CN113604475A - Application of cotton GH _ D03G1517 gene in drought resistance promotion and salt tolerance promotion - Google Patents

Abstract

Translated fromChinese

本发明公开了棉花GH_D03G1517基因在促进植物抗旱和耐盐中的应用，属于植物基因工程技术领域。GH_D03G1517基因具有SEQ ID NO:1所示的核苷酸序列并可编码SEQ ID NO:2所示氨基酸序列。利用本发明，可为植物尤其是棉花的抗逆分子改良进行技术支持。

The invention discloses the application of cottonGH_D03G1517 gene in promoting plant drought resistance and salt tolerance, and belongs to the technical field of plant genetic engineering.The GH_D03G1517 gene has the nucleotide sequence shown in SEQ ID NO:1 and can encode the amino acid sequence shown in SEQ ID NO:2. Using the present invention, technical support can be provided for the improvement of plants, especially cotton, for the improvement of stress resistance molecules.

Description

Application of cotton GH _ D03G1517 gene in drought resistance promotion and salt tolerance promotion

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a cotton GH _ D03G1517 gene in drought resistance promotion and salt tolerance promotion.

Background

The major stresses affecting crop production worldwide are abiotic stresses, such as: high temperature, salt and water deficit. The most important of these stresses are drought and salt stresses, which have a significant impact on agriculture and food supply and cause significant losses in crop productivity. Plants successfully achieve metabolic, physiological and morphological changes by using specific and complex signaling mechanisms to deal with external stresses, including accumulation of metabolites, stress-related gene expression, synthesis of osmotic pressure and antioxidants, development and transpiration of root systems, and regulation of water lost to the atmosphere.

Cotton is an important fiber crop and is widely used in the global textile industry. In regions of the world affected by abiotic stress, one of the major methods to maintain the growing cotton yield is to mine key genes to improve stress tolerance. However, current research on stress resistance of cotton, particularly drought and salt tolerance genes, is still insufficient.

Disclosure of Invention

The inventor shows that the GH _ D03G1517 gene plays an important role in cotton drought resistance and salt tolerance by identifying and analyzing the GH _ D03G1517 gene of a MAPK group A member in cotton and combining the results of fluorescence quantification, Arabidopsis transformation, VIGS test and the like, and can be used for cotton stress-resistant molecule improvement. Thus, the present invention has been completed.

The invention provides application of a GH _ D03G1517 gene in promoting drought resistance and salt tolerance of plants, wherein the GH _ D03G1517 gene has a nucleotide sequence shown in SEQ ID NO. 1.

The open reading frame of the GH _ D03G1517 gene is 1128 bp.

In some embodiments of the invention, the nucleotide sequence set forth in SEQ ID NO. 1 is capable of encoding the amino acid sequence set forth in SEQ ID NO. 2. The protein comprising the amino acid sequence has the relative molecular weight of 43.018kDa and the isoelectric point of 5.677.

In some embodiments of the invention, the expression level of the GH _ D03G1517 gene is increased in plants to promote drought resistance and salt tolerance of the plants.

In some embodiments of the present invention, the increase of the expression level of the GH _ D03G1517 gene in the plant is achieved by: increasing expression of a plant endogenous GH _ D03G1517 gene, or overexpressing an exogenous GH _ D03G1517 gene in a plant.

In a specific required embodiment of the invention, the overexpression of the exogenous GH _ D03G1517 gene refers to the expression of the GH _ D03G1517 gene in a plant through agrobacterium-mediated transformation by using a plant expression vector.

Further, the GH _ D03G1517 gene is introduced into a plant cell, tissue or organ via a plant expression vector.

Further, the plant expression vector drives the expression of the GH _ D03G1517 gene through a constitutive or inducible promoter.

Still further, the constitutive promoter is a 35S promoter.

In the present invention, the promotion of flowering refers to promotion of the flowering phase of plants to be advanced.

In the present invention, the plant is cotton, corn, rice, wheat or Arabidopsis.

The invention has the advantages of

According to the invention, the result of silencing the GH _ D03G1517 gene in cotton shows that the GH _ D03G1517 gene may play a key role in promoting drought resistance and salt tolerance of cotton. The invention can be used for carrying out technical support on the improvement of stress-resistant molecules of plants, particularly cotton.

Drawings

Fig. 1 shows: evaluation of physiological parameters of GH _ D03G1517 overexpression lines under drought and salt stress conditions: phenotypic characteristics of overexpression lines and WT plants before and after drought and salt treatment. After 8D stress, WT and over-expression strain leaves (A) Relative Leaf Water Content (RLWC), (B) in vitro leaf water loss (ELWL), (C) ion leakage, (D) quantitative determination of chlorophyll II content. Each experiment was repeated three times with error bars indicating Standard Deviation (SD) and different letters on the column indicating statistically significant differences (ANOVA, P < 0.05). WT: wild type, L2, L6, L8: overexpression lines.

Fig. 2 shows: determination of levels of oxidant and antioxidant concentrations in GH _ D03G1517 transgenic lines under drought and salt stress conditions: leaves of wild type and overexpressing lines after 8d stress (A) quantitative determination of Malondialdehyde (MDA), (B) Hydrogen peroxide (H)₂O₂) Quantitative determination of concentration, (C) quantitative determination of Catalase (CAT) content, and (D) quantitative determination of Peroxidase (POD). Each experiment was repeated three times, error bars indicate Standard Deviation (SD), different letters on the column indicate statistically significant differences (ANOVA, P)<0.05). WT: wild type, L2, L6, L8: overexpression lines.

