CN118272388A - Application and acquisition method of GhXTH gene in cold stress resistance of cotton - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to an application and an acquisition method of GhXTH gene in cold stress resistance of cotton. The invention analyzes the expression pattern of GhXTH gene in leaf blade after stress treatment for 0,1, 3, 6, 12, 24 and 48 hours at 5594 ℃ of cotton cold sensitive variety YM21 and cotton cold resistant variety H, and the result shows that the expression level of the gene in cold resistant variety is obviously increased compared with YM21 along with the extension of treatment time. To further investigate the effect GhXTH on plant stress resistance, the present invention heterologously expressed GhXTH in Arabidopsis to obtain transgenic plants. Cold stress treatment of wild type and overexpressed Arabidopsis found that the overexpressed strain grew better than the wild type, indicating that GhXTH gene responded to cold stress in cotton.

Description

Application and acquisition method of GhXTH gene in cold stress resistance of cotton

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an application and an acquisition method of GhXTH gene in cold stress resistance of cotton.

Background

Cotton is a globally essential crop that has been grown for thousands of years, providing the textile industry with the greatest proportion of natural fibers, and enjoying the reputation of "platinum". Cotton fibers for the manufacture of textiles, apparel, and other products; cottonseed oil extracted from the seeds of cotton plants is also used in the cooking, cosmetic and other industries. Various extreme environmental stresses may affect the normal growth and physiological metabolism of cotton, resulting in damage and even death of the plant. In recent years, with the gradual migration of cotton planting to northwest inland other areas, the influence of the weather of spring cold autumn frost on cotton is more and more serious. Therefore, identifying the cold-resistant related genes of cotton, clarifying the regulation mechanism thereof and creating excellent germplasm resources of cold-resistant cotton is an important task facing the current genetic improvement of cotton.

Many studies indicate that XTH plays an important role in coping with a variety of abiotic stresses. XTH17 and XTH31 were reported to regulate aluminum toxicity tolerance of arabidopsis roots. XTH31 affects the sensitivity of aluminum by modulating the cell wall xyloglucan content and the ability to bind to aluminum. BES1 acts on the expression of the regulatory genes upstream of XTH19 and XTH23, promoting lateral root development, and thus affecting enhancement of adaptation to salt stress. In addition, in low temperature environment, XTH19 negatively regulates cold tolerance by regulating concentration of xyloglucan, thereby affecting plant cell wall remodeling. XTH22 can control plant growth by modulating cell wall homeostasis under low boron conditions. In leaf mustard, as cadmium concentration in the soil increases gradually from 10-50mg/kg, XTH18, XTH22 and XTH23 are down-regulated, but PME17 and PME14 are up-regulated, which may help maintain cell wall integrity. In summary, the findings suggest that XTH may play a role in structural modification of plant cell walls under stress conditions, enabling improvement of plant structural adaptability and plant stress resistance.

There is no report related to the cold stress resistance of GhXTH gene in cotton in the prior art.

Disclosure of Invention

In order to solve the problems, the invention provides an application and an acquisition method of GhXTH gene in cold stress resistance of cotton, and GhXTH gene responds to cold stress in cotton.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of GhXTH gene in cold stress resistance of cotton.

Preferably, the nucleotide sequence of GhXTH gene is shown as SEQ ID No. 1.

Preferably, the amino acid sequence of GhXTH gene is shown in SEQ ID No. 2.

Preferably, the temperature of the cold stress is-8 ℃ to 4 ℃.

The invention also provides a method for obtaining the cold-resistant arabidopsis plant, which comprises the following steps:

1) The GhXTH gene in the technical proposal is connected into a PBI121 vector to obtain a plant expression vector;

2) Transforming the plant expression vector obtained in the step 1) into escherichia coli to obtain escherichia coli transformed bacteria;

3) Transforming the escherichia coli transformed bacteria obtained in the step 2) into agrobacterium tumefaciens to obtain agrobacterium tumefaciens transformed bacteria;

4) Infecting the arabidopsis inflorescence with the agrobacterium tumefaciens transformation bacteria obtained in the step 3), and harvesting arabidopsis seeds after maturation;

5) And 4) planting the arabidopsis seeds obtained in the step 4), vernalizing for 2d at the temperature of-8-4 ℃, and culturing to obtain cold-resistant arabidopsis plants.

Preferably, the nucleotide sequence of the upstream primer used in the amplification of GhXTH gene in the step 1) is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.

