CN110396510B - Drought-resistant protein, coding gene and application thereof - Google Patents

Abstract

A drought-resistant protein is the following protein 1) or 2): 1) protein consisting of an amino acid sequence shown by SEQ ID No. 2 in a sequence table; 2) protein which is derived from 1) and is related to plant drought resistance by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence of SEQ ID No. 2 in the sequence table.

Description

Drought-resistant protein, coding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a highly drought-resistant protein, a coding gene thereof and application thereof.

Background

Corn is the cereal crop with the widest planting range and the largest yield in the world and is the first crop of the three major food crops. China is a big country for corn production and consumption, and the sowing area, the total yield and the consumption are second to the United states and all live in the second place of the world. The cultivation of new varieties of crops with high, high and stable yield is the best way for sustainable development of grain production. The biggest threat of food production in China is drought, and corn is a hydrophilous plant and is most easily affected by drought, so that the growth and development of corn plants are seriously affected, and the yield per unit is reduced. Therefore, the excavation of the gene of the corn drought stress response has very important significance for the elucidation of the molecular mechanism of the corn drought stress response formation and the genetic improvement of the corn drought tolerance.

Several studies have now shown that parts of the gene encoding protein kinases in plants can respond to abiotic stress such as drought. Several molecular and biochemical studies have demonstrated that mitogen-activated protein kinase (MAPK) cascade pathway can respond to drought stress. The cascade pathway comprises three protein kinases: MAPKKK (mitogen-activated protein kinase kinase), MAPKK (mitogen-activated protein kinase) and MAPK (mitogen-activated protein kinase). They are serine/threonine phosphatases that are activated stepwise by phosphorylation of their specific amino acids, transmitting a signal that specifically senses upstream stimuli and cascades to the target protein. MAPKKKs are located upstream of the MAPK cascade pathway and are a class of Ser/Thr protein kinases. It is activated by phosphorylation by direct sensing of extracellular stimuli through signaling molecule receptors or itself.

A series of studies have demonstrated the significance of the MAPK pathway in drought stress (Morris, 2001; Ye et al, 2017). Alfalfa can activate the expression of p44MMK4 by drought in ABA-independent situations (Jonak et al, 1996); whereas a gene with high homology in Arabidopsis and tobacco, AtMPK3, was also up-regulated under drought induction (Mizoguchi et al, 1996); the MAPKKK gene AtMEKK1 in arabidopsis was activated 5 minutes after drought treatment, followed by a sustained increase in expression over 24 hours (Tsuneaki et al, 2002). OsMKK1, OsMSRMK2, OsMAPKK44 and OsMAPK5 in rice express significant enhancement of water average under drought conditions (Kumar et al, 2008; Agrawal et al, 2002; Jeong et al, 2006; Xiong et al, 2003) and overexpression of DSM1 genes of the MAPKKKK family can significantly enhance drought resistance of transgenic rice lines at the seedling stage (Ning et al, 2010). In maize, however, it has also been found that the transcriptional level of ZmMPK3 rapidly increases after drought treatment (Wang et al, 2010 a). In addition, MAPK can play a certain role in regulating and controlling the resistance to oxidative damage of corn leaves under water stress (Zhangxianhua et al, 2009; Zhang Alying, 2006). The study of Shou et al (2004) showed that maize plants of the MAPKKK gene NPK1 of tobacco showed significantly better drought resistance than wild type. In addition, after the NPK1 gene is transferred into rice by Xiao et al (2009), the yield of the transgenic rice is increased by 23% under the drought condition.

Disclosure of Invention

The invention aims to provide a synthetic drought-resistant protein and a coding gene thereof, which are named as ZmMAPKKK 18.

The protein provided by the invention is the protein of the following 1) or 2):

1) protein consisting of an amino acid sequence shown in SEQ ID No. 2;

2) the protein which is related to plant drought resistance and is composed of an amino acid sequence derived from the amino acid sequence shown in SEQ ID No. 2 through substitution and/or deletion and/or addition of one or more amino acid residues.

The nucleotide sequence of SEQ ID No. 1 is 1440bp, which is as follows:

