CN109837296B - New salt-tolerant drought-tolerant function of corn gene ZmNAC77 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioscience, and particularly relates to application of a corn gene ZmNAC77 in simultaneously improving drought and salt stress of arabidopsis thaliana. The invention separates and clones genes from cornZmNAC77Will beZmNAC77The cDNA sequence of the gene is connected with 35S-1300-flag, Arabidopsis thaliana is infected by utilizing an agrobacterium inflorescence method, and the experimental result shows thatZmNAC77The gene is over-expressed in the arabidopsis thaliana, and the drought and salt stress tolerance of the transgenic arabidopsis thaliana is obviously improved. The invention separates and clones corn gene for the first timeZmNAC77，And over-expressed for the first time in Arabidopsis thalianaZmNAC77The gene provides a new idea for cultivating drought-resistant and salt-resistant transgenic plants.

Description

A new function of maize gene ZmNAC77 in salt tolerance and drought tolerance and its application

technical field

The invention belongs to the technical field of biological sciences, and particularly relates to the application of a maize gene ZmNAC77 in simultaneously improving drought and salt stress of Arabidopsis thaliana.

Background technique

Saline soil is distributed all over the world, covering an area of about 1 × 109 hm2. Among them, about half of the irrigated land in arid and semi-arid areas is affected by salinization, while non-irrigated areas are also severely salinized. China's land ^salinization accounts for about ^1/10 of the global salinization area, and the land salinization is gradually showing an upward trend. 25%. China's saline land is widely distributed, large in area and many types, among which the North China Plain, the Northeast Plain, the Northwest Plain and the coastal areas are relatively concentrated. In recent years, due to people's unreasonable farming and further industrial development, the salinity content in the soil has greatly increased, and the secondary salinization of the soil has been aggravated. Therefore, the degree of soil salinization and drought in China is becoming more and more serious, which has become one of the important factors restricting plant cultivation. Studying the regulation mechanism of plants under drought and salt stress, and breeding salt- and drought-resistant plants is beneficial to agricultural production and yield increase, as well as the management and improvement of drought and saline-alkali land.

Maize is very sensitive to adversity stress such as salt tolerance and drought tolerance. Therefore, it is very important to study stress-related genes, clarify the molecular mechanism of stress resistance, and improve maize stress resistance. With the development of molecular biology technology, it has become the focus of current research to transfer stress resistance genes into related crops and make them highly expressed in the target crops, thereby improving the adaptability of crops to adverse environments and improving the quality of crop yields. However, the related research on NAC genes in maize is not perfect, and there is no method to improve plant stress resistance by using maize NAC genes.

SUMMARY OF THE INVENTION

In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of a maize Zm00001d049540 gene (named ZmNAC77 ) in simultaneously improving drought and salt stress in Arabidopsis thaliana.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical scheme to realize:

Application of the maize gene ZmNAC77 to simultaneously improve drought and salt stress in Arabidopsis.

The application method of maize gene ZmNAC77 in improving drought and salt stress in Arabidopsis thaliana at the same time, the cDNA fragment of maize gene ZmNAC77 is used as the application gene, the gene is forwardly transferred into Arabidopsis thaliana, the expression level of ZmNAC77 gene is improved, and maize is obtained Transgenic Arabidopsis plants with increased ZmNAC77 gene expression.

The above method firstly amplifies the full-length coding region cDNA of the maize ZmNAC77 gene by PCR method, then connects the cDNA sequence of ZmNAC77 with 35S-1300-flag, and uses the Agrobacterium inflorescence method to infect Arabidopsis thaliana to improve the expression of the ZmNAC77 gene, Arabidopsis plants with increased expression of the maize ZmNAC77 gene were obtained.

The nucleotide sequence of the above-mentioned maize gene ZmNAC77 is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 3.

The above-mentioned primer sequences of the maize gene ZmNAC77 that regulate the fatty acid and starch content of Arabidopsis are:

ZmNAC77-F: 5‵-CGG GGTACC TCGATGGTGGAGATGTCTGTG-3‵;

ZmNAC77-R: 5‵-GCG GGATCC GCACCGCACCAGGATAGATTT-3‵;

The underline is the restriction site, the restriction site with the primer on it is Kpn1, and the restriction site of the downstream primer is BamH1.

beneficial effect

(1) The experiments of the present invention prove that the present invention finds the maize gene ZmNAC77 in maize, and overexpresses this gene in Arabidopsis, and the drought and salt stress tolerance of the transgenic Arabidopsis is significantly improved.

(2) The present invention isolates and clones the maize gene ZmNAC77 for the first time, and overexpresses the ZmNAC77 gene in Arabidopsis for the first time, which provides a new idea for cultivating drought-resistant and salt-resistant transgenic plants. It has important production and life significance for crop breeding and production application.