Fig. 3 shows: expression levels of abiotic stress response genes (ABF4, KIN1, RAB18 and RD22) in GH _ D03G1517 overexpressing strains (L2, L6 and L8) and wild type arabidopsis thaliana. AtACTIN2 served as the housekeeping gene. The letters a/b indicate statistically significant differences (ANOVA, P < 0.05). Error bars represent Standard Deviation (SD) of 3 biological replicates.

Fig. 4 shows: germination rate and root growth of wild type and GH _ D03G1517 overexpression lines under PEG treatment. The seed germination was quantified (a)1/2MS agar, (B) 8% PEG plates, (C) 10% PEG plates, (D) 15% PEG plates, and each experiment was repeated three times, each measurement representing the average germination ± SD of 50 seeds. (E) Germination of seeds after 7 days germination on 1/2MS supplemented with 0, 8%, 10% and 15% PEG. Seedlings grown for 3 days on 1/2MS medium were transferred to medium containing 0, 8%, 10% and 15% PEG for 7 days (F) root growth of wild type and GH _ D03G1517 overexpression lines, (G) comparison of root elongation, three replicates per experiment, scale bar 2cm, each measurement representing the average root length of 50 seedlings, error bars indicate Standard Deviation (SD), different letters on the column indicate statistically significant differences (ANOVA, P < 0.05).

Fig. 5 shows: germination rate and root growth of wild type and GH _ D03G1517 overexpression lines under salt treatment. The seed germination was quantified (a)1/2MS agar, (B)50mM plates, (C)75mM plates, (D)100mM plates, and each experiment was repeated three times, each measurement representing the average germination ± SD of 50 seeds. (E) Germination of seeds after 7 days germination on 1/2MS supplemented with 0, 50mM, 75mM and 100mM NaCl. Seedlings grown for 3 days on 1/2MS medium were transferred to media containing 0, 50mM, 75mM and 100mM NaCl (F) root growth of wild type and GH _ D03G1517 overexpressing lines, (G) comparison of root elongation after 7 days of growth, three replicates per experiment, scale bar 2cm, each measurement representing the average root length of 50 seedlings, error bars indicate Standard Deviation (SD), different letters on the column indicate statistically significant differences (ANOVA, P < 0.05).

Fig. 6 shows: GH _ D03G1517 improves drought tolerance throughout the growth cycle of Arabidopsis. (A-B) drought tolerance analysis of transgenic GH _ D03G1517 gene Arabidopsis thaliana in one complete growth cycle,scale bar 4 cm. (C) The GH _ D03G1517 transgene and the pod length of wild type Arabidopsis thaliana under drought treatment and blank control conditions. (D-F) Stem length, pod length and pod number per plant of 35S: GH _ D03G1517 and WT plants were measured under drought-treated and blank control conditions. Each experiment was repeated three times. Error bars indicate standard error (SD), different letters on the bars indicate statistically significant differences (ANOVA, P < 0.05).

Fig. 7 shows: evaluation of physiological parameters of VIGS-treated cotton plants: (I) (A-C) PSD, null and phenotype of GH _ D03G1517 silenced plants, (D) expression levels of GH _ D03G1517 in placebo and GH _ D03G1517 silenced plants. (II) after 8 days of drought and control treatment, the leaves of idling and GH _ D03G1517-VIGS plants (A) RLWC, (B) ELWL, (C) ion leakage, and (D) chlorophyll concentration were determined. Each experiment was repeated three times. Error bars represent SD of three biological replicates. Different letters on the bars indicate statistically significant differences (ANOVA, P < 0.05).

Fig. 8 shows: under the drought stress condition, the expression levels of oxidant and antioxidant enzyme in leaves of GH _ D03G1517-VIGS and VA cotton plants are analyzed and stress response genes are expressed. (IA-D) quantitative determination of (A) MDA, (B) H2O2, (C) CAT and (D) POD 7 days after drought and control treatment. (II) relative expression quantity of stress response gene (A) DXT/MATE, (B) GhmeKk24, and (C)Ghraf 4. Error bars represent SD of three biological replicates. Different letters on the bars indicate statistically significant differences (ANOVA, P < 0.05).

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Example 1

1. Cotton material

The cotton material selected in this example is upland cotton TM-1, Zhongmiao No. 10, Zhonghan 177. Tissue or organ expression was assessed using Gossypium hirsutum TM-1. Plants for vegetative tissue were planted in a 25 ℃ growth chamber 16 hours light and 8 hours dark. Plants for obtaining reproductive tissues were planted in the cotton research institute field (Anyang City, Henan, China). In the house, tissues like young leaves were sampled at the early stage of planting, and stalks, true leaves and roots were sampled at two weeks after sowing. Flowers were harvested from thefield 10 days after flowering.

Salt stress and drought stress tests were performed using Zhongmian institute No. 10 and ZhongH 177, respectively. Cotton seedlings from medium cottonship nos. 10 and H177 were grown in a 25 ℃ laboratory growth room with a 16 hour light/8 hour dark cycle. Seedlings were treated with 15% polyethylene glycol 6000(PEG-6000) to induce drought stress, and plants were treated with 200mM sodium chloride to induce salt stress. Samples were taken at 0h, 2h, 4h, 6h, 12h, 48h and 72h after treatment, respectively. Each experiment was performed 3 times.

2. Reagent and consumable

2.1 enzymes and kits:

GXL DNA Polymerase high-fidelity enzyme, gel recovery kit and PCR product purification kit were purchased from Takara; RNA reverse transcription kit, KOD FX Neo enzyme (code. No. KFX-201) purchased from Toyobo Co;

the Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small quantity extraction kit is purchased from magenta company; restriction enzymes (BamH I, Sac I) were purchased from NEB; the DNA Marker III and the plant total RNA extraction kit are purchased from TIANGEN company; fluorescent quantitation TransStart Top Green qPCR SuperMix was purchased from TransGen.