Preferably, the conditions for the connection in the step 1) include: the temperature is 37 ℃ and the time is 30min;

the PBI121 vector was digested with XbaI and SacI.

Preferably, the E.coli of step 2) comprises E.coli DH 5. Alpha.

Preferably, the agrobacterium tumefaciens in the step 3) includes agrobacterium tumefaciens GV3101, and the agrobacterium tumefaciens is transformed by a freeze-thawing method.

Preferably, the number of infections in step 4) is 2, the interval is 7d, and the time for each infection is 60s.

The beneficial effects of the invention are as follows:

The invention analyzes the expression pattern of GhXTH gene in leaf blade after stress treatment for 0, 1, 3, 6, 12, 24 and 48 hours at 5594 ℃ of cotton cold sensitive variety YM21 and cotton cold resistant variety H, and the result shows that the expression level of the gene in cold resistant variety is obviously increased compared with YM21 along with the extension of treatment time. To further investigate the effect GhXTH on plant stress resistance, the present invention heterologously expressed GhXTH in Arabidopsis to obtain transgenic plants. Cold stress treatment of wild type and overexpressed Arabidopsis found that the overexpressed strain grew better than the wild type, indicating that GhXTH gene responded to cold stress in cotton.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 shows the expression pattern of GhXTH after cold treatment at YM21 and H5594 ℃;

FIG. 2 is an analysis of the expression level of GhXTH in overexpressed Arabidopsis;

FIG. 3 is a diagram showing the growth of GhXTH transgenic Arabidopsis and wild type stress treated;

FIG. 4 is a plot of survival statistics of GhXTH transgenic Arabidopsis and wild type stress treatments.

Detailed Description

The invention provides an application of GhXTH gene in cold stress resistance of cotton. In the present invention, the temperature of the cold stress is preferably-8℃to 4 ℃.

In the invention, the nucleotide sequence of GhXTH gene is shown as SEQ ID No.1, and is specifically as follows:

ATGCCCAGCTCTACAATTCCTATCAATCACATAAATTCTCTGTTTTATCTCTGCTCTGCAAAATTATCAAAATACAAAATGATCAGTTTTTCGAGTTTCAAGTTTACCTTGCTTCTGCCTCTTATGGTTGGCTGTCTTATGGCAGCAGCAGCGAGCAACTTCAACAATGATTTTGACATCACATGGGGAGACGGGCGTGGTAAGATTGGCAACAATGGAGAGGTCCTCACTCTCTCCCTCGATAAATCTTCTGGTTCTGGTTTCCAGTCTAAGAATGAGTATCTCTTCGGCAAGATTGATATGCAGCTCAAGCTTGTCCCTGGAAACTCTGCTGGCACTGTCACTGCCTACTATCTTTCTTCGAAAGGATCAACATGGGATGAAATTGATTTCGAGTTCCTTGGAAATCTAAGTGGTGATCCTTACATTCTTCACACAAATGTGTTCAGTCAAGGCAAGGGTAACAGAGAGCAACAATTCTATCTCTGGTTTGACCCAACTGCAGATTTCCACACTTATTCCATCCTCTGGAGCCCCCAAAGTATCATATTCTCTGTGGACGGTACACCCATTAGAGAGTTCAAGAACATGGAGTCAATTGGTGTCCCATTTCCTAAAAATCAGCCGATGAGAATTTACTCTAGCTTGTGGAATGCTGATGACTGGGCTACAAGGGGTGGTCTGGTTAAGACTGACTGGACCCTAGCTCCTTTCACTGCCTCTTACAGGAACTTCAATGCCGATGCCTGTGTGTGGTCCAATGGAGCCTCTTCCTGCAAATCGACTGCTACATCAACAAACAGTGCATGGTTCTCTCAAGAGATGGATTCCGCAAAGCAGCAAAGGTTGCAATGGGTGCAGAAGAACTACATGATCTACAACTACTGCAATGACGCTAACAGATTTCCTCAAGGTCTACCGCCTGAGTGCAGTATGTCTTAA.