Description of drawings

Figure 1 shows the result of DNA verification (DNA maker: DL2000, target band length is 500bp);

Fig. 2 is the phenotype diagram of overexpression line and wild type under different conditions;

Figure 3 shows the comparison of root lengths of Arabidopsis plants under different stress conditions;

Figure 4 is a graph showing the comparison of fresh weight of Arabidopsis plants under different stress conditions;

Figure 5 shows the comparison of the number of lateral roots of Arabidopsis plants under different stress conditions.

Detailed ways

The technical solutions of the present invention are further explained in the following examples. According to the above description and these examples, those skilled in the art can determine the basic characteristics of the present invention, and can understand the present invention without departing from the spirit and scope of the present invention. Various changes and modifications are made to make it suitable for various uses and conditions; the present invention introduces the above-mentioned expression vector into the model plant Arabidopsis thaliana cells, and the introduction method is the transformation method mediated by Agrobacterium. The above-mentioned expression vector is introduced into plant cells, and the introduction methods are well known to those skilled in the art, and these methods include but are not limited to: Agrobacterium-mediated transformation, biolistic method, electrical stimulation, ovary injection and the like.

Example 1 Construction of Arabidopsis overexpression lines

The Arabidopsis thaliana overexpression vector uses 35S-1300-flag, the vector recombination method uses enzyme cleavage ligation, and the method for infecting Arabidopsis thaliana is the floral dip method. In the Arabidopsis overexpression experiment, in order to facilitate later experimental screening, the infected wild-type col Arabidopsis is usually called the T ₀ generation, and the seeds obtained after the infection are the T ₁ generation seeds, and the T ₁ generation seeds _The seedlings that germinate after the seeds are planted are the T1 generation seedlings, the seeds harvested from the T1 generation seedlings are the _T2 generation seeds, and the _T2 generation seeds have genotype separation, that is, the homozygous positive seedlings are screened at the T2 generation, and the expression of different genes is determined. Overexpression lines with different gene expression levels were obtained.

(1) Construction of recombinant vector

(1) Gene amplification and restriction enzyme ligation

By designing primers at the initiation site (ATG) of the ZmNAC77 gene to introduce the Kpn1 restriction site and the BamH1 restriction site at the 3' end, the full-length cDNA sequence of the gene was obtained by PCR amplification. The primers used for gene amplification The sequence is:

ZmNAC77-Kpn1 F: CGG GGTACC TCGATGGTGGAGATGTCTGTG

ZmNAC77-BamH1R: GCG GGATCC GCACCGCACCAGGATAGATTT

The obtained amplification product was recovered by gel, and the concentration was determined. The vector and the target gene must use the same restriction site, and then use Kpn1 and BamH1 double restriction enzyme to digest the target gene fragment and vector, and then recover it. Use NEB's T4 ligase to connect the target gene with the overexpression vector 35S-1300-flag. The connection system is as follows:

Content Volume(μL)

ddH2O 9

Vector 2

ZmNAC77 gene fragment 4

T4 ligase 1

5xT4 buffer 4

The above reaction solution was placed in a PCR apparatus to react overnight at 16° C. to obtain a recombinant vector, which was used for the transformation of Escherichia coli.

(2) Preparation of E. coli competent

1) Escherichia coli (E.coli) DH5α strain was streaked on the LB medium plate with an inoculation loop, and cultured at 37°C overnight;

2) Pick a fresh single colony and inoculate it into 5M1LB liquid medium, 37℃, 250rpm shaking culture overnight;

3) Diluted 100 times, inoculated into 50mL LB liquid medium, cultivated at 37℃, 250rpm for 3~5h to the logarithmic phase of bacterial growth;

4) Transfer the bacterial solution to a 50mL centrifuge tube and place it on ice for 10min;

5) 4℃, 4000rpm, centrifuge for 10min, discard the supernatant;

6) Resuspend the cells with 50mL of 0.1M CaCl (pre-cooled at 24°C), centrifuge at 4°C, 4000rpm for 10min, and discard the supernatant;

7) Add 25mL of 0.1M CaCl to the pellet (pre-cooled at 24°C), resuspend the pellet, 4°C, 4000rpm, 10min, discard the supernatant;

8) Add 2.5mL of 0.1MCaCl2 (containing 15% glycerol) to each tube, and resuspend the pellet;

9) Dispense into 1.5ml EP centrifuge tubes, 100μL/tube;

10) Store in -70℃ refrigerator for later use.

(3) Transformation of recombinant vector

1) Thaw E. coli competent cells on ice;

2) Add the ligation product to the competent cells, add about 10 μL of the recombinant vector to 50 μL of the competent cells, gently rotate and mix, and place on ice for 30 minutes;

3) Heat shock in a water bath at 42°C for 90sec, do not shake, immediately put it back on ice to cool for 2min;

4) Add 600 μL LB medium, 37℃, 150rpm shaking culture for 60~90min;

5) Centrifuge at 4000rpm for 5min at room temperature, aspirate part of the LB medium, and the remaining volume is about 200μL;

6) Mix the remaining liquid and the precipitate, and spread it evenly on the LB medium plate containing 100mg/mL Spec+ resistance;

7) Invert at 37°C for 12~16h until colonies appear.