2.2 other drugs: agarose is Spanish original product, peptone, yeast extract, chloroform, isoamylol, ethanol, isopropanol, sodium chloride, sucrose, silwet L-77, phloroglucinol and the like are domestic analytical purities, kanamycin, streptomycin sulfate, ampicillin and the like are purchased from Botryobo bioengineering (Dalian) Co., Ltd., Escherichia coli competent cell Trans5 alpha is purchased from Beijing holotype gold biotechnology Co., Ltd., and Agrobacterium competent cell LBA4404 is purchased from Shanghai Weiji Biotechnology Co., Ltd.

2.3 culture Medium: LB liquid medium: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), and 10g/L sodium chloride (NaCl); LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of Agar powder (Agar), and the volume is fixed to 1L; LB selective medium: before LB plate, adding antibiotic with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 deg.C, shaking up and plating. The reagent solutions mentioned but not listed here were prepared according to the method of the third edition of the molecular cloning instructions, and the biochemical reagents were analytically pure or of higher grade.

2.4 Main instruments: PCR amplification instrument (Eppendorf), high-speed centrifuge (Eppendorf 5427R), electrophoresis equipment (six Beijing), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), fluorescence microscope (Olympus BX43), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test box (Saifu) and the like.

Experimental methods and results

1 bioinformatics analysis and cloning of Cotton GH _ D03G1517 Gene

1.1 obtaining gene sequence of GH _ D03G1517 from NCBI, designing Primer by using Primer Premier 5.0 software, amplifying from TM-1 in upland cotton by using PCR (polymerase Chain reaction) method, wherein the open reading frame is 1128bp, 375 amino acids are encoded, the relative molecular weight of protein is 43.018kDa, and the isoelectric point is 5.677. The gene open reading frame sequence is (SEQ ID NO: 1):

ATGGCTGACGTCGCTCCGGGAAACGCCGGCGGTCAATTTGGAGATTTTCCGACGATTCATACACATGGAGGTCAGTTTATTCAGTATAATATTTTTGGAAATTTGTTCGAGGTGACGTCTAAGTATCGGCCTCCGATCATGCCGATCGGTCGTGGAGCCTACGGCATCGTTTGCTCGGTGTTGAATTCGGAGACAAACGAGATGGTTGCGGTAAAGAAAATCGCCAACGCTTTTGATAATCACATGGATGCTAAGCGCACGCTTCGTGAGATTAAACTCCTTCGACATTTGGATCACGAAAACGTTATTGGAATCAAAGATGTGATTCCTCCGCCTTTAAGGAGGGAATTTACTGATGTTTACATTGCGACTGAGCTCATGGATACCGATCTTCACCAAATCATTCGCTCTAATCAGAGTTTATCGGAGGAGCATTGCCAGTATTTCTTGTATCAAATTCTTCGAGGACTGAAGTACATACATTCTGCCAATGTCATTCATAGAGATTTGAAACCCAGCAACCTCTTGCTGAATGCTAATTGTGATCTTAAGATTTGCGACTTTGGTCTCGCTCGGCCTACTGCTGAGAATGAGTTTATGACTGAATATGTTGTCACGAGGTGGTATCGGGCACCGGAGATATTGCTAAACTCTTCAGACTACACCGCTGCCATAGATGTCTGGTCTGTTGGTTGCATCTTCATGGAGCTCATGAATAGGAAGCCTCTGTTTCCAGGCAAAGATCATGTACATCAAATGCGTTTATTAACTGAGCTGCTCGGCACACCAACTGAATCCGATCTTGGATTTCTCCGGAACGAGGATGCAAGGAGATATATCAGGCAGCTCCCAGCACATCCGCGCCAATCACTAGCAGAAGTTTTCCCACATGTTCATCCATTGGCCATTGATCTCATTGACAGAATGTTGACATTTGATCCGACCAGAAGGATTACTGTTGAAGAAGCATTGGCACATCCTTACCTCGAAAGATTACACGACATATCTGATGAACCAGTCTGCCCCGAACCGTTTTCTTTCGACTTTGAGCAGCAACCATTGGGAGAAGAACAGATGAAGGACATGATTTACCAAGAGGCCTTGGCTCTGAATCCAACTTATGCTTAA

the amino acid sequence is (SEQ ID NO: 2):

MADVAPGNAGGQFGDFPTIHTHGGQFIQYNIFGNLFEVTSKYRPPIMPIGRGAYGIVCSVLNSETNEMVAVKKIANAFDNHMDAKRTLREIKLLRHLDHENVIGIKDVIPPPLRREFTDVYIATELMDTDLHQIIRSNQSLSEEHCQYFLYQILRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLARPTAENEFMTEYVVTRWYRAPEILLNSSDYTAAIDVWSVGCIFMELMNRKPLFPGKDHVHQMRLLTELLGTPTESDLGFLRNEDARRYIRQLPAHPRQSLAEVFPHVHPLAIDLIDRMLTFDPTRRITVEEALAHPYLERLHDISDEPVCPEPFSFDFEQQPLGEEQMKDMIYQEALALNPTYA

1.2 the process of cloning genes specifically is:

(1) in the seedling stage of cotton, the young and tender cotton leaves are taken, quickly frozen in liquid nitrogen and stored in a refrigerator of 80 ℃ below zero for later use. The total RNA of the plants is extracted by a TIANGEN reagent kit.

(2) The RNA extraction steps are as follows:

all centrifugation steps below were performed at room temperature.

1) And (3) homogenizing treatment: 100mg of plant leaves were rapidly ground to a powder in liquid nitrogen, 700. mu.L SL (beta-mercaptoethanol added before use) was added and the sample was mixed by shaking vigorously immediately.