ATGGCAACAGCGGCGCCGGTCAGCTGCCGGTGGACGCGCGTCCTCACGCTCGGCCGCGGCGCGTCGGGCGCGGTGGTGTCGCTCGCGGCCGACGCCGCCTCGGGCGCGCTCTTCGCGGTCAAGTCCGCCCCCGCGGGGACGCGGGCCGCGGAGAGCCTGCGGCGCGAGGGGAGCATCCTGTCCGCGCTCCGGTCCCCGCACGTGGTCCCCTGCCTGGGCCTCCGCGCCGCGGCGGACGGCGGGTGCGAGCTGCTCCTCGAGTTCGCGCCGGGCGGCTCGCTGGCGGACGTGGCGGCGAGGAGGAGCGGGCGCGACGAGCGCGCGGTCGCCGCGTACGCCGCGGACGTGGCGCGGGGCCTGGCGTACCTCCACGGGCGCTCGGTCGTGCACGGCGACGTCAAGGCGCGGAACGTCGTGGTGGGCGCCGACGGCCGCGCCATGCTCGCCGACTTCGGCTGCGCCAGGGCCGCGGCGGGCGGCGCCGACCCCGGGCGCCCCGTCGGCGGCACGCCGGCGTTCATGGCGCCCGAGGTGCTGCGCGGCGAGGGGCAGGGCCCCGCCGCCGACGTCTGGGCGCTGGGCTGCACCGTCGTCGAGATGGCCACGGGCCGCGCGCCGTGGAGCGACCTGGACGGCCTCCCCGCGGCCGTGCTCCGGGTCGGGTACACCGACGCCGTGCCCGAGGCGCCCCGCTGGATGTCGCCGGAGGCCAAGGACTTCTTGGCCCGGTGCTTCGCGAGGGACCCGCGCGAGCGGTGCACCGCGGCGCAGCTGTTGGAGCACCCGTTCCTGGCGTCGGCTGGCTGCGGCGCCATGGCGGAGTGGGTGTCTCCCAAAAGCACGCTGGACGCCGCGCTCTGGGAATCCGACGCCGACGATGGCTCCGATGACGAGGGGGACGTGTCAGAGAGCCCCGCCCAGAGAATCAAGGCGTTGGCCTGCCCCTGCTGCTCGGCCTTGCCGGACTGGGATTCCGAAGAAGGCGACTGGATTGAGGTGCTCGGTGAGCAATGTGAGGCCAACGGTTTGGTACCACCGACCAAAGAGGTGGCCAAGGAGACGGCCAGCGAAGACGAGTGCCAGCTCCTGATCCTGAGTGGGGTGTTGGAAACAGAGGTTGACTTCGTCGACGCCGATGCAGAGGGTGATCATCGTGCTAGGTGTTCTGTAGATGTAGGATTAGCTACTGTTCCGTCAGTTGAGCAGCTGCAGGAAGAGCAGCCAACTGTTTTTCTTACGGAGGCTTGTCATAACAATACTGAAATGTCGAAGTCATTTTTACTGCAAAATCGTCCCTTCGTTGCTGTGTCTTCTGTTCTCCTACTATTCGTTCTACTATTCGTCCACAAAAGAAAATCTAGAACTGCCAAACTGGTGCGATGCGACAATTCCAGAAACGTTGTTCTAGTTCTAGACCGAGGCGGCGACGGCGAGGCGTGA

the amino acid sequence shown in SEQ ID No. 2 consists of 479 amino acid residues as follows:

MATAAPVSCRWTRVLTLGRGASGAVVSLAADAASGALFAVKSAPAGTRAAESLRREGSILSALRSPHVVPCLGLRAAADGGCELLLEFAPGGSLADVAARRSGRDERAVAAYAADVARGLAYLHGRSVVHGDVKARNVVVGADGRAMLADFGCARAAAGGADPGRPVGGTPAFMAPEVLRGEGQGPAADVWALGCTVVEMATGRAPWSDLDGLPAAVLRVGYTDAVPEAPRWMSPEAKDFLARCFARDPRERCTAAQLLEHPFLASAGCGAMAEWVSPKSTLDAALWESDADDGSDDEGDVSESPAQRIKALACPCCSALPDWDSEEGDWIEVLGEQCEANGLVPPTKEVAKETASEDECQLLILSGVLETEVDFVDADAEGDHRARCSVDVGLATVPSVEQLQEEQPTVFLTEACHNNTEMSKSFLLQNRPFVAVSSVLLLFVLLFVHKRKSRTAKLVRCDNSRNVVLVLDRGGDGEA。

the invention identifies all 71 MAPKKK family members in B73 maize genome by bioinformatics technology, finds 8 genes with significant differences in expression under drought through RNA-Seq, then finds that relative expression of one gene in Zhengdan 619 and 8 inbred lines (comprising J24, J853, X178, E28, C8605-2, 200B, Qiqi 319 and B73) under drought stress are significantly up-regulated in 9 samples in total, and verifies that 7 SNPs in the gene are significantly related to drought tolerance through maize natural variation group association analysis. The gene is deduced to play an important role in drought tolerance of maize seedling stage, therefore, the new gene (GRMZM2G305066) which is positioned in chromosome 8 and the expressed protein is positioned in chloroplast is named ZmMAPKK18, the codon of the new gene is optimized, the optimized fragment which is artificially synthesized is transferred into arabidopsis thaliana and maize, and the transgenic plant shows stronger drought tolerance than the wild type.

Drawings

FIG. 1 PCR amplification result of ZmKKK 18 gene; m is DNAmarker; 1-4 is ZmKKK 18 target gene amplification product;

FIG. 2 is a schematic representation of a ZmKKK 18 overexpression vector;

FIG. 3 shows the detection result of ZmKKK 18 overexpression vector double digestion (BamHI/AscI); m is DNAmarker; 1-10 is ZmKKK 18 expression vector enzyme digestion product;

FIG. 4 direct PCR results of leaf blades of ZmKKK 18 transformed plants; m is DNAmarker; 1-4 is the direct PCR product of the leaf of the transformed plant, + is a positive control, -is a negative control;

FIG. 5 shows a process of Agrobacterium-mediated genetic transformation of maize embryos;

FIG. 6 screening ZmMAPKKK18 transgenic Arabidopsis thaliana T1 generation positive plants; no plants on the left were grown as wild type controls and transgenic plants on the right;

FIG. 7 molecular assay of ZmMAPKKK18 transgenic Arabidopsis plants; m represents a DNAmarker; -represents using wild type arabidopsis genomic DNA as a template; + represents plasmid DNA as positive control template; 2.5, 11 and 17 are respectively the transgenic arabidopsis plant genome DNA with corresponding numbers as templates;

FIG. 8 phenotypic identification of ZmMAPKKK18 transgenic T3 plants; WT is wild type Arabidopsis thaliana, vi8 is transgenic plant; CG is a normal water application treatment group; EG is a 10% PEG6000 solution stress treatment group;

FIG. 9 ZmMAPKKK18 overexpression and the seedling phenotype of non-transgenic maize lines; ZPM9 is a control plant, and V18 is a transgenic plant; CK is a normal water application treatment group; MD is moderate stress treatment group; SD is severe stress treatment group;

FIG. 10 identification of drought resistance related indexes of transgenic T3 generation plants of ZmMAPKKK 18.