(4) Screening and sequencing of positive colonies

1) Pick a single colony into a 1.5mL centrifuge tube containing 0.6mL LB (Spec+resistance), 37 ℃, 220rpm shaking culture for about 6h;

2) PCR amplification with universal primers T7/SP6. Take 1 μL of bacterial solution as a template, and the annealing temperature is 52 °C. The PCR product of the positive clone should be about 1kb; if the clone is false positive (vector self-ligation), the PCR product fragment is about 300bp;

3) Pick 3 positive colonies, send them to the company for sequencing, and select the correctly sequenced strains for subsequent experiments.

(5) Extraction of plasmids

1) Pick a single colony of Escherichia coli containing the vector plasmid, and culture it in LB liquid medium containing Spec+ resistance at 37°C and 200rpm with shaking overnight;

2) Pour the bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and collect the bacterial pellet;

3) Aspirate the supernatant with a pipette, add 100 μL of pre-cooled Solution I to the tube and shake well, and bath on ice for 5 minutes;

4) Add 200 μL of newly prepared Solution II (room temperature), close the mouth of the tube tightly, and gently invert it 2~3 times to mix it evenly.

5) Add 150 μL of pre-cooled Solution III, mix well, ice bath for 5 minutes; centrifuge at 12000rpm for 5 minutes at 4°C;

6) Transfer the supernatant to a centrifuge tube, add an equal volume of phenol and chloroform (1:1) for extraction, centrifuge at 12,000 rpm for 5 min, and take the supernatant;

7) Extract once again with an equal volume of phenol and chloroform (1:1); transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, invert and mix, and let it settle for 2 hours at room temperature;

8) 4℃, 12000rpm, centrifuge for 15min, discard the supernatant, and collect the precipitate; wash the precipitate twice with 1 mL of 70% ethanol;

9) 4°C, 12000rpm, centrifuge for 2min, blot dry the liquid; dry the precipitate at room temperature, dissolve the precipitate in 50μL ddH2O, and store at -20°C.

(6) Preparation of Agrobacterium competent

1) Agrobacterium EHA105 strain was streaked on a YEB plate containing 100μg/mLRif+, and cultured at 28°C for 36-48h until colonies grew;

2) Pick a single colony and inoculate it in 5 mL of YEB liquid medium containing 100 μg/mL mL Rif+, and inoculate it with shaking at 28 °C and 250 rpm for 16-24 h;

3) Transfer 0.5mL of bacterial liquid to 50mL of YEB liquid medium containing 50μg/mL Rif+, 28°C, 250rpm, shake to culture to logarithmic phase, OD600=0.5, about 8~16h;

4) Transfer the bacterial liquid to a 50mL centrifuge tube, ice bath for 30min, and centrifuge at 5000rpm for 10min to pellet the cells;

5) Add 10 mL of pre-cooled 0.15M NaCl, gently suspend the cells, centrifuge at 4°C, 5000 rpm for 5 min, and remove the supernatant;

6) Repeat step (5) once;

7) Resuspend the cells with 1 mL of pre-chilled Cacl ₂ containing 15% glycerol, aliquot into 1.5 mL centrifuge tubes, 100 μL/tube, snap-frozen in liquid nitrogen, and store at -70°C for later use.

(7) Agrobacterium transformation and positive colony screening

1) Take the competent cells EHA105 frozen at -70℃ and thaw on ice; add 5-10 μl of the plasmid to be transformed into 50-100 μl of competent cells, mix gently, and take an ice bath for 30 minutes;

2) Quick-freeze the centrifuge tube in liquid nitrogen for 1min, immediately put it at 37°C for 3min,

3) Immediately add 800ml of YEP medium, mix well, and incubate at 28°C with shaking at 180rpm for 3h;

4) Centrifuge at 5000 rpm for 1 min at room temperature to pellet the cells;

5) Aspirate the excess medium and keep about 200 μL to spread on YEB (containing 100 mg/L rifampicin, 50 mg/L kanamycin) plate;

6) Invert at 28°C for 2~3 days until colonies appear

7) The method of screening positive colonies refers to the above screening of Escherichia coli positive colonies to obtain the positive colony ZmNAC77-35s-1300flag. The ZmNAC77-35S -1300flag positive Agrobacterium obtained by the above experiments is stored in a -80°C ultra-low temperature refrigerator for later use.

(2) Agrobacterium infection of Arabidopsis thaliana

(1) Activation and expansion of Agrobacterium

1) Take 500ul of ZmNAC77-35S -flag positive Agrobacterium and add it to 10ml of YEP liquid medium containing Rif ⁺ and Kan ⁺ , and culture at 29°C, 200rpm overnight.

2) Take 1ml of ZmNAC77-35S -flag Agrobacterium cultured overnight, add it to 50ml of YEP liquid medium containing Rif ⁺ and Kan ⁺ , cultivate at 29°C and 200rpm, and continuously monitor the OD value of the bacterial solution during the culture process. Shake to OD600=1.2.