2) Centrifuge at 12,000rpm for 2 min.

3) The supernatant was transferred to the filtration column CS and centrifuged at 12,000rpm for 2min, and the supernatant from the collection tube was carefully pipetted into a new RNase-Free centrifuge tube, the tip being kept from touching the cell debris in the collection tube.

4) Adding 0.4 times volume of anhydrous ethanol, mixing, transferring the mixture into adsorption column CR3, centrifuging at 12,000rpm for 15sec, discarding the waste liquid in the collection tube, and returning the adsorption column CR3 to the collection tube.

5) 350. mu.L of deproteinizing solution RW1 was added to the adsorption column CR3, and centrifuged at 12,000rpm for 15sec, thereby discarding the waste liquid in the collection tube and returning the adsorption column CR3 to the collection tube.

6) DNase I working solution: mu.L of DNase I stock and 70. mu.L of RDD solution were gently mixed.

7) 80. mu.L of DNase I working solution was added to CR3 and allowed to stand at room temperature for 15 min.

8) After standing, 350. mu.L of deproteinizing solution RW1 was added to CR3, centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

9) To the adsorption column CR3, 500. mu.L of rinsing solution RW (ethanol was added before use), centrifuged at 12,000rpm for 15sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

10)Step 9 is repeated.

11) Centrifuging at 12,000rpm (13,400 Xg) for 2min, placing adsorption column CR3 into a new RNase-Free centrifuge tube, and adding 30-50 μ L RNase-Free ddH dropwise into the middle part of the adsorption membrane₂O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution. Note that: the volume of elution buffer should not be less than 30. mu.L, and too small a volume affects the recovery efficiency. The RNA samples were stored at-70 ℃. If the expected RNA yield is more than 30. mu.g, the RNA solution obtained by centrifugation in step 11 may be added to an adsorption column CR3, and left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.

To prevent RNase contamination, precautions:

1) the gloves are often replaced with new ones. Because the skin is often bacteria-bearing, RNase contamination may result;

2) the RNase-free plastic product and the gun head are used to avoid cross contamination;

3) RNA is not degraded by RNase while in lysate SL. However, after extraction, plastics and glassware without RNase should be used in the further processing.

4) The preparation solution should use RNase-Free ddH₂O。

(3) And (3) synthesizing cDNA. And (2) carrying out reverse transcription on 500ng of RNA into cDNA by adopting a reverse transcription kit FSQ-201 of Toyobo, wherein a reverse transcription system is as follows:

RT reaction liquid is prepared according to the following components (the reaction liquid is prepared on ice):

the reverse transcription reaction conditions were as follows:

15min at 37 ℃ (reverse transcription reaction),

5s at 98 ℃ (inactivation reaction of reverse transcriptase);

the reverse transcription product cDNA solution was diluted 6-fold as a template for PCR reaction.

(4) PCR reaction system, program and product detection for gene cloning

1) PCR reaction system according to TaKaRa

GXL DNA Polymerase high-fidelity enzyme instruction, and a PCR reaction system is as follows:

2) the PCR amplification procedure was:

the primer sequence is as follows:

upstream primer F

5′-ATGGCTGACGTCGCTCCGGGA-3′(SEQ ID NO:3)

Downstream primer R

5′-TTAAGCATAAGTTGGATTCAG-3′(SEQ ID NO:4)

3) Detection of PCR products

And adding 11 mu L of 6 XLoading Buffer into 1 mu L of PCR product, mixing uniformly, spotting on 1% agarose gel, and carrying out electrophoresis detection.

And (3) cutting and recovering the target fragment by using a gel recovery kit.

The product recovered from the above gum is

The Ultra One Step Cloning Kit is used for constructing a connection T vector and transforming Escherichia coli.

After picking single clone from the resistant LB culture medium overnight at 37 ℃, the culture was carried out with shaking at 37 ℃.

And (3) carrying out PCR verification on bacterial liquid, selecting a positive clone sample, sending the sample to Jinzhi biotechnology limited for sequencing, and adding a certain amount of glycerol into the bacterial liquid with correct sequencing to ensure that the final concentration of the glycerol is about 20 percent and storing the glycerol at-70 ℃.

Construction of 2PBI121-GH _ D03G1517 plant expression vector

The test vector is constructed by

The Ultra One Step Cloning Kit is suitable for connection of any vector and any gene fragment, the reaction time only needs 15min, and the insertion fragment and the linearized vector are required to have 15bp overlapping regions at the 5 'end and the 3' end respectively.

The amplification primers are as follows:

upstream primer F (SEQ ID NO. 5: 5 '-3')

CACGGGGGACTCTAGAATGGCTGACGTCGCTCC

Downstream primer R (SEQ ID NO. 6: 5 '-3')

GATCGGGGAAATTCGAGCTCTTAAGCATAAGTTGGATTCAGAGCCAAGG

The operation procedure is as follows: taking a cloning vector of a target gene GH _ D03G1517 as a template, and purifying an insert after amplification; the obtained insert GH _ D03G1517 and pBI121 linearized vector were arranged in a molar ratio of 2: 1.

And (3) uniformly mixing the solutions, reacting for 10min at 50 ℃, then placing on ice, transforming Trans5 alpha competent cells, picking monoclonals, and carrying out sample sequencing to obtain an overexpression vector containing the correct target gene.

The process uses a plate as kanamycin-resistant LB resistance medium, prepared kanamycin 50mg/mL, diluted 1000 times in use, i.e. 100mL of medium added with 100. mu.L of 50mg/mL kanamycin.