In the invention, the amino acid sequence of GhXTH gene is shown as SEQ ID No.2, and is specifically as follows:

MPSSTIPINHINSLFYLCSAKLSKYKMISFSSFKFTLLLPLMVGCLMAAAASNFNNDFDITWGDGRGKIGNNGEVLTLSLDKSSGSGFQSKNEYLFGKIDMQLKLVPGNSAGTVTAYYLSSKGSTWDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPTADFHTYSILWSPQSIIFSVDGTPIREFKNMESIGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWTLAPFTASYRNFNADACVWSNGASSCKSTATSTNSAWFSQEMDSAKQQRLQWVQKNYMIYNYCNDANRFPQGLPPECSMS.

the invention also provides a method for obtaining the cold-resistant arabidopsis plant, which comprises the following steps:

1) The GhXTH gene in the technical proposal is connected into a PBI121 vector to obtain a plant expression vector;

2) Transforming the plant expression vector obtained in the step 1) into escherichia coli to obtain escherichia coli transformed bacteria;

3) Transforming the escherichia coli transformed bacteria obtained in the step 2) into agrobacterium tumefaciens to obtain agrobacterium tumefaciens transformed bacteria;

4) Infecting the arabidopsis inflorescence with the agrobacterium tumefaciens transformation bacteria obtained in the step 3), and harvesting arabidopsis seeds after maturation;

5) And 4) planting the arabidopsis seeds obtained in the step 4), vernalizing for 2d at the temperature of-8-4 ℃, and culturing to obtain cold-resistant arabidopsis plants.

The GhXTH gene in the technical scheme is connected into the PBI121 vector to obtain the plant expression vector.

In the invention, the nucleotide sequence of an upstream primer used for amplifying GhXTH gene is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID No.4, and the specific steps are as follows:

SEQ ID No.3：

CACGGGGGACTCTAGAATGCCCAGCTCTACAATTCCTAT；

SEQ ID No.4：

GATCGGGGAAATTCGAGCTCTTAAGACATACTGCACTCAGGCG。

in the present invention, the conditions of the connection preferably include: the temperature is 37 ℃ and the time is 30min; the PBI121 vector is preferably digested with XbaI and SacI. The method of amplifying GhXTH gene of the present invention is not particularly limited, and those skilled in the art can use the method conventionally. The method for double digestion of the PBI121 vector by the XbaI and SacI is not particularly limited, and conventional methods are adopted.

The plant expression vector obtained by the invention is transformed into escherichia coli to obtain escherichia coli transformed bacteria. In the present invention, the E.coli preferably includes E.coli DH 5. Alpha. The method for transforming the plant expression vector into the escherichia coli is not particularly limited, and a person skilled in the art can adopt a conventional method.

The invention transforms the obtained escherichia coli transformed bacteria into agrobacterium tumefaciens to obtain agrobacterium tumefaciens transformed bacteria. In the present invention, the agrobacterium tumefaciens preferably includes agrobacterium tumefaciens GV3101, and the agrobacterium tumefaciens is transformed by a freeze-thawing method. The method for transforming the escherichia coli transformed bacteria into the agrobacterium tumefaciens is not particularly limited, and a conventional method is adopted.

The invention infects the arabidopsis inflorescence with the obtained agrobacterium tumefaciens transformation bacteria, and the arabidopsis seeds are harvested after the arabidopsis inflorescence is mature. In the present invention, the number of infections is preferably 2, the interval is preferably 7d, and the time per infection is preferably 60s. The method for infecting the arabidopsis inflorescence by the agrobacterium tumefaciens transformation bacteria is not particularly limited, and can be carried out by a person skilled in the art according to the routine.

The invention cultures the obtained arabidopsis seeds after planting for vernalization for 2d at the temperature of-8 ℃ to 4 ℃ to obtain the cold-resistant arabidopsis plants. The method of the present invention is not particularly limited, and conventional methods for culturing Arabidopsis thaliana may be used.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.

Example 1

1. Test materials

1.1 Cotton Material

The cotton materials selected in this example were cotton cold sensitive variety Hemsleya 21 and cotton cold resistant variety H559, where Hemsleya 21 and H559 were different in cold resistance (Table 1), were planted in cotton culture room of cotton institute of national academy of agricultural sciences, and were managed under normal greenhouse conditions of 25℃in daytime and 22℃at night, and were subjected to light/dark cycle for 16 hours/8 hours with relative humidity of 60-70%. The cold treatment condition is that the temperature is 4 ℃ in daytime and 4 ℃ at night, the light/dark cycle is 16 hours/8 hours, and the relative humidity is 60-70%. The sampling mode is that the leaves of two cotton varieties are subjected to cold treatment for 0h, 1h, 3h, 6h, 12h and 24h, and the leaves are placed at-80 ℃ for retention before extracting sample RNA in liquid nitrogen.