3) Transfer the bacterial liquid to a 50ml centrifuge tube, centrifuge at 10,000 rpm for 10 min, and collect the bacterial liquid for later use.

(2) Infection of Arabidopsis by inflorescence method

1) Prepare 600mL of Insalts sucrose solution with distilled water: Insalts 1.32g, Sucrose 30g, Silwet L-770.18mL (add it during infection, and add it now); first take 30mL of Insalts sucrose solution and resuspend the centrifuged cells, pour into In a clean beaker, dilute with Insalts sucrose solution to an OD value of 1.1-1.2; before infection, adjust the OD value between 0.7-0.9 with the remaining Insalts sucrose solution, and carry out infection after standing for 20 min.

2) Arabidopsis thaliana is self-pollinating, and the plants have been pollinated before flowering. Therefore, to obtain more transgenic seeds, it is best to prune fruit pods and pods one day before the transformation of Arabidopsis thaliana before Agrobacterium infection. Flowers that have already opened are then infected with Agrobacterium.

3) Soak the flower branches with unopened buds in Insalts sucrose solution containing Agrobacterium for 30s-60s, take them out to dry, bind them and place them in the dark at 22°C for 24h, take them out and put them in a light culture room for normal growth. Can.

(3) Screening of Arabidopsis overexpression homozygous lines

After the infected Arabidopsis seeds are mature and harvested, the T1 generation seeds can be obtained. The T1 generation seeds are placed in a 28°C oven to dry for 3 days, and then placed at room temperature for 10 days. Germination can increase the rate of seed germination.

Generation of positive seedling screening

(1) Hygromycin screening

1) Disinfect and sterilize the post-ripening T1 generation seeds. First put the T1 generation seeds into 75% ethanol for 5 minutes, then use 75% ethanol + Tration 100 for 5 minutes, and finally resuspend them with absolute ethanol and spread them on the sterilizer. air-dry naturally on the filter paper;

2) Prepare 1/2 MS solid medium, after sterilization, when the temperature is about 50-60 ℃, add hygromycin to make the final concentration 30mg/L, and pour it into a sterilized petri dish after mixing. Blow dry in the clean bench for about 2h;

3) Place the sterilized Arabidopsis seeds evenly on the surface of the medium, seal the petri dish with parafilm, place it in a refrigerator at 4°C for 3 days, and then place it in a light culture room at 22°C for 16 hours of light and 8 hours of dark culture. ;

4) After about 10 days of growth, the transgenic Arabidopsis seedlings will grow well in hygromycin medium, with green leaves and larger plants; Arabidopsis seedlings without transgenics will have yellow leaves and smaller plants;

5) Transfer the well-grown transgenic Arabidopsis seedlings to normal 1/2 MS solid medium without hygromycin, grow normally for about a week, and then move to soft soil for growth.

(2) DNA verification

The 98 positive seedlings screened were sampled and DNA was extracted. Using DNA as a template and the universal primer on the 35s-1300-flag vector as a template, PCR amplification was performed to verify whether the primary screening positive seedlings contained the transferred recombinant vector. DNA was extracted by CTAB method, and the experimental method was as follows:

1) Put a few corn leaves into a 2 mL centrifuge tube, put steel balls with a diameter of 4 mm, freeze them in liquid nitrogen, and use a high-throughput tissue grinder to crush them into powder.

2) Add 500 mL of CTAB extraction buffer (1.17M NaCl, 0.0016M EDTA-8.0, 0.835M Ttis-7.5, 1.6% CTAB, 1% β-mercaptoethanol) preheated to 65°C and mix well.

3) React in a 65°C water bath for 45 minutes, and shake the centrifuge tube carefully every 15 minutes.

4) Take out the centrifuge tube, let it cool to room temperature (25°C), add an equal volume of chloroform:isoamyl alcohol (24:1), shake the tube carefully for 50min, until the organic phase changes from colorless → green → black (dark green), Let stand for 10 minutes.

5) Centrifuge at 8000rpm for 10min at room temperature (≥25℃), transfer the supernatant to another 1.5mL centrifuge tube with a pipette tip with the head cut off, and repeat the previous step if the supernatant is still green.

6) Add 2/3 volume of isopropanol (-20℃), invert up and down several times and mix carefully, 12000rpm, 4℃, 10min.

7) Pour off the supernatant, add 400 μL of 70% ethanol, wash twice, suck off the remaining ethanol with a pipette tip, and dry at room temperature until the ethanol evaporates completely.

8) After the DNA is dry until transparent, add 20 μL of TE (pH 8.0) to dissolve the DNA.

9) Aspirate 2 μL of DNA, add Loading buffer, use 1.0% agarose gel to detect DNA quality, and NANO to detect DNA concentration.

10) DNA is stored in a -20℃ refrigerator for later use.

11) The extracted DNA is used as a template, and PCR amplification is carried out through the universal primer on the 35s-1300-flag vector. After agarose gel electrophoresis, the target band is obtained. The target band is the plant containing the recombinant vector. , and delete plants that do not contain the recombinant vector.