Detailed Description

ZmMAPKKKK 18 gene function verification

1 materials and methods

1.1 Experimental materials

In the experiment, Arabidopsis thaliana wild type Columbia subtype (Columbia-0) is selected for genetic transformation.

1.2 Experimental reagents

Endonuclease(s): thermo corporation;

green clean PCR Mix: thermo corporation;

plant RNA kit extraction kit: omega corporation;

e, coli plasmid extraction kit: axygen corporation;

herbicide (glyphosate isopropylamine salt): monsanto Co Ltd

1.3 test strains

Plant expression vector VK 011-zmmkkkk 18:

agrobacterium tumefaciens GV3101 shocked to transform competent cells: beijing Quanjin Co Ltd

1.4 culture media and solutions

MS solid medium (MS 4.43g, sucrose 30g, agar powder 9g, distilled water constant volume to 1L, pH 5.8)

Dipping flower infection transformation buffer (MS 2.21g, sucrose 50g, distilled water constant volume to 1L, pH 5.8)

1.5 Experimental methods

1.5.1 construction of VK 011-ZmKKK 18 overexpression vector

Construction of a VK011-ZmMKKK18 (hereinafter referred to as V18) overexpression vector was carried out using a monocot transgenic vector construction kit (Catalog. No. VK011-04) (purchased from Shangrid Biotechnology Ltd., only).

1.5.1.1 ZmMKKK18 gene PCR primer

The PCR primers are designed according to the requirements of the vector construction kit. The primer information is as follows:

TABLE 1 ZmKKK 18 Gene PCR primer information

1.5.1.2 PCR reaction of target gene

The PCR reaction was carried out using the PCR primers shown in Table 1 for the target gene ZmKKK 18, and the reaction system is shown in Table 2 below.

TABLE 2 ZmKKK 18 PCR reaction System (50. mu.l)

The PCR amplification procedure was: pre-denaturation at 94 ℃ for 2min → (denaturation at 98 ℃ for 10s → annealing at 56 ℃ for 30s → extension at 72 ℃ for 1min) × 35 cycles → overextension at 72 ℃ for 7min → storage at 4 ℃.

1.5.1.3 gel electrophoresis of PCR products and recovery

And (3) performing gel electrophoresis on the ZmKKK 18 gene PCR product, and recovering after the electrophoresis is finished.

And (3) adding 50 mu l of PCR product and 40 mu l of enzyme digestion product into 1% of gel holes for electrophoresis, setting the voltage to be 10V/cm, carrying out electrophoresis for 30min, observing and analyzing the result by using a gel imager, and selecting gel strips with the sizes of 1437bp and 4000bp for gel cutting and recovery.

Agarose gel recovery was performed using the AxyPrep DNA gel recovery kit (from Axygen):

1) putting the cut gel into 1.5ml centrifuge tubes respectively, weighing and recording the total weight, and subtracting the weight of the 1.5ml centrifuge tube to obtain the weight of the gel (taking 1g weight as 1ml volume);

2) adding 3 volumes of Buffer DE-A (melting agent), heating in 75 deg.C metal bath, mixing uniformly by turning over every 2min, and completely melting gel after about 8 min;

3) adding 0.5 volume of Buffer DE-A of Buffer DE-B (binding solution), and fully and uniformly mixing;

4) transferring all the solution in the step 3 into a DNA adsorption column of the kit, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

5) adding 500ul Buffer W1 (washing solution), centrifuging at 12000rpm for 1min, and removing the filtrate;

6) adding 700ul Buffer W2 (desalting solution), centrifuging at 12000rpm for 1min, discarding the filtrate, wherein the step needs to be performed twice, and adding absolute ethanol before the washing solution is used;

7) the adsorption column was placed in a sterile, clean 1.5ml centrifuge tube, allowed to stand for 5min, 25. mu.l of deionized water was added, allowed to stand again for 5min, and centrifuged at 12000rpm for 2min to elute DNA. Heating deionized water extraction to 65 ℃, and eluting for 2 times can effectively increase the recovery amount of DNA.

8) The product was finally quantitatively diluted to 15 ng/. mu.l.

1.5.1.4 connection of target gene and product

1) Taking out Easy Assembly mix in the kit, placing the Easy Assembly mix on ice for ice bath melting, and carrying out low-speed centrifugation on the melted reagent to enable residual liquid on the tube cover and the tube wall to be centrifuged to the bottom of the tube;

2) mixing 0.5. mu.l of the quantitatively diluted recovered product with 2. mu.l of Plant Transgenic Vector in a 0.2ml centrifuge tube, sucking out all the recovered product, adding the mixture into Easy Assembly mix in an ice bath, and gently stirring and mixing the mixture by using a gun head;

3) the mixture was immediately reacted on a 50 ℃ metal bath for 25min for the next conversion experiment.