TABLE 1 Cold sensitivity cotton variety Hemsleya 21 and Cotton Cold tolerance variety H559 Cold damage index

Material name	Cold injury index CI (%)
		H559	4.47％
Yu cotton 21Yumian and 21	43.17％

1.2 Reagents and consumables

Restriction endonuclease, modification enzyme, related enzyme of PCR reaction system, homologous recombination enzyme, gel recovery kit, cloning kit, plasmid small extraction kit are purchased from Novamat biotechnology Co., ltd, fluorescent quantitative kit is purchased from century biotechnology Co., ltd, and RNA extraction kit is purchased from Beijing-day root biochemical technology Co., ltd

Other drugs: agarose is spanish original product, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are domestic analytically pure, kanamycin, etc. Soilebao biological Co., ltd, and E.coli competent cells DH5 alpha and Agrobacterium competent cells are purchased from Optimago biological Co., ltd

Culture medium: LB liquid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L; 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, and fixing the volume to 1L; LB selection Medium: before LB plate paving, adding antibiotics with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 ℃, shaking uniformly, and then paving the plate; 1/2MS solid medium: 1/2MS22g/L, agar powder (agar powder) 8g/L, sucrose (sucrose) 30g/L.

The main instrument is as follows: PCR amplification apparatus (BIO-RAD), high-speed centrifuge (HettichMIKRO R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR apparatus (ABI 7500), electrothermal constant temperature incubator (Shanghai Simpson), constant temperature culture oscillator (Shanghai Zhi Cheng), artificial climate test box (Saifu), artificial climate chamber.

2. Test methods and results

2.1 Gene cloning and sequence analysis

The CDS sequence and the encoded amino acid sequence of the GhXTH (Gh_D03G0483) gene, whose CDS sequence is 942bp, encoding 313 amino acids, were obtained from CottonFGD (http:// www.cottonfgd.org /), named GhXTH, and their functions were studied.

GhXTH22 CDS sequence (SEQ ID No. 1):

ATGCCCAGCTCTACAATTCCTATCAATCACATAAATTCTCTGTTTTATCTCTGCTCTGCAAAATTATCAAAATACAAAATGATCAGTTTTTCGAGTTTCAAGTTTACCTTGCTTCTGCCTCTTATGGTTGGCTGTCTTATGGCAGCAGCAGCGAGCAACTTCAACAATGATTTTGACATCACATGGGGAGACGGGCGTGGTAAGATTGGCAACAATGGAGAGGTCCTCACTCTCTCCCTCGATAAATCTTCTGGTTCTGGTTTCCAGTCTAAGAATGAGTATCTCTTCGGCAAGATTGATATGCAG CTCAAGCTTGTCCCTGGAAACTCTGCTGGCACTGTCACTGCCTACTATCTTTCTTCGAAAGGATCAACATGGGATGAAATTGATTTCGAGTTCCTTGGAAATCTAAGTGGTGATCCTTACATTCTTCACACAAATGTGTTCAGTCAAGGCAAGGGTAACAGAGAGCAACAATTCTATCTCTGGTTTGACCCAACTGCAGATTTCCACACTTATTCCATCCTCTGGAGCCCCCAAAGTATCATATTCTCTGTGGACGGTACACCCATTAGAGAGTTCAAGAACATGGAGTCAATTGGTGTCCCATTTCCTAAAAATCAGCCGATGAGAATTTACTCTAGCTTGTGGAATGCTGATGACTGGGCTACAAGGGGTGGTCTGGTTAAGACTGACTGGACCCTAGCTCCTTTCACTGCCTCTTACAGGAACTTCAATGCCGATGCCTGTGTGTGGTCCAATGGAGCCTCTTCCTGCAAATCGACTGCTACATCAACAAACAGTGCATGGTTCTCTCAAGAGATGGATTCCGCAAAGCAGCAAAGGTTGCAATGGGTGCAGAAGAACTACATGATCTACAACTACTGCAATGACGCTAACAGATTTCCTCAAGGTCTACCGCCTGAGTGCAGTATGTCTTAA.

GhXTH the amino acid sequence encoded by GhXTH is SEQ ID No.2:

MPSSTIPINHINSLFYLCSAKLSKYKMISFSSFKFTLLLPLMVGCLMAAAASNFNNDFDITWGDGRGKIGNNGEVLTLSLDKSSGSGFQSKNEYLFGKIDMQLKLVPGNSAGTVTAYYLSSKGSTWDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPTADFHTYSILWSPQSIIFSVDGTPIREFKNMESIGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWTLAPFTASYRNFNADACVWSNGASSCKSTATSTNSAWFSQEMDSAKQQRLQWVQKNYMIYNYCNDANRFPQGLPPECSMS.