12) The plants containing the recombinant vector continue to grow until the seeds are mature, and the T2 generation seeds are collected.

, Identification of homozygous strains with different expression levels

(1) Identification of homozygous strains

1) Screen homozygous without segregation

The T2 generation seeds harvested from a single plant were air-dried for 10 days, sterilized, and germinated in 1/2 MS medium containing hygromycin. 200 seeds were selected for each positive single plant. After germination was completed, the germination rate was calculated (due to the T2 generation seeds Separation has occurred, and only all germinated seeds are homozygous positive lines), and the lines with a germination rate close to 1 are selected as candidate homozygous lines;

(2) Screening of homozygous strains with different expression levels

Pick 6 homozygous lines of Arabidopsis thaliana leaves, select 9 samples from each line, take the leaves to extract RNA, reverse transcribed into cDNA, and use RT-PCR experiment to determine the gene expression of different lines respectively. Homozygous lines with different expression levels were screened, and three lines with low, medium and high expression levels were selected, namely OE1, OE2, and OE3.

Example 2 Salt tolerance treatment

When the mutant seeds were germinated to a plant height of about 1 cm, they were transferred to 100 mmol/L NaCl medium and grown for 10 days. The amount of mutant lines was also different, the higher the overexpression amount, the more obvious the growth advantage under the stress treatment. The results are shown in Fig.

Example 3 Drought tolerance treatment:

When the mutant seeds were germinated to a height of about 1 cm, they were transferred to 200 mmol/L mannitol medium and grown for 10 days. The expression levels of mutant lines were also different. The higher the overexpression level, the more obvious the growth advantage under the stress treatment.

sequence listing

<120> A new function of maize gene ZmNAC77 in salt tolerance and drought tolerance and its application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4052

