6-OE showed significantly reduced biomass (fresh weight of aerial parts) compared to B73-329 under 100mM NaCl treatment (FIG. 3B) and significantly increased Na ⁺ levels both in aerial and underground parts (FIG. 3C). All data were obtained as three independent replicates, plotted using GRAPHPAD PRISM software, and error analyzed using SPSS software.

Example III determination of DAB and NBT content of Zmgpat mutant Material and ZmGPAT-OE overexpression Material under salt stress

Maize WT, B73-329, zmgpat mutant material and ZmGPAT-OE overexpressing material were water cultured for one week, treated with 125mM NaCl salt for 48 h, and the untreated control group and salt stress treated leaves were immersed in DAB/NBT dye solution under vacuum for 1h and then stained overnight at room temperature in the absence of light. After the dyeing is completed, decolorization is carried out in boiling water ethanol (90%, V/V) and the phenotype is recorded by photographing. Wherein 0.1% dab was dissolved in 10mM MES (ph=5.8) buffer. 0.05% nbt was dissolved in 50 mM HEPES (ph=7.8) buffer. The results are shown in fig. 4: under salt stress, zmgpat mutant plants had significantly reduced hydrogen peroxide and superoxide anion content compared to WT, no oxidative stress was generated (fig. 4A), zmGPAT-OE plants had significantly increased content compared to B73-329, and oxidative stress was generated (fig. 4B). The results show that: under salt stress, zmgpat mutant plants showed reduced ROS content in vivo compared to WT, exhibiting a salt tolerant phenotype; whereas ZmGPAT-OE plants have increased ROS content in vivo compared to B73-329, and the plant growth is inhibited, exhibiting a salt-sensitive phenotype.

Example IV determination of ROS scavenging enzyme Activity under salt stress, zmgpat mutant Material and ZmGPAT6-OE overexpressing Material

After corn WT, B73-329, zmgpat mutant material and ZmGPAT-OE over-expression material are cultured in water for one week, 48 h is treated with 125mM NaCl salt, 0.2g fresh leaves or roots of the untreated control group and the salt stress treated material are weighed respectively, placed in a mortar, 2mL of phosphate buffer solution (containing 1% polyvinylpyrrolidone and having a molecular weight of about 30000,0.2 mmol/L disodium ethylenediamine tetraacetate and 10 mmol/L magnesium chloride) with a precooling concentration of 50 mmol/L and pH 7.0 is added, a small amount of quartz sand is added and ground into homogenate, transferred into a 2.0 mL centrifuge tube, after standing at 4 ℃ for 10min, 15000 rpm centrifuge 25 min is carefully sucked up to a new 2.0 mL, the supernatant is crude enzyme solution, and the corresponding reaction mixture is added to measure absorbance values at wavelengths nm, 560 nm and 240 nm, respectively, and POD, CAT and activity are analyzed. The results are shown in fig. 5: CAT, POD and SOD enzyme activities of Zmgpat6 mutant plants were significantly higher than that of WT under 125mM NaCl salt stress treatment (FIG. 5A); CAT, POD and SOD enzyme activities were significantly lower in ZmGPAT-OE plants than in B73-329 (FIG. 5B). The results show that: under salt stress, CAT, POD and SOD enzyme activities in Zmgpat mutant plants are increased, so that hydrogen peroxide and superoxide anions generated by salt stress can be timely removed, cells are prevented from being subjected to oxidative stress, the plants grow normally, and the phenotype of salt tolerance is obtained; however, the enzyme activities of CAT, POD and SOD in ZmGPAT-OE over-expression plants are reduced, hydrogen peroxide and superoxide anions generated by salt stress cannot be removed in time, so that cells are subjected to oxidative stress, the plants are normally inhibited, and the phenotype shows a salt-sensitive phenotype.