3. Transforming GH _ D03G1517 gene into Arabidopsis by Agrobacterium-mediated method

3.1 transforming agrobacterium tumefaciens GV3101 competent cells by a freeze-thaw method, wherein the specific transformation process is as follows:

(1) adding 1 mu g (2-10 mu L) of constructed target gene overexpression vector plasmid into 100 mu L of agrobacterium tumefaciens GV3101 competent cells, uniformly mixing, and carrying out ice bath for 30 min; quick freezing with liquid nitrogen for 2-3min, and heat-shocking at 37 deg.C for 90 s;

(2) ice-bath for 5min, and adding 800 μ L LB liquid culture medium;

(3) culturing at 190rpm and 28 ℃ for 4h, centrifuging at 4000rpm for 5min, sucking the supernatant until 500 mu L of the residual 400-plus is obtained, repeatedly sucking, uniformly mixing, taking 200 mu L of bacterial liquid, coating the bacterial liquid on a three-antibody screening culture medium containing kanamycin, streptomycin sulfate and rifampicin, culturing at 28 ℃ for about 36-48h, and enabling resistant colonies to be visible;

(4) selecting a single colony, and culturing the single colony in 1mL of LB liquid culture medium containing the three antibiotics for about 16h until the single colony is turbid;

(5) and (3) carrying out colony PCR and enzyme digestion identification, screening out positive agrobacterium tumefaciens strains, and storing 20% glycerol bacterial liquid at the temperature of minus 80 ℃.

3.2 transformation of Arabidopsis thaliana by inflorescence Dipigmentation

(1) Inoculating 20 mu L of agrobacterium tumefaciens liquid stored atminus 80 ℃ to 1mL of LB liquid culture medium, carrying out shaking culture at 28 ℃ and 180rpm overnight, adding 200 mu L of activated bacterium liquid to 20mL of LB liquid culture medium, carrying out shaking culture at 28 ℃ and 180 rpm;

(2) when the OD value of the bacterial liquid is about 1.2-1.6, centrifuging the bacterial liquid at 3000rpm, and collecting thalli;

(3) the formula of the transformation medium is as follows: 5% sucrose, 0.03% silwet L-77(Steven J, 1998);

(4) suspending the cells in the above transformation medium, and adjusting OD₆₀₀Starting padding when the dye value is 0.8;

(5) placing the arabidopsis inflorescence in a transformation medium for 30-50s, wrapping the arabidopsis by using a preservative film after dip dyeing, culturing in the dark for 24h, then culturing under normal conditions, and harvesting seeds after maturation.

4. Phenotypic identification of transgenic Arabidopsis plants

4.1 stress treatment of overexpression and wild Arabidopsis, measurement of physiological parameters, determination of oxidants and antioxidants

Seeds of overexpression T3 generation Arabidopsis lines L2, L6, L8 and wild type were surface-sterilized and planted in a greenhouse by the inventors. After 7 days, the seedlings were transplanted into small pots filled with vermiculite and humus mixed in a ratio of 1: 1. To induce drought stress, water was cut off for 8 and 14 days. At the same time, salt stress was induced using 250mM NaCl solution. For drought stress, plants were re-watered to measure drought resistance. And measuring physiological parameters such as the water content of the relative leaves of the leaves, the water loss of the leaves in vitro, ion leakage, chlorophyll content and the like. The method for measuring the relative water content of the leaves comprises the steps of weighing the leaves of the same part of a treated plant, weighing the leaves, putting the leaves into a culture dish containing tap water, absorbing water for 12h, wiping the surface water of the leaves, weighing the leaves (TW) immediately, putting the leaves into an oven, baking the leaves at 65 ℃ for 12h, weighing the leaves (DW), and calculating the relative water content of the leaves, which is { (FW-DW)/(TW-DW) } × 100. The in vitro leaf water loss measuring method comprises the steps of taking off leaves, weighing Fresh Weight (FW), then placing in a culture room with constant temperature, humidity and illumination intensity for 24 hours, weighing (WW), then uniformly placing the leaves in an oven, baking for 12 hours at 65 ℃, and weighing (DW), wherein the in vitro leaf water loss is (FW-WW)/DW). The ion leakage of the leaf blade takes about 0.1g of cotton leaf blade, the cotton leaf blade is washed by double distilled water for several times, absorbent paper slightly absorbs the water on the surface of the leaf blade, the leaf blade is cut into pieces and placed in 25mL of double distilled water, air is pumped in a vacuum drier for 20min under 0.05MPa, the mixture is slowly oscillated for 2h at 25 ℃, then the conductivity S1 is measured, and simultaneously the blank conductivity C1 of the water is measured. And treating the sample in a boiling water bath for 20min, cooling to room temperature, metering to 25mL to determine the conductivity S2, treating the water in the same manner, and determining the blank conductivity C2. Ion leakage ═ S1-C1)/(S2-C2) × 100%. Measuring chlorophyll content 0.1g of leaves are taken, chlorophyll is extracted by an 80% acetone extraction method, and the chlorophyll content is calculated according to the following formula: c_T＝20.29D₆₄₅+8.05D₆₆₃。

4.2 Germination and root growth tests under drought and salt treatment

For germination rates of transgenic and wild type seeds, the inventors surface-sterilized the seeds and sown them on 1/2MS plates supplemented with PEG (8%, 10%, 15%) and salt (50mM, 75mM and 100mM), respectively. The plates were placed in layers in the dark at 4 ℃ for 2 days, then placed in a growth chamber under controlled conditions of 22 ℃, 70% relative humidity, and 16/8 hour light/dark cycle. Germination was recorded daily for 7 days. Each experiment was performed 3 times for a total of 3 technical replicates, with 50 seeds per plate. In the root growth test, seeds were sterilized and spread on 1/2MS and placed in a growth chamber, then three-day-old seedlings were transferred to 1/2MS plates added with PEG (8%, 10% and 15%) solvent for vertical growth to simulate water deficit under drought stress, three-day-old seedlings were transferred to 1/2MS plates added with salt (50mM, 75mM and 100mM) for vertical growth for salt stress, and after one week, the length of roots was measured.