1.5.1.5 transformation

Vector transformation experiments were performed using DH5 α e.coli competent and kanamycin resistant LB medium. The specific operation steps are as follows:

1) taking a plurality of competent cells stored in an ultra-low temperature refrigerator at minus 80 ℃, and immediately placing 100 mu l of each competent cell on ice for melting for later use;

2) and (3) slowly adding the ligation products into the competent cells, gently stirring the competent cells uniformly by using a gun head, strictly forbidding the blow-beating of a pipette in the process, and immediately placing the competent cells on ice for 30min after the sample addition. Note that positive and negative controls need to be set separately during the experiment: positive control set to 100 u l competence with 0.1L Plant Transgenic Vector, its main purpose for testing competent cell activity; negative controls were set up to add an appropriate volume of deionized water to 100 μ l of competence, which was used primarily to test whether the LB medium during transformation had a resistant selection;

3) the ice-bath-terminated competent cells were immediately removed and placed in a metal bath at 42 ℃ for 30s by heat shock, after which the competence was quickly transferred to ice for a further 2min of ice-bath. Multiple experiments show that the conversion efficiency of heat shock for 30s is higher than that of 45s and 90 s;

4) to the heat shock-terminated competence, 800. mu.l of non-resistant sterilized LB medium was added, and cultured at 37 ℃ and 200rpm for 1 hour to sufficiently recover it. Meanwhile, placing an LB culture medium flat plate containing the resistance of the kanamycin antibiotic in an incubator at 37 ℃ for preheating;

5) the competence after the addition of the LB medium for the recovery culture was all added to the preheated LB plate, and the cells were spread evenly on the plate. And placing the sealed mixture in an incubator at 37 ℃ for 12-14h after inversion and sealing.

1.5.1.6 enzyme digestion identification

Selecting positive single colonies on the plate, inoculating the positive single colonies in LB culture solution containing kanamycin resistance for propagation, taking bacteria liquid after propagation for plasmid extraction, and specifically performing experimental operation steps as follows:

1) placing 50ml of sterilized LB culture solution into a sterilized 100ml conical flask, adding 50 mul of kanamycin, adding 40 mul of Escherichia coli solution containing V18 carrier, sealing, placing on a shaker, shaking at 37 deg.C and 200rpm for 12-14 h;

2) placing 2ml of the fully cultured bacterial liquid in a 1.5ml centrifuge tube, taking a plurality of tubes, centrifuging at 12000rpm for 1min, and removing the supernatant as far as possible;

3) 250. mu.l of Buffer P1 (bacterial suspension) was added, and the mixture was repeatedly blown up with a tip until the cells were completely lysed and suspended in the solution. If the thalli is not completely cracked, the final plasmid yield can be influenced;

4) add 250. mu.l Buffer P2 (bacterial lysate) and mix gently until the solution turns pale red. The mixture can not be shaken forcibly during mixing so as to avoid the pollution of genome DNA;

5) add 350. mu.l Buffer P3 (neutralization solution), mix gently immediately, when a white filamentous precipitate is formed, let stand for 5min, and centrifuge at 12000rpm for 10 min. Too much force is also avoided in this step when shaking, otherwise the fragmentation of the circular plasmid is likely to occur;

6) and (4) completely sucking out the supernatant in the step (5), transferring the supernatant into a centrifugal adsorption column in the kit, and standing for 5min, wherein in the process, the plasmids in the solution can be fully combined with a silica gel membrane in the adsorption column. Centrifuging at 12000rpm for 1min, and removing waste liquid;

7) mu.l Buffer PD (deproteinized solution) was added to the adsorption column, and the column was centrifuged at 12000rpm for 1min, and the waste solution was discarded. This step can further eliminate the nuclease in the host bacteria, preventing it from degrading the plasmid.

8) Mu.l of Buffer PW (washing solution) was added to the adsorption column, and the column was centrifuged at 12000rpm for 1min, and the waste solution was discarded. This step is performed twice. Before the washing liquid is used, absolute ethyl alcohol is added according to requirements;

9) placing the adsorption column in a sterilized 1.5ml centrifuge tube, standing for 5min, air drying, adding 50 μ l deionized water, standing for 5min, centrifuging at 12000rpm for 2min, and collecting eluate in the tube.

Experiments show that the deionized water for eluting plasmids is placed on a 60 ℃ metal bath in advance for heating, so that the elution efficiency can be obviously improved; the elution efficiency can also be increased to a certain extent by using deionized water for repeated elution; the dosage of the deionized water is not too much or too little, if too much, the final concentration of the plasmid is reduced, and if too little, the plasmid on the silica gel membrane cannot be sufficiently eluted, and the plasmid yield is also reduced.

And carrying out double enzyme digestion identification on the extracted plasmid, wherein the enzyme digestion sites are BamH I and Asc I. The enzyme digestion system is prepared as follows:

TABLE 3V 18 vector restriction system (10. mu.l)

Preparing an enzyme digestion system in a 0.2ml centrifugal tube according to the above table, shaking and centrifuging after the completion, placing on a metal bath, and carrying out enzyme digestion for 1h at 37 ℃.

And performing gel electrophoresis on the enzyme digestion product, if two bands appear in the electrophoresis result, one band is 10.4kb, and the other band is 1440bp, indicating that the bacterial colony is a positive bacterial colony, taking partial bacterial liquid or plasmid for sequencing, and verifying that the V18 expression vector is successfully constructed. And preserving the positive bacteria liquid as glycerol bacteria.

1.5.2 Agrobacterium-mediated transformation of maize

1.5.2.1 preparation of Agrobacterium-infected competent cells

1) Taking out a tube of EAH105 agrobacterium strains, unfreezing the strains on ice, taking a small amount of strains after thawing, inoculating the strains into a YEP culture medium containing kanamycin resistance, putting the strains in an incubator at 28 ℃, and culturing for 48 hours under dark conditions;

2) picking single colony in 50ml YEP culture solution containing kanamycin resistance (containing 50mg/L Rif), shaking on a shaker at 28 ℃ for 12-14h at 200rpm under dark condition;

3) carrying out ice-bath on the cultured bacterial liquid for 10min, centrifuging at 4 ℃ and 5000rpm for 5min, removing supernatant, and collecting cells at the bottom of a centrifugal tube;

4) resuspending cells in liquid MS medium and adjusting OD using spectrophotometer₅₅₀To 0.4 to 0.6;

5) the agrobacterium-infected cells were split into 50. mu.l/tube and stored in an ultra-low temperature refrigerator at-80 ℃ for further use.