2.2 Pattern analysis

In order to further study whether the expression patterns of the gene in cold-resistant and cold-sensitive varieties are different, cotton cold-sensitive varieties, namely Yu cotton 21 and cotton cold-resistant varieties H559, are selected, leaf RNA of cold treatment for 0H, 1H, 3H, 6H, 12H and 24H is extracted, and the expression pattern analysis is carried out on GhXTH by using qRT-PCR technology.

2.2.1 Sampling and grinding

The cold sensitive variety Hei 21 and the cold resistant variety H559 were selected, and the leaves were cold treated for 0H, 1H, 3H, 6H, 12H, 24H in liquid nitrogen, ground to a powder using a mortar and pestle, and about 1g of the sample was placed in a 1.5ML centrifuge tube.

2.2.2 Extraction

RNA extraction was performed using kit FastPure Universal PlantTotal RNAIsolationKit (novalun, south kyo, china), comprising the following steps:

(1) The experiment was performed at normal temperature, 600. Mu.l Buffer PSL (polyphenol polysaccharide plant) was immediately added to a centrifuge tube with plant tissue, vigorously vortexed and oscillated for 30sec, the sample was thoroughly and uniformly mixed with the lysate, centrifuged at 12,000rpm (134,00 ×g) for 5min, and immediately followed.

(2) About 500. Mu.l to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III)

Has been placed in a collection tube), centrifuged at 12,000rpm (13,400 Xg) for 30sec, and FastPure gDNA-Filter Columns III was discarded and the filtrate was collected.

(3) Anhydrous ethanol (about 250 μl, adjusted according to the actual condition of the supernatant) was added to the collection tube in an amount of 0.5 times the volume of the filtrate, and mixed by shaking for 15sec. The above mixture was transferred to FastPure RNA Columns V (FastPure RNAColumns V was placed in a collection tube, centrifuged at 12,000rpm (13,400 Xg) for 30sec, and the filtrate was discarded.

(4) To FastPure RNA Columns V, 700. Mu.l Buffer RWA was added, and the mixture was centrifuged at 12,000rpm (13,400 Xg) for 30sec, and the filtrate was discarded.

(5) To FastPure RNA Columns V was added 500. Mu.l Buffer RWB (please check if 48ml absolute ethanol had been added before use), centrifuged at 12,000rpm (13,400 Xg) for 30sec, and the filtrate was discarded.

(6) And (6) repeating the step 6.

(7) FastPure RNA Columns V was placed back in the collection tube and centrifuged at 12,000rpm (13,400 Xg) for 2min.

(8) FastPure RNA Columns V was transferred to a fresh RNase-free Collection Tubes 1.5.5 ml centrifuge tube, 30-100. Mu.l of RNase-free ddH2O was suspended in the center of the column, and centrifuged at 12,000rpm (13,400 Xg) for 1min.

The elution volume of (2) is not less than 30. Mu.l, and too small a volume may affect the recovery efficiency of nucleic acid.

Can help to increase the concentration of RNA products by: dropwise adding RNase-free ddH2O, and standing at room temperature for 5min; and (3) adding the first eluent into the adsorption column again for eluting.

(9) The extracted RNA can be directly used for downstream experiments or stored at-85 to-65 ℃.

2.2.3 Synthesis of reverse transcribed cDNA

Kit for synthesis and utilization of reverse transcription cDNAIIQ RT SuperMix for qPCR (+ GDNA WIPER) (Norvain, nanjing, china) can be divided into two parts, removal of genomic gDNA and reverse transcription of RNA, the reaction is carried out on ice, and the reaction steps are as follows:

(1) Removal of genomic gDNA

TABLE 2 reaction system configuration

Reagent(s)	Dosage of
		RNase-freeddH2O	to16μl
4×gDNAwiperMix	4μl
		Template RNA	1pg-1μg

Gently beating and mixing by a pipette. 42 ℃ for 2min.

(2) Preparation of reverse transcription reaction System

TABLE 3 reaction system

Reagent(s)	Dosage of
		Reaction solution of the first step	16μl
5×HiScriptIIqRTSuperMixII	4.0μl

The mixture was gently stirred with a pipette, and 