4.3 expression of stress-responsive genes in WT and GH _ D031517 under drought and salt stress

The inventors selected four stress response genes ABF4, KIN1, RD22 and RAB18 to determine their expression in GH _ D03G1517 overexpressing arabidopsis and wild type under drought and salt stress. RNA was extracted from mature leaves of GH _ D031517 overexpressing arabidopsis thaliana and Wild Type (WT) seedlings cultured under control, drought and salt stress conditions using a Takara extraction kit. RNA was converted to cDNA (TaKaRa, China) using the Prime Script RT kit. qPCR experiments were performed using SYBR-Green Master fluorescent intercalating dyes.

4.4 overexpression of GH _ D03G1517 improves drought and salt tolerance of transformed plants

The relative water content of the wild type and the transgenic line is evaluated by the inventor, and the relative water content in the overexpression line is obviously higher than that of the wild type under drought and salt stress. The over-expressed strains had the same concentration of relative water content. The excised leaf water loss (EWL) of the transgenic lines was significantly lower compared to the wild type. In the wild type, the ion leakage was also higher than in the transgenic lines, indicating that the transgenic lines had higher Cell Membrane Stability (CMS) than the wild type (fig. 1). The high CMS of the GH _ D03G1517 transgenic line indicates that this gene regulates drought and salt stress conditions. Chlorophyll content was the same in the transgenic lines but was significantly reduced in the wild type. This indicates that, in contrast to the transgenic lines, the wild type has a higher level of oxidative stress. Drought treatment for 14 days resulted in extreme wilting of wild type plants compared to the transgenic lines. Under normal conditions, wild type and transgenic lines have similar growth conditions.

In addition, the inventors analyzed the enzymatic activity of the transgenic line and the wild type. Two antioxidants (CAT and POD) and two oxidants (MDA and H) were tested before and after treatment, respectively₂O₂). The antioxidant concentration of the transgenic lines was significantly higher than the wild type (fig. 2). The higher antioxidant level in the transgenic lines indicates that the transgenic lines can greatly reduce the ROS to a normal level under salt and drought stress. Wild-type oxidants (H) under drought and salt stress treatment₂O₂And MDA) concentration, while the oxidant (H) of the transgenic line is significantly increased₂O₂And MDA) concentration was significantly reduced (fig. 2). When plants are in adverse environments such as lack of water and high salt, the cellular functions and different biochemical parameters are significantly changed.

To further investigate the role of GH _ D03G1517 in cotton drought and salt stress, the inventors investigated the expression levels of 4 stress-responsive genes. The four STRESS response genes used are ABRE BINDING FACTOR4(ABF4), STRESS-INDUCED PROTEIN (KIN1), RAS-RELATED PROTEIN (RAB18) and DESCCATION-RESPONSIVE (RD 22). In the control, the expression profiles of the wild type and the transgenic GH _ D03G1517 plants were identical. However, all transgenic lines showed up-regulation after drought and salt treatment compared to the wild type (fig. 3). This indicates that overexpression of GH _ D03G1517 has a beneficial effect on the expression profile of stress response genes. These gene modulations are implicated to play an important role in abiotic stress tolerance in these plants. These genes are considered stress tolerance genes.

4.5 overexpression of GH _ D03G1517 enhances seed germination and root growth to tolerate drought and salt stress

Under the PEG treatment, the germination rates of the transgenic line and the wild type seed have significant difference. The sensitivity of wild type seed germination to PEG is higher than that of the transgenic line. Under the treatment of 8% PEG, the germination rate of the transgenic arabidopsis seeds is 84-85%, and the germination rate of the wild type seeds is 67-69%. The germination rate of wild type seeds was delayed compared to the transgenic lines at 10% and 15% PEG treatment (fig. 4, a-D). To evaluate the root length, the inventors measured the root growth of transgenic lines and wild type under drought stress, and found that there was a large difference in root length between the transgenic lines and the wild type (FIG. 4, F-G).

In the salt stress environment, the germination rates of the wild type and the transgenic line are both obviously reduced, and the germination rate of the transgenic line is inhibited to a lower degree. The germination rate was lower for the wild type, but much higher for the over-expressed lines (FIG. 5, A-D). Under salt stress conditions, the transgenic lines had longer root elongation, while the wild type had shorter root length (FIG. 7, E-F).

4.6 GH _ D03G1517 maintained drought tolerance throughout the growth phase of Arabidopsis lines

In the seedling stage of transgenic arabidopsis, the overexpression of GH _ D03G1517 obviously improves the drought tolerance of arabidopsis. An increase in drought resistance is also observed throughout the entire growth cycle, thus resulting in an increase in seed yield and dry matter. This increase was maintained throughout the growth cycle of the plants and ultimately increased yield of dried seeds (FIG. 6, A-B). With the continuation of drought stress, the plant height, pod length and number of seeds per pod of the transgenic lines were significantly higher than the wild type (fig. 6, C-F). Both GH _ D03G1517 overexpressing arabidopsis and the control wild type showed normal growth. The results show that the over-expression of GH _ D03G1517 in plants improves the tolerance of plants to drought stress and seed yield.