1.5.2.2 Freeze-thawing method for transforming Agrobacterium

1) Taking the prepared agrobacterium tumefaciens competence, immediately placing the agrobacterium tumefaciens competence on ice to melt, adding 2 mu l V18 vector plasmid, gently stirring and uniformly mixing, and immediately carrying out ice bath for 30 min;

2) treating with liquid nitrogen for 1min, performing heat shock on a constant temperature metal bath at 37 deg.C for 5min, and immediately performing ice bath for 2min without shaking the centrifuge tube;

3) adding 900 mul of non-resistant LB culture solution, and shaking at 200rpm for 3h at the temperature of 28 ℃;

4) centrifuging at 8000rpm for 1min, pouring out supernatant, and dissolving thallus with 100 μ l LB;

5) adding all the bacterial liquid to a YEP culture medium (containing 100mg/L Rif) containing kanamycin resistance, uniformly coating, inverting, sealing and putting in an incubator at 28 ℃ for 2-3 days;

6) and (3) selecting a single colony, verifying a positive single colony by using the PCR primers in the table 1, and performing amplification propagation and preserving the bacterial liquid for later use after verification is correct.

1.5.2.3 Agrobacterium-mediated transformation of maize

1) Taking ZPM9 inbred line young ears pollinated for 10 days by field inbred pollination, removing the top and bottom parts of the young ears, soaking the young ears in a mixed solution of sodium hypochlorite and 0.1% Tween20 for 30min, and sterilizing;

2) and taking out the young ears, thoroughly cleaning the young ears by using sterile water, and stripping embryos in an ultra-clean workbench, wherein the size of the young embryos is 1.0-1.5 mm, and the young ears are beneficial to agrobacterium infection. Placing the immature embryos into a 1.5ml centrifuge tube filled with Inf staining solution for later use;

3) taking the bacterial liquid stored in 1.5.2.2, and infecting the immature embryos for 15-20 min at the temperature of 20 ℃;

4) sucking out the staining solution by using a liquid transfer gun, slowly sucking out the residual staining solution on the young embryo by using sterile filter paper, flatly placing the young embryo on a Coc culture medium, and co-culturing for 3d in a dark culture box at 28 ℃;

5) transferring the young embryo after the co-culture to an I-type selective culture medium, culturing for 14 days in a dark incubator at 28 ℃, and growing the young embryo into callus;

6) transferring the callus which normally grows after the type I selective culture is finished to a type II selective culture medium, and culturing for 14 days in a dark incubator at the temperature of 28 ℃;

7) transferring the callus with good growth vigor to a type I regeneration culture medium after the type II selective culture is finished, and culturing in a dark incubator at 26 ℃ until sprouts grow out;

8) transferring the buds after the regeneration culture of the type I to a regeneration culture medium of the type II, and performing photoperiod culture in an incubator at 26 ℃ until green buds grow;

9) transferring the green bud after the type II regeneration culture to a type III regeneration culture medium, and continuing performing photoperiod culture in an incubator at 26 ℃ until young roots grow to form seedlings;

10) transferring the seedlings after the type III regeneration culture to a rooting culture medium, and performing photoperiod culture in an incubator at 26 ℃ for 10-14 days;

11) regenerated Plant small leaves were taken and subjected to Direct PCR using the Phore Plant Direct PCR Master Mix kit (purchased from Thermo Scientific) for rapid transgenic detection of the plants.

Direct PCR reaction procedure: 98 ℃ -pre-denaturation 5min → (98 ℃ -denaturation 5s → 62 ℃ -annealing 5s → 72 ℃ -extension 20s) × 40 cycles → 72 ℃ -overextension 1min → 4 ℃ -preservation.

Performing gel electrophoresis on the PCR product, and judging whether the detection material is a positive plant;

12) taking small regenerated plant leaves, detecting Bar protein expression in the plants by using a Bar transgenic rapid detection kit (purchased from Aoxing biotechnology limited), and judging whether the detection material is a positive plant.

13) And further carrying out RT-PCR detection on the detected positive plants, and selecting 2 transgenic plants with high expression quantity of the target gene ZmMKKK18 for subsequent drought resistance function identification.

1.5.2.4 transformation of Arabidopsis thaliana

1) Taking wild arabidopsis thaliana which grows for three weeks and grows more undeveloped flowers for standby;

2) culturing the transformed agrobacterium until the OD value is about 1.2-1.6, centrifuging for 15min at the room temperature of 4000rpm, and removing the supernatant to collect thalli;

3) resuspending the thallus by using a flower-dipping infection transformation buffer solution, and diluting until the OD value is 0.8;

4) adding 10 mu L of Silweet into every 20mL of bacterial liquid, inverting and immersing the arabidopsis into the bacterial liquid, and uniformly infecting for 1 min;

5) wrapping the infected plant with preservative film, and culturing in 16 deg.C incubator in dark for 24 hr;

6) taking out and slightly stripping the preservative film, and placing the preservative film on a culture rack for continuous culture until the preservative film is solid.

1.5.2.5 screening of Arabidopsis Positive plants

1) Part of T1 generation transgenic seeds are uniformly and equivalently planted in the seeds respectively containing

Testing the concentration of the herbicide which can be tolerated on the MS culture medium plate;

2) and then uniformly scattering the rest seeds on the soil-containing vermiculite in the ratio of 1: 1, covering a preservative film in the nutrition pot, and thoroughly watering;

3) after two green cotyledons grow out of arabidopsis, spraying herbicide once in three days according to the tested tolerance concentration, wherein after about one week, the non-positive plants wither and die, and the positive plants grow normally;

4) taking positive plant leaves to extract DNA as a template for PCR identification.