Example five Zmgpat mutant materials and ZmGPAT6-OE overexpressing materials cereal Gu Tanju disease infection phenotype and physiological analysis

Corn grass Gu Tanju pathogenic bacteria are inoculated on an oat culture medium, the oat culture medium is incubated for 10 days at the constant temperature of 28 ℃, grass Gu Tanju pathogenic bacteria spores are washed out by 0.1% Tween-20 solution, and the concentration is regulated to about 10 ⁵/mL. The conidia of pathogenic bacteria are washed off, a spore suspension with the concentration of 1 multiplied by 10 ⁵ is prepared by using 0.1 percent Tween-20 sterile water, a second leaf of corn leaves growing for ten days is taken, the spore suspension is inoculated on the leaves of corn WT, B73-329, zmgpat mutant materials and ZmGPAT-OE over-expression materials, and each liquid drop is 10 mu L. The inoculation results were investigated after 4 d.

In this embodiment, the statistics of the area of the disease spots is carried out by placing corn leaves treated by the pathogenic bacteria of grass Gu Tanju on A4 paper, taking a photo, placing the photo in the software of Image J, carrying out treatment, setting a scale, then circling the pathogenic bacteria infected part of the leaves, carrying out click measurement, and recording the area of the disease spots, wherein the unit is mm ² (square millimeter). The different corn lesions were sampled according to the three-extracted RNA example, and cDNA was obtained by reverse transcription, and RT-PCR was performed using CgTubulin primers F (GCAAACCATTCACGGCGAG), R (GGGCTCAAGGTCAACCAGGA), and the relative biomass of the bacteria was expressed by the expression level of CgTubulin. The internal reference is ZmActinF (GGTTTCGCTGGTGATGATGC), R (CAATGCCATGCTCAATCGGG). Three seedlings of each of maize WT, B73-329, zmgpat mutant material and ZmGPAT-OE overexpressing material were assayed and subjected to three biological replicates, the results of which are shown in FIG. 4: after inoculation of the grass Gu Tanju bacteria, the Zmgpat6 mutant showed disease resistant phenotype (fig. 6A), the area of lesions was significantly smaller than WT (fig. 6B), the biomass of the grass Gu Tanju bacteria was significantly lower than WT (fig. 6C); zmGPAT6-OE-1 and ZmGPAT6-OE-4 overexpressing materials exhibited a phenotype of susceptibility to Gramineae Gu Tanju (FIG. 6D), gramineae Gu Tanju had a significantly higher plaque area than B73-329 (FIG. 6E) and a significantly higher biomass than B73-329 (FIG. 6F). The results show that: zmgpat6 mutant plants are materials resistant to Gramineae Gu Tanju disease, while ZmGPAT6-OE-4 overexpressing plants are materials susceptible to Gramineae Gu Tanju disease.

Example six Zmgpat mutant Material and ZmGPAT-OE overexpression Material assayed by chitin-induced ROS burst assay

Culturing Zmgpat mutant material and ZmGPAT-OE over-expression material according to the method of example one, taking holes and sampling from corn leaf discs of 2 week old WT, B73-329, zmgpat mutant material and ZmGPAT-OE over-expression material along both sides of the leaf vein with a hole puncher of appropriate size one night in advance, taking down the punched leaf samples, and incubating overnight in a 90mm dish with sterile water in the dark; every three corn leaves were stacked on the following day for ROS assay into a reaction system containing 100. Mu. L Immunstar-HRP substrate and 1. Mu.L HRP (reaction system placed in a 1.5mL centrifuge tube). Then sterile water and chitin were added separately and the burst of ROS was immediately detected using a Glomax20120luminometer luminometer (Promega) with luminometer set parameters of KINETICCYCLE set to 60, interval time to 1min, and total time per sample detection of 25min. The data are obtained from three independent replicates. The results show that: after induction by chitin, the burst of Zmgpat mutant ROS was significantly increased compared to WT (fig. 7A); following induction by chitin, zmGPAT-OE material ROS burst was significantly reduced compared to B73-329 (fig. 7B). The massive accumulation of ROS at the site of pathogen infection directly plays a role in the inhibition and poisoning of pathogens, and the results indicate that: after being induced by chitin, zmgpat mutant plants obviously improve ROS burst and have stronger capability of resisting cereal Gu Tanju disease infection; whereas ZmGPAT-OE over-expressed plants significantly reduced ROS bursts with weaker resistance to infestation by Gramineae Gu Tanju.