5. Silencing of GH _ D03G1517 in cotton

5.1 the detailed operation process of cotton VIGS is described as follows:

1) construction of the vector: avoiding the conserved domain, based on the CDS sequence of GH _ D03G1517, primer design was performed by Oligo7, linker sequences (Spe I and Asc I) were added, and amplification of the target fragment was performed using TM-1 leaf sample cDNA as a template. And recovering and purifying the amplified product, and then connecting the product with the pCLCrVA vector after enzyme digestion.

Upstream primer F (SEQ ID NO. 7: 5 '-3')

ATGCCTGCAGACTAGTCCGCTGCCATAGATGTCTGGTCT

Upstream primer R (SEQ ID NO. 8: 5 '-3')

AGACCTAGGGGCGCGCCCTGGTTCATCAGATATGTCGTGTAATCTTTCG

2) And (3) transformation: the recombinant vector was transformed into E.coli competent DH5 α, and after cultivation, single clones were picked for sequencing verification. And (3) propagating the positive clones with correct sequences, and then extracting plasmids. The recombinant plasmid is electrically transformed into an agrobacterium competent strain LBA 4404.

3) Preparing plants: the method comprises the steps of selecting plump TM-1 cotton seeds, planting the cotton seeds in an artificial climate chamber, culturing the cotton seeds for about 10 days under the conditions of illumination/darkness growth at 25 ℃ for 16/8h, and carrying out follow-up experiments when the cotton cotyledon is spread and the first true leaf does not appear or the leaf core just appears.

4) Bacterial liquid activation: the strain LBA4404 containing pCLCrVA-GH _ D03G1517, pCLCrVA-PDS, No-load pCLCrVA and pCLCrVB was added to the liquid LB medium containing the three antibodies (kanamycin, rifampicin and streptomycin, 50mg/l), and cultured at 28 ℃ at 180r/min for 14-16 h.

5) Expanding and shaking a bacterial liquid: 100 mu L of the activated bacterial suspension is added into 50mL of liquid LB culture medium containing the three antibiotics, and the mixture is cultured for 16-20h at 28 ℃ and 180r/min, so that the OD600 value of the bacterial suspension is between 1.5 and 2.0 (the bacterial suspension is generally orange yellow). 5000g, centrifuging for 10min, and recovering the thallus.

6) Preparing a transformation medium: the collected cells were suspended in a prepared transformation medium to an OD600 value of about 1.5, and then left to stand at room temperature in the dark for 3 hours or more. The conversion medium formula is as follows:

7) infection: the medium of pCLCrVB and pCLCrVA (no load), pCLCrVA-PDS and pCLCrVA-GH _ D03G1517 are mixed uniformly according to the ratio of 1:1 respectively. The back of the cotton cotyledon was lightly scratched with a sterile syringe needle, and the mixed bacterial solution was aspirated with a 1mL syringe (needle removed) and injected into the cotyledon until the cotyledon was completely infiltrated. The injected seedlings are cultured for 1d in the dark and then cultured in an artificial climate chamber under the conditions of light/dark growth at 22 ℃ for 16/8 h.

8) Observation and sampling: after the positive control plant (pCLCrVA-PDS) shows albino phenotype, DNA extraction is carried out on each plant, and PCR detection is carried out by using primers (VA-F/R and VB-F/R). And transplanting the positive plants into a large pot to be cultured until the positive plants bloom, observing the phenotype of the flower organs of each individual plant, taking anther tissues of each plant, extracting RNA (ribonucleic acid), and detecting the expression change of each gene.

5.2 silencing of GH _ D03G1517 in Cotton increases susceptibility of Cotton to drought

pCLCrVA-PDS showed aalbino phenotype 2 weeks after infecting cotton plants. The albino phenotype is the result of loss of chlorophyll content of the leaves (figure 7). The effect of VIGS on GH _ D03G1517 expression was examined using qRT-PCR. GH _ D03G1517 expression levels were significantly lower in the silencing plants (pCLCrVA-G _ D03G1517) compared to the control plants (pCLCrVA) (fig. 7). These findings indicate that the gene of interest was successfully knocked out in cotton plants. Physiological parameters such as RLWC, ELWL, chlorophyll II content and ion leakage were examined for silenced cotton (pCLCrVA-GH _ D03G1517) and control plants (pCLCrVA). The results show that the RLWC and chlorophyll content of the silenced plants is lower than the control, and the ELWL and ion leakage are higher than the control. Excessive ion leakage was due to loss of cell membrane stability due to stress exposure (figure 7).

The inventors determined two oxidants (MDA and H) for GH _ D03G1517 silenced and pCLCrVA control plants₂O₂) And the enzymatic activity of two antioxidants (CAT and POD). The antioxidant concentrations (MDA and H2O2) of the silenced plants were significantly higher than those of the control plants. The concentration of antioxidants (CAT and POD) in GH _ D03G1517 silenced plants was significantly lower than in control plants (FIG. 8). This indicates that the gene has been successfully repressed. The antioxidant capacity of different cotton varieties measures their tolerance to drought stress (Ullah et al 2017). In addition, the results of virus-induced gene silencing (VIG) indicate that the water loss and drought sensitivity are high in GH _ D031517 silencing cotton strains, and that the gene plays a role in the tolerance of cotton to drought stressAnd (4) acting.