1.5.2.6 DNA extraction of positive plants

1) All the operations were carried out at room temperature in accordance with the instructions.

2) Adding liquid nitrogen into the whole plant, and grinding into powder;

3) adding 500 μ L of lysis solution per 50-100mg, vortex shaking to mix well, centrifuging at 14000g speed for 5 min;

4) transferring the supernatant to a collecting column, standing for 5min, centrifuging at 14000g for 5min, and discarding the solution;

5) adding 500 mu L of Wash I lotion into the collection column, and centrifuging for 1min at the rotating speed of 12000 g;

6) adding 500 mu L of Wash II washing solution into the collection column again, centrifuging at the rotating speed of 12000g for 1min, and repeating once;

7) discarding the collected liquid, and centrifuging for 2min in an empty tube;

8) adding 30 μ L DEPC water into the collection column, and centrifuging for 1 min;

9) the total DNA of the plants is collected in the collecting tube, and the concentration is measured and recorded.

1.5.2.7 PCR identification of positive plants

1 mu L of the extracted DNA template is taken for PCR identification. The following reagents were added in order:

and (2) slightly and uniformly mixing the reagents, and then carrying out PCR reaction under the following reaction conditions:

remove 5. mu.L running agarose gel electrophoresis for identification.

1.5.3 drought stress treatment of Positive plants

Respectively planting wild Arabidopsis and positive transgenic plants, selecting 6 pots of wild Arabidopsis and Arabidopsis plants with 4 weeks of growth and basically consistent growth vigor, randomly selecting two groups of a normal water application control group and a treatment group irrigated by 10% PEG6000 solution, carrying out 3 times of treatment, measuring equal volume of ddH during the period, and respectively planting the wild Arabidopsis and the positive transgenic plants, wherein each group is selected as a normal water application control group and a treatment group irrigated by 10% PEG6000 solution₂Seedlings were irrigated 3 times with O and 10% PEG6000 solution. Phenotypes were observed after 14 days of treatment.

1.5.4 drought-resistant biological function identification of transgenic corn plant

Carrying out continuous two-generation selfing seed reproduction on the obtained transgenic maize plant to obtain T₂The generation seeds are used as treatment materials in drought stress experiments, and the control material is ZPM 9. The experiment is carried out in a drought shed of corn research center of agriculture and forestry academy of sciences of Beijing, and the specific experimental scheme is as follows:

1) experimental materials are named as ZPM9, V18-1 and V18-2 are respectively provided with 3 different water treatments of normal irrigation (CK), Moderate Drought (MD) and Severe Drought (SD), and each treatment is provided with 10 repetitions;

2) germinating the experimental material, spreading clean culture box and sterilized filter paper in the culture box, soaking in sterile water, placing ZPM9, V18-1, and V18-2 seeds in the culture box, germinating at 30 deg.C in dark condition, and supplementing water;

3) meanwhile, preparing drought pots for sowing, filling a proper amount of seedling-stage nutrient soil (nutrient soil and slow release fertilizer) into each pot, and watering until the soil surface just submerges for later use;

4) after 2-3 days of seed germination, selecting seeds with uniform germination in a drought pot, sowing 4 seeds in each drought pot, placing the germinated seeds in a hole with downward roots and upward buds, and slightly covering with nutrient soil;

5) when the seedlings grow to the two-leaf stage, taking a small number of leaves, carrying out transgenic detection on the seedlings by using a Bar transgenic rapid detection kit, removing non-transgenic hybrid plants, and leaving positive plants;

6) the transgenic plants screened by the test paper strip continue to grow, water is normally poured in the transgenic plants, and drought treatment is carried out when the plants grow to three leaves and one core;

7) 9 parts per day: 00 and 15: 00 Water content measurement is carried out twice by using a soil water content measuring instrument and recorded, water content among treatment groups is strictly controlled, and the control standard of the water content of each treatment group is as follows:

normal irrigation treatment (CK): 16-18 (70% -80% of field water capacity)

Moderate drought treatment (MD): 12-14 (50% -60% of field water capacity)

Moderate drought treatment (SD): 8-10 (30-40% of field water capacity)

8) Drought stress treatment was continued for 14 days, and the growth of each group of materials was photographed. Taking 10 pieces of each processed leaf and stem, immediately freezing with liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 deg.C for next determination of related physiological indexes;

9) the method for extracting the crude enzyme liquid of the plant proposed by Grace and Logan in 1996 is referred to for optimization and improvement, and the extraction of the crude enzyme liquid of the sample is carried out. Taking about 0.3g of the freeze-dried sample preserved in the previous step, adding a small amount of liquid nitrogen, slightly grinding, putting the mortar on ice, adding 2ml of extraction buffer solution and quartz sand, grinding until the mixture is homogenized, putting the homogenized mixture into a plurality of 2.0ml centrifuge tubes, centrifuging at 12000rpm for 10min at 4 ℃, taking the supernatant, and preserving the supernatant in an ultra-low temperature refrigerator at-80 ℃ for later use.

1.5.4.1 photosynthetic rate, stomatal conductance and transpiration rate

And (3) measuring photosynthetic rate, stomatal conductance and transpiration rate indexes by using an LI-6400XT photosynthetic apparatus. The determination was made on day 14 of drought stress at 9-11 am. Is provided with an air inlet chamber CO₂The concentration is 400ppm, and the light intensity of the illumination chamber is 1200 Lux. And selecting the same part of the completely unfolded new leaf for measurement, and recording data after the numerical value to be measured is stable.