<212> DNA

<213> Corn

<400> 1

gaggcactcg ctttcttcca caccacgtct cctccgcctg cgtctgcgtc ccttcctcac 60

gcggacgccc tcctaaaatc cccaaaatcc gatccgacca ccaaacccta accccatccc 120

gcactccccc cctccccccc ccccccaaca cgcgcgcgct ttcttagccg ctaagcatat 180

ccctctccgc cacgatctcg atggtggaga tgtctgtggt ggagctcagg acgctgccgc 240

tcggcttccg cttccacccc accgacgagg agctcgtcac ccactacctc aagggcaaga 300

tcaccggcag gatcaactcc gagtccgagg tcatccccga gatcgacgtc tgcaagtgcg 360

agccgtggga cctcccaggt aatacttgct tctgctaact gcccgcgtcg cttgatcgcc 420

cctctctgct ctcgattttt tatccgttcc ttcaagtaat tttgtgctcg ctggtcgtac 480

acggtaatct ctactgggat tgctgagttc ttcttgttct ggggctactc gttgcttcga 540

gttttttctg cgattttctt tcatcgggat ttgcttcgat gtacttttgc gagagtgtga 600

cgcgtttgtg ctgggaaaaa aagccgagca gatcacaacc cgctgcttcc tctgttctga 660

ttcgcgactg tttgcactag cggttcctca cgagatattt ctccgtgggc ggggcgggac 720

gggacgccgt gctgtgctgg ttcgatgcga ttcctcgaac tccgctgaat cctttcttta 780

ataccgcgtc ccccctttcc aagatgcgtt ttgattttgt gcgatgcagc tgaacaaaat 840

ccttgcgctg atccattttg ttgttgatcc gcgtgtcgag tcgagcaaac tcccgggaaa 900

gattattcag ttttcctgat cagatcctgg gtactgatga cgtgactgtg tatatgcaga 960

taagtccctg atccggtccg atgatcccga gtggttcttc ttcgccccca aggaccgcaa 1020

gtaccccaac ggtagcaggt ctaacagggc caccgaggcg ggatactgga aggctactgg 1080

caaggacagg ataatcaagt ccaagggcga caagcgcaag cagcatacca tcggtatgaa 1140

gaagaccctc gtcttccacc gcggtcgtgc ccccaagggc gagcgcacgg ggtggattat 1200

gcacgagtac cgcaccaccg agcctgagtt cgagtccggc gagcaggtat gaagctatta 1260

ttctcggtgc agcttatggt tgtcaagttc tctagtgtta gtatctgtac agagatgtgt 1320

gctttgtact tgagaactta agtttgtctt ggataaatat aacatagtag tctgttcact 1380

gaagaatgtg cccctaaaca ctagtttgat ttctggtggt taaaagaatt gagttctaat 1440

catcttcaca attagttctg tacagtataa gcacatagta aatgattttt ttgtgttgtg 1500

tctcatggca aatgtttact aaaatcacct ggtgcttgtg tatctgattt tccagggtgg 1560

ttatgttctt tatcgcctct tccgtaagca agaggagaaa actgagcgct ccagcccaga 1620

ggaagtggat agaagtggct actcacctac tccttctcgt tcttcacctg acaacctaga 1680

ggcgaatgag gaagctaata caccattaaa taaggaatct ccagaatctg ctctgcatga 1740

aagcccgatt gagctgccaa aatctgttga aaccaatggt ggttcaatga caaggtggct 1800

ggcggacaga aatgataact tgatggttac tgcaccagat gtttcccata tacctttcca 1860

cggacatgct gctggtgtag ctaaggtgag catcagagta aagagcctat taacatatca 1920

gcattcaaca tttgtctgta tgctatccac catcataatt ataattttat aactgaattt 1980

ctcctaacaa ttccctcatc tttattatta caagcaggtt gatccttctg ttggtgcttc 2040

aggccactta gttaatcccc acaatggaaa tgatgattat aacaaatttg tgtctgattt 2100

cactcccatt ctaccacttg aaaatgcatt cttcactgat atacaacaag gagcttttgg 2160

ttttgatggc atcatgaatc ctcttgatcc ccttgatgct ttcttgaatc aaacacttgt 2220

tgatcctgat gaacattcat caacaacatc aaaagttcag tatgattctg acattccaac 2280

agaatttgag aaccattgga atatgaaggt actggttttc tattcatatt gagtatttga 2340

gtttgccatc cgatgttttg gttatggtta atccagcaat ccttatattt tgctatattt 2400

gcaggttgag cctcaagatg atcaatgctg gtgggcaaac ataggatttg agccagacga 2460

accaaatcct ctgcttcctt gtgacaccac tgaccaagac atactttctg ttgactctgg 2520

tgctgattcc ttcaatgagc ttttcaatag tatggaagag actggaatta ttgttaggcc 2580

tcaacagttg gactccactg tgcaaccaaa ccatgtgttc gctagccagg gcaatgcagc 2640

acggaggctg cggctacagg ttgagtctag ggagatcata actaaagatg agagtgaaga 2700

tgaagtatca tgtgttctaa ctccagactg tttgaacgat tctgttgagg agtccactgc 2760

agagaaggat gtggcttctg atggggatga ggctgagtca acagggattg ttattagaag 2820

ccatcaccct gctccaagat caagctcaga gagttcattc actcaacagg gaactgctat 2880

gcaaaggctg cggctacagt caggccttaa caaaggccag cgtcctagta ctgatgattc 2940

atcaagctgc attatagacg aaccaggaag tcagcacaaa gcagaaaaag cagaggtgat 3000

tgttcttttt gttacccaga agctaaattt acatctgttt tttatgctag tgatgatcgg 3060

tatctaggat attccataga tactagttct gttagtgtac tgctactatt ttgcagtgtt 3120

ttcttaaatc ttcattgatc ctggccctgg gtgatctatt ttagtaaatg ataccatctg 3180

acctttttta tgttatgtca gattgaagag gatgcgagta caaacctcgc tggaagtgct 3240

gatgatctac ctggtaatat ccatgatgat gagcaaaaga acatccctga acatggtaat 3300

atgctttgga acttctgtat attgtgtctc ttgtgatgac ttctgttttt gctaaactag 3360