Reference is made to:

[1] Guo, R., Shi, L., Yan, C., et al. Ionomic and metabolic responses to neutral salt or alkaline salt stresses in maize (Zea mays L.) seedlings.BMC Plant Biology, 2017, 17(1): 41.

[2] Hsu, F., Yan, J., Song, Y., et al. Sarracenia purpurea glycerol-3-phosphate acyltransferase 5 confers plant tolerance to high humidity in Arabidopsis thaliana. Physiologia Plantarum, 2021, 173(3): 1221-1229

[3] Liang, X., Li, J., Yang, Y., et al. Designing salt stress-resilient crops: Current progress and future challenges.Journal of Integrative Plant Biology, 2024, 66(3), 303-329.

[4] Ohlrogge, J., Browse, J. Lipid biosynthesis.Plant Cell, 1995, 7(7): 957-970

[5] Park, J., Kim, Y., Yun, J. A new insight of salt stress signaling in plant.Molecules and Cells, 2016, 39(6), 447-459.

[6] Torres, M., Cuadros, D., Vaillancourt, L. Evidence for a diffusible factor that induces susceptibility in the Colletotrichum–maize disease interaction.Molecular Plant Pathology, 2014, 15(1): 80-93.

[7] Yang, W., Pollard, M., Li-Beisson, Y., et al. A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.Proceedings of the National Academy of Sciences, 2010,107(26), 12040-12045.

Claims (8)

1. Application of corn ZmGPAT gene or biological material containing ZmGPAT gene in regulating plant salt tolerance and cereal Gu Tanju disease resistance; wherein the cDNA sequence of ZmGPAT gene is shown as SEQ ID NO.1, and the amino acid sequence of ZmGPAT gene coded protein is shown as SEQ ID NO. 2.

2. The ZmGPAT gene as defined in claim 1, wherein the nucleotide sequence also includes the nucleotide sequence shown in SEQ ID No.1, substituted, deleted and/or added with one or more nucleotides and expressing the same functional protein.

3. The biomaterial of claim 1 comprising ZmGPAT genes including, but not limited to, expression cassettes, vectors, host cells and recombinant bacteria.

4. The use of claim 1, including but not limited to the following:

(1) Improving plant salt tolerance and cereal anthracnose resistance germplasm resources;

(2) Selecting transgenic plants with salt tolerance and grass Gu Tanju disease resistance;

(3) The survival rate of the plants in a high-salt environment is improved;

(4) The survival rate of the plant cereal anthracnose in the environment is improved;

(5) The survival rate of the plants in the high-salt and cereal anthracnose environment is improved.

5. The use according to claim 1, wherein the plant is a monocot or dicot plant, including but not limited to maize, rice, wheat, soybean, sorghum, millet, cotton, barley.

6. A method for improving salt tolerance and standing grain Gu Tanju disease resistance of plants, which is characterized in that expression of ZmGPAT gene of the plants is inhibited or silenced by a method of transgene, hybridization, backcross, selfing or asexual propagation.

7. A method for obtaining plant salt-sensitive and seedling-sensing Gu Tanju disease, which is characterized in that the expression of ZmGPAT gene of plants is improved by a method of transgene, hybridization, backcross, selfing or asexual propagation.

8. The method of claim 6 or 7, wherein said transgenes include, but are not limited to, the introduction of recombinant expression vectors comprising said ZmGPAT gene into different plants using Ti plasmids, plant viral vectors, direct DNA transformation, microinjection, gene gun, conductance, agrobacterium-mediated methods.