1.5.4.2 PEPC carboxylase

The reaction mixture (0.5ml 70mM MgSO. RTM.) was added to the cuvette₄，0.5ml 70mM NaHCO₃1ml of 14mM PEP) and 1ml of enzyme extract, comparing the color at 340nm, reading the OD value every 20s, obtaining the consumption rate of NADH according to the change of the OD value, and defining the PEPC activity according to the consumption rate of NADH per milligram of protein.

1.5.4.3 chlorophyll

0.2g of the leaf blades were cut into pieces and placed in a 50ml centrifuge tube, and 0.5ml of 100% acetone and 15ml of 80% acetone were added to the tube for extraction. After the leaves are whitened, leaching is finished, centrifuging is carried out at 12000rpm for 5min, supernatant is taken and added into a cuvette, and the wavelengths of 645nm, 663nm and 652nm are subjected to color comparison according to a formula: chlorophyll a concentration: ca 12.72 × a663-2.59 × a 645; chlorophyll b concentration: cb ═ 22.88 × a663-4.67 × a 645. The sum of the two is the total chlorophyll concentration.

2 results and analysis

2.1 PCR amplification of the ZmMKK 18 Gene

The ZmKKK 18 gene was PCR amplified using the primers in Table 1, and the product was subjected to gel electrophoresis, the results of which are shown in FIG. 1. And (3) taking 1Kb DNA Marker as a reference, wherein the amplification product is about 1400bp and is consistent with the known target gene 1440bp, and the product is cut and recovered for later use.

2.2 construction of VK 011-ZmKKK 18 vector

Construction of V18 overexpression vector was performed using monocot transgenic vector construction kit (Catalog. No. VK011-04), which is schematically shown in FIG. 2. The obtained plasmid was double digested with BamHI and AscI endonucleases as shown in FIG. 3. The plasmid extracted from 10 single colonies is subjected to double enzyme digestion, the enzyme digestion electrophoresis result shows that two bands are provided, one band is 10.4kb, the other band is 1440bp, the sequencing result is completely matched with the ZmKKK 18 gene sequence, and the construction of the V18 overexpression vector is successful.

2.3 genetic transformation of maize

Transforming an agrobacterium-competent EAH105 into a V18 expression vector, taking a maize receptor material ZPM9 as a young embryo subjected to self-pollination for 10 days, carrying out agrobacterium-mediated transformation, and carrying out co-culture, resting culture, selective culture and regeneration culture for four periods to obtain a transformed plant, which is detailed in a figure 5. The transformed plant is detected by a direct PCR method (detecting a target gene) and Bar transgenic rapid detection test paper (detecting Bar protein), and a transgenic positive plant is obtained by screening. Bar transgenic rapid detection test paper is used for detection, 4 plants with positive transgenic test paper are obtained through screening, small leaves of the 4 transformed plants are further taken, direct leaf PCR reaction is carried out on the obtained small leaves of the transformed plants, the result shows that the detailed figure 4 shows that 1440bp of a target gene strip is amplified in the 4 transformed plants, and the target gene is integrated into a corn genome.

2.4 identification of transgenic Arabidopsis positive plants by ZmMAPKKK18

The pCAMBIA 3301-ZmMAPKKKK 18 expression vector is transferred into arabidopsis thaliana by an agrobacterium flower dipping infection method, and the whole process is finishedSequences incorporated into the arabidopsis genome include the 35s promoter, the ZmMAPKKK18 gene, and the herbicide resistance gene. Transgenic Arabidopsis thaliana T1 generation plants

After the herbicide spraying screening at the concentration, as shown in fig. 6, the first and second rows on the left side had half of 6 pots in total of wild type arabidopsis thaliana. After the herbicide is sprayed, basically no plant individuals capable of normally growing seedlings exist in the nutrition pot planted with wild type arabidopsis, and partial plants grow normally in the nutrition pot planted with T1 transgenic plants in the second row and the third and fourth rows of 10 total pots, so that the plants are green and show the resistance to the herbicide. These plants that survive herbicide stress are likely to be positive transgenic plants.

And then after fructification, picking the blades of the herbicide-resistant plants with normal growth vigor and numbering according to the picking sequence. Extracting genome DNA, amplifying ZmMAPKKK18 gene segment, and the size of the target segment should be 1.4 kb. As shown in FIG. 7, the randomly selected positive clones 2, 5, 11 and 17 are shown to be subjected to PCR amplification, and the results show that the four clones can amplify target gene segments with correct sizes, and all the target gene segments are transgenic positive plants. Finally, 31 positive plants capable of amplifying target segments are screened from 68T 1 transgenic plants. After seed harvesting, the T2 generation transgenic plants were subjected to segregation ratio identification on herbicide-containing plates, wherein the segregation ratio of 14 lines was 3:1, and the 14 lines were selected for screening of homozygous lines.

The arabidopsis transgenic plant over-expressing the ZmMAPKKK18 gene is obtained through an experiment, as shown in FIG. 8, after drought stress treatment, the wild type plant shows a large area of withering, yellowing and wilting or even necrosis. However, only part of leaves of the transgenic plant overexpressing ZmMAPKKKK 18 have slight withering and yellow wilting, no plant dies basically, and most of the transgenic plants are not greatly influenced by drought stress, which indicates that the transgenic plant overexpressing ZmMAPKKKK 18 shows stronger drought resistance under the drought stress.