The inventors analyzed the expression levels of cotton Raf kinase (GhRAF4), mitogen-activated protein kinase (GhMEKK12) and DXT/MATE (GhDTX2) in silenced cotton using qRT-PCR. The results show that under stress conditions, expression is down-regulated in GH _ D03G1517 gene-silenced leaf tissue and up-regulated in control plants (fig. 8).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

Application of <120> cotton GH _ D03G1517 gene in promoting drought resistance and salt tolerance

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aagtatcggc ctccgatcat gccgatcggt cgtggagcct acggcatcgt ttgctcggtg 180

ttgaattcgg agacaaacga gatggttgcg gtaaagaaaa tcgccaacgc ttttgataat 240

cacatggatg ctaagcgcac gcttcgtgag attaaactcc ttcgacattt ggatcacgaa 300

aacgttattg gaatcaaaga tgtgattcct ccgcctttaa ggagggaatt tactgatgtt 360

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ttatcggagg agcattgcca gtatttcttg tatcaaattc ttcgaggact gaagtacata 480

cattctgcca atgtcattca tagagatttg aaacccagca acctcttgct gaatgctaat 540

tgtgatctta agatttgcga ctttggtctc gctcggccta ctgctgagaa tgagtttatg 600

actgaatatg ttgtcacgag gtggtatcgg gcaccggaga tattgctaaa ctcttcagac 660

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aagcctctgt ttccaggcaa agatcatgta catcaaatgc gtttattaac tgagctgctc 780

ggcacaccaa ctgaatccga tcttggattt ctccggaacg aggatgcaag gagatatatc 840

aggcagctcc cagcacatcc gcgccaatca ctagcagaag ttttcccaca tgttcatcca 900

ttggccattg atctcattga cagaatgttg acatttgatc cgaccagaag gattactgtt 960

gaagaagcat tggcacatcc ttacctcgaa agattacacg acatatctga tgaaccagtc 1020

tgccccgaac cgttttcttt cgactttgag cagcaaccat tgggagaaga acagatgaag 1080

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<210> 2

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

65 70 75 80

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85 90 95

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115 120 125

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130 135 140

His Cys Gln Tyr Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile

145 150 155 160

His Ser Ala Asn Val Ile His Arg Asp Leu Lys Pro Ser Asn Leu Leu

165 170 175

Leu Asn Ala Asn Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg

180 185 190

Pro Thr Ala Glu Asn Glu Phe Met Thr Glu Tyr Val Val Thr Arg Trp

195 200 205

Tyr Arg Ala Pro Glu Ile Leu Leu Asn Ser Ser Asp Tyr Thr Ala Ala

210 215 220

Ile Asp Val Trp Ser Val Gly Cys Ile Phe Met Glu Leu Met Asn Arg

225 230 235 240

Lys Pro Leu Phe Pro Gly Lys Asp His Val His Gln Met Arg Leu Leu

245 250 255

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Gln Ser Leu Ala Glu Val Phe Pro His Val His Pro Leu Ala Ile Asp

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<400> 4

ttaagcataa gttggattca g 21

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<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacgggggac tctagaatgg ctgacgtcgc tcc 33

<210> 6

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatcggggaa attcgagctc ttaagcataa gttggattca gagccaagg 49

<210> 7

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgcctgcag actagtccgc tgccatagat gtctggtct 39

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<213> Artificial Sequence (Artificial Sequence)

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Claims (9)

Translated fromChinese

1.GH_D03G1517基因在促进植物抗旱和耐盐中的应用，其特征在于，所述GH_D03G1517基因具有SEQ ID NO: 1所示的核苷酸序列。1. the application ofGH_D03G1517 gene in promoting plant drought resistance and salt tolerance, it is characterised in that theGH_D03G1517 gene has the nucleotide sequence shown in SEQ ID NO: 1.2.权利要求1所述基因编码的多肽在促进植物抗旱和耐盐中的应用，其特征在于，所述多肽具有SEQ ID NO: 2所示氨基酸序列。2. The application of the polypeptide encoded by the gene of claim 1 in promoting drought resistance and salt tolerance of plants, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO: 2.3.根据权利要求1所述的应用，其特征在于：在植物中提高GH_D03G1517基因的表达量，以促进植物抗旱和耐盐。3. The application according to claim 1, wherein the expression of theGH_D03G1517 gene is increased in the plant to promote drought resistance and salt tolerance of the plant.4.根据权利要求3所述的应用，其特征在于，所述的在植物中提高GH_D03G1517基因的表达量是通过如下方法实现：提高植物内源GH_D03G1517基因的表达，或在植物中过表达外源GH_D03G1517基因。4. application according to claim 3 is characterized in that, the described expression that improvesGH_D03G1517 gene in plant is realized by following method: improve the expression of plant endogenousGH_D03G1517 gene, or in plant overexpress exogenousGH_D03G1517 gene.5.根据权利要求4所述的应用，其特征在于，所述过表达外源GH_D03G1517基因是指将所述GH_D03G1517基因利用植物表达载体，经农杆菌介导转化到植物中进行过表达。5. The application according to claim 4, wherein the overexpression of the exogenousGH_D03G1517 gene means that theGH_D03G1517 gene is transformed into a plant through Agrobacterium-mediated overexpression using a plant expression vector.6.根据权利要求5所述的应用，其特征在于，所述GH_D03G1517基因通过植物表达载体导入植物细胞、组织或器官。6. The application according to claim 5, wherein theGH_D03G1517 gene is introduced into a plant cell, tissue or organ through a plant expression vector.7.根据权利要求6所述的应用，其特征在于，所述植物表达载体通过一种组成型或诱导型启动子驱动所述GH_D03G1517基因的表达。7. The use according to claim 6, wherein the plant expression vector drives the expression of theGH_D03G1517 gene through a constitutive or inducible promoter.8.根据权利要求7所述的应用，其特征在于，所述组成型启动子是35S启动子。8. The use according to claim 7, wherein the constitutive promoter is a 35S promoter.9.根据权利要求5-8任一所述的应用，其特征在于，所述植物是棉花、玉米、水稻、小麦或拟南芥。9. The application according to any one of claims 5-8, wherein the plant is cotton, corn, rice, wheat or Arabidopsis thaliana.

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