ttaaatatgt tgttgaactg aaggtgctga aatgacttct ccagaagcca aatctgttct 3420

gaggttgcgg aagacttctg aggaaggcaa caaggatgtc aagcaggaga gttgtctcga 3480

gccacacgtg agagcaccaa tgcagaaggg aggcttccaa tcatacatca tctggctggt 3540

tcttccggtg gctctgctcc tgcttctctg tgttgggaca tatggatggg tatgaaatct 3600

atcctggtgc ggtgcaaggc atgacatttt gcgcacatgg gttcctggcg tcaacctggt 3660

gtgcggtctt ctgtacataa cctgtggcat cctttcaaca gccgtaccct gcagtaagac 3720

atcccggcat tgccaaggca gctgaaaaat aaataaaaaa acaagtggta gaaatgacaa 3780

ggtggaaaag agcgaggtgt gttattgttg gttttagtac atcgtagtct tcttttgctg 3840

ttgttgtaga tgtcttgcta atgttgtcgc tgttgtgtag ttgcaattca agcccagggc 3900

tgccagtttt atttcctaat gtaatgtagc aagagcgaga ccctgttaga cgggtgcgct 3960

gaaattctga acctctgtaa tatctgatgt tcttgtggat acctgtttca gaattttcgg 4020

ttcctgaacc cagttactga tggcttgttg at 4052

<210> 2

<211> 1983

<212> DNA

<213> Corn

<400> 2

atggtggaga tgtctgtggt ggagctcagg acgctgccgc tcggcttccg cttccacccc 60

accgacgagg agctcgtcac ccactacctc aagggcaaga tcaccggcag gatcaactcc 120

gagtccgagg tcatccccga gatcgacgtc tgcaagtgcg agccgtggga cctcccagat 180

aagtccctga tccggtccga tgatcccgag tggttcttct tcgcccccaa ggaccgcaag 240

taccccaacg gtagcaggtc taacagggcc accgaggcgg gatactggaa ggctactggc 300

aaggacagga taatcaagtc caagggcgac aagcgcaagc agcataccat cggtatgaag 360

aagaccctcg tcttccaccg cggtcgtgcc cccaagggcg agcgcacggg gtggattatg 420

cacgagtacc gcaccaccga gcctgagttc gagtccggcg agcagggtgg ttatgttctt 480

tatcgcctct tccgtaagca agaggagaaa actgagcgct ccagcccaga ggaagtggat 540

agaagtggct actcacctac tccttctcgt tcttcacctg acaacctaga ggcgaatgag 600

gaagctaata caccattaaa taaggaatct ccagaatctg ctctgcatga aagcccgatt 660

gagctgccaa aatctgttga aaccaatggt ggttcaatga caaggtggct ggcggacaga 720

aatgataact tgatggttac tgcaccagat gtttcccata tacctttcca cggacatgct 780

gctggtgtag ctaaggttga tccttctgtt ggtgcttcag gccacttagt taatccccac 840

aatggaaatg atgattataa caaatttgtg tctgatttca ctcccattct accacttgaa 900

aatgcattct tcactgatat acaacaagga gcttttggtt ttgatggcat catgaatcct 960

cttgatcccc ttgatgcttt cttgaatcaa acacttgttg atcctgatga acattcatca 1020

acaacatcaa aagttcagta tgattctgac attccaacag aatttgagaa ccattggaat 1080

atgaaggttg agcctcaaga tgatcaatgc tggtgggcaa acataggatt tgagccagac 1140

gaaccaaatc ctctgcttcc ttgtgacacc actgaccaag acatactttc tgttgactct 1200

ggtgctgatt ccttcaatga gcttttcaat agtatggaag agactggaat tattgttagg 1260

cctcaacagt tggactccac tgtgcaacca aaccatgtgt tcgctagcca gggcaatgca 1320

gcacggaggc tgcggctaca ggttgagtct agggagatca taactaaaga tgagagtgaa 1380

gatgaagtat catgtgttct aactccagac tgtttgaacg attctgttga ggagtccact 1440

gcagagaagg atgtggcttc tgatggggat gaggctgagt caacagggat tgttattaga 1500

agccatcacc ctgctccaag atcaagctca gagagttcat tcactcaaca gggaactgct 1560

atgcaaaggc tgcggctaca gtcaggcctt aacaaaggcc agcgtcctag tactgatgat 1620

tcatcaagct gcattataga cgaaccagga agtcagcaca aagcagaaaa agcagagatt 1680

gaagaggatg cgagtacaaa cctcgctgga agtgctgatg atctacctgg taatatccat 1740

gatgatgagc aaaagaacat ccctgaacat ggtgctgaaa tgacttctcc agaagccaaa 1800

tctgttctga ggttgcggaa gacttctgag gaaggcaaca aggatgtcaa gcaggagagt 1860

tgtctcgagc cacacgtgag agcaccaatg cagaagggag gcttccaatc atacatcatc 1920

tggctggttc ttccggtggc tctgctcctg cttctctgtg ttgggacata tggatgggta 1980

tga 1983

<210> 3

<211> 660

<212> PRT

<213> Corn

<400> 3

Met Val Glu Met Ser Val Val Glu Leu Arg Thr Leu Pro Leu Gly Phe

1 5 10 15

Arg Phe His Pro Thr Asp Glu Glu Leu Val Thr His Tyr Leu Lys Gly

20 25 30

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

35 40 45

Asp Val Cys Lys Cys Glu Pro Trp Asp Leu Pro Asp Lys Ser Leu Ile

50 55 60

Arg Ser Asp Asp Pro Glu Trp Phe Phe Phe Ala Pro Lys Asp Arg Lys

65 70 75 80

Tyr Pro Asn Gly Ser Arg Ser Asn Arg Ala Thr Glu Ala Gly Tyr Trp

85 90 95

Lys Ala Thr Gly Lys Asp Arg Ile Ile Lys Ser Lys Gly Asp Lys Arg

100 105 110

Lys Gln His Thr Ile Gly Met Lys Lys Thr Leu Val Phe His Arg Gly

115 120 125

Arg Ala Pro Lys Gly Glu Arg Thr Gly Trp Ile Met His Glu Tyr Arg

130 135 140