2.5 drought stress treatment results in transgenic maize

Drought stress treatment is carried out on the selected transgenic plants in a dry shed, the materials of experimental groups are V18-1 and V18-2, the material of a contrast material is ZPM9, the drought stress treatment is carried out on the materials through seedling stage water control, and the growth conditions of the materials of each group are photographed after stress for 14 days. As can be seen in FIG. 9, the MD group and SD group were less vigorous than the CK group under drought stress conditions, indicating that different degrees of drought stress had different degrees of influence on the growth of the plants. Meanwhile, the phenomena of obvious leaf rolling, withered old leaves and the like of MD and SD processing materials under the drought condition of ZPM9 are found; the MD and SD treated materials of V18-1 and V18-2 have no stress expression such as leaf rolling and leaf withering. The ZmMAPKKKK 18 transgenic plant phenotype is less affected by drought stress under moderate and severe drought stress.

The control experiment of FIG. 10 shows that the photosynthetic rates of the three materials treated with CK, MD and SD are reduced, however, the decrease of ZPM9 is much higher than that of V18-1 and V18-2. Under MD and SD treatment, the photosynthetic rate of V18-1 and V18-2 is higher than that of ZPM9 under the same treatment, especially under SD treatment, it can be seen that under severe drought stress, the photosynthetic rate of V18-1 and V18-2 can still be kept higher, while the photosynthetic rate of ZPM9 is reduced to below 10. Therefore, the photosynthetic rate of the V18 plants under drought is higher than that of the ZPM9 plants.

Three groups of materials, namely ZPM9, V18-1 and V18-2, show extremely remarkable increase in PEPC carboxylase under MD treatment: ZPM9 has 60% fluctuation, V18-1 85% fluctuation and V18-2 47% fluctuation. Under SD treatment, the PEPC carboxylase of ZPM9 decreased significantly, by 24%, compared to MD treatment, whereas V18-1, V18-2 did not change significantly compared to MD treatment, i.e., PEPC carboxylase activity of V18 plants under SD treatment was higher than that of ZPM9 under the same treatment. Therefore, the V18 transgenic plant has the possibility of ensuring the normal photosynthesis under severe drought by maintaining higher PEPC carboxylase activity.

And the transpiration rate change of the three materials under the three treatments is basically consistent with the porosity conductivity change trend. Compared with ZPM9 plants, the transgenic plants of V18 have lower transpiration rate under the same drought conditions, which is particularly obvious under SD treatment. By reducing the transpiration rate, V18 plants were better able to adapt to drought environments.

Chlorophyll of CK-MD-SD of ZPM9 shows a gradient descending trend, MD treatment is reduced by 28% compared with CK treatment, and SD treatment is reduced by 23% compared with MD treatment; the chlorophyll content of the V18-1 treated by MD is not obviously changed compared with that of the CK treated by SD, and the chlorophyll content of the SD treated by SD is reduced by 22% compared with that of the MD treated by MD; the chlorophyll content of V18-2 was also not significantly changed in MD compared to CK-treated, and 23% decreased in SD compared to MD. Overall, the chlorophyll content of V18-1MD treatment is higher than that of ZPM9 which is also MD treatment, and the chlorophyll content of V18-1 and V18-2 is obviously higher than that of ZPM9 under SD treatment. Therefore, V18 plants had a higher chlorophyll content than ZPM9 plants under drought.

In conclusion, all drought resistance indexes of the ZmMAPKKK18 transgenic lines are superior to those of the control lines.

Summary of the invention

Because the drought resistance of the plant is a more complex character, after the over-expression of part of the drought resistance genes, although the over-expression of the part of the drought resistance genes can show better drought resistance under drought stress, the growth of the over-expressed transgenic plant can be inhibited under the condition of normal water application, and the over-expression of the part of the drought resistance genes is also an important reason that a plurality of the drought resistance genes cannot be applied to the actual production. However, the research finds that under the condition of normal water application, the phenotype of the transgenic plant over-expressing ZmMAPKKK18 is basically consistent with that of a wild plant, the normal growth and development of the plant are not influenced, and the transgenic plant shows excellent drought resistance only under drought stress.

The experimental results show that the maize gene ZmMAPKKK18 can obviously enhance the drought resistance of maize, and is a drought-resistant gene which can be applied to the drought resistance improvement and breeding of maize excellent germplasm resources.

Claims (1)

1. An application of drought-resistant protein in drought resistance of plants, wherein the drought-resistant protein is protein consisting of an amino acid sequence shown in SEQ ID No. 2,

the amino acid sequence shown in SEQ ID No. 2 consists of 479 amino acid residues as follows:

MATAAPVSCRWTRVLTLGRGASGAVVSLAADAASGALFAVKSAPAGTRAAESLRREGSILSALRSPHVVPCLGLRAAADGGCELLLEFAPGGSLADVAARRSGRDERAVAAYAADVARGLAYLHGRSVVHGDVKARNVVVGADGRAMLADFGCARAAAGGADPGRPVGGTPAFMAPEVLRGEGQGPAADVWALGCTVVEMATGRAPWSDLDGLPAAVLRVGYTDAVPEAPRWMSPEAKDFLARCFARDPRERCTAAQLLEHPFLASAGCGAMAEWVSPKSTLDAALWESDADDGSDDEGDVSESPAQRIKALACPCCSALPDWDSEEGDWIEVLGEQCEANGLVPPTKEVAKETASEDECQLLILSGVLETEVDFVDADAEGDHRARCSVDVGLATVPSVEQLQEEQPTVFLTEACHNNTEMSKSFLLQNRPFVAVSSVLLLFVLLFVHKRKSRTAKLVRCDNSRNVVLVLDRGGDGEA。

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