Thr Thr Glu Pro Glu Phe Glu Ser Gly Glu Gln Gly Gly Tyr Val Leu

145 150 155 160

Tyr Arg Leu Phe Arg Lys Gln Glu Glu Lys Thr Glu Arg Ser Ser Pro

165 170 175

Glu Glu Val Asp Arg Ser Gly Tyr Ser Pro Thr Pro Ser Arg Ser Ser

180 185 190

Pro Asp Asn Leu Glu Ala Asn Glu Glu Ala Asn Thr Pro Leu Asn Lys

195 200 205

Glu Ser Pro Glu Ser Ala Leu His Glu Ser Pro Ile Glu Leu Pro Lys

210 215 220

Ser Val Glu Thr Asn Gly Gly Ser Met Thr Arg Trp Leu Ala Asp Arg

225 230 235 240

Asn Asp Asn Leu Met Val Thr Ala Pro Asp Val Ser His Ile Pro Phe

245 250 255

His Gly His Ala Ala Gly Val Ala Lys Val Asp Pro Ser Val Gly Ala

260 265 270

Ser Gly His Leu Val Asn Pro His Asn Gly Asn Asp Asp Tyr Asn Lys

275 280 285

Phe Val Ser Asp Phe Thr Pro Ile Leu Pro Leu Glu Asn Ala Phe Phe

290 295 300

Thr Asp Ile Gln Gln Gly Ala Phe Gly Phe Asp Gly Ile Met Asn Pro

305 310 315 320

Leu Asp Pro Leu Asp Ala Phe Leu Asn Gln Thr Leu Val Asp Pro Asp

325 330 335

Glu His Ser Ser Thr Thr Ser Lys Val Gln Tyr Asp Ser Asp Ile Pro

340 345 350

Thr Glu Phe Glu Asn His Trp Asn Met Lys Val Glu Pro Gln Asp Asp

355 360 365

Gln Cys Trp Trp Ala Asn Ile Gly Phe Glu Pro Asp Glu Pro Asn Pro

370 375 380

Leu Leu Pro Cys Asp Thr Thr Asp Gln Asp Ile Leu Ser Val Asp Ser

385 390 395 400

Gly Ala Asp Ser Phe Asn Glu Leu Phe Asn Ser Met Glu Glu Thr Gly

405 410 415

Ile Ile Val Arg Pro Gln Gln Leu Asp Ser Thr Val Gln Pro Asn His

420 425 430

Val Phe Ala Ser Gln Gly Asn Ala Ala Arg Arg Leu Arg Leu Gln Val

435 440 445

Glu Ser Arg Glu Ile Ile Thr Lys Asp Glu Ser Glu Asp Glu Val Ser

450 455 460

Cys Val Leu Thr Pro Asp Cys Leu Asn Asp Ser Val Glu Glu Ser Thr

465 470 475 480

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

485 490 495

Ile Val Ile Arg Ser His His Pro Ala Pro Arg Ser Ser Ser Glu Ser

500 505 510

Ser Phe Thr Gln Gln Gly Thr Ala Met Gln Arg Leu Arg Leu Gln Ser

515 520 525

Gly Leu Asn Lys Gly Gln Arg Pro Ser Thr Asp Asp Ser Ser Ser Ser Cys

530 535 540

Ile Ile Asp Glu Pro Gly Ser Gln His Lys Ala Glu Lys Ala Glu Ile

545 550 555 560

Glu Glu Asp Ala Ser Thr Asn Leu Ala Gly Ser Ala Asp Asp Leu Pro

565 570 575

Gly Asn Ile His Asp Asp Glu Gln Lys Asn Ile Pro Glu His Gly Ala

580 585 590

Glu Met Thr Ser Pro Glu Ala Lys Ser Val Leu Arg Leu Arg Lys Thr

595 600 605

Ser Glu Glu Gly Asn Lys Asp Val Lys Gln Glu Ser Cys Leu Glu Pro

610 615 620

His Val Arg Ala Pro Met Gln Lys Gly Gly Phe Gln Ser Tyr Ile Ile

625 630 635 640

Trp Leu Val Leu Pro Val Ala Leu Leu Leu Leu Leu Cys Val Gly Thr

645 650 655

Tyr Gly Trp Val

660

<210> 4

<211> 30

<212> DNA

<213> Corn

<400> 4

cggggtacct cgatggtgga gatgtctgtg 30

<210> 5

<211> 30

<212> DNA

<213> Corn

<400> 5

gcgggatccg caccgcacca ggatagattt 30

Claims (3)

1. the application of maize gene ZmNAC77 in improving Arabidopsis drought and salt stress simultaneously, it is characterized in that, its nucleotide sequence of described maize gene ZmNAC77 is as shown in SEQ ID NO: 1, CDS sequence is as shown in SEQ ID NO: 2, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 3. 2. the application method of maize gene ZmNAC77 in improving drought and salt stress in Arabidopsis thaliana simultaneously, it is characterized in that, the cDNA fragment of maize gene ZmNAC77 is used as application gene, this gene is forwardly transferred in Arabidopsis thaliana, improves ZmNAC77 gene The expression level of the maize ZmNAC77 gene was increased to obtain a transgenic Arabidopsis thaliana plant; The nucleotide sequence of the maize gene ZmNAC77 is shown in SEQ ID NO: 1, the CDS sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 3. 3. application method according to claim 2 is characterized in that, at first amplify the full-length coding region cDNA of maize ZmNAC77 gene by PCR method, then the cDNA sequence of ZmNAC77 is connected with 35S-1300-flag, utilizes Agrobacterium The inflorescence method was used to infect Arabidopsis thaliana to increase the expression of ZmNAC77 gene to obtain Arabidopsis thaliana plants with increased expression of maize ZmNAC77 gene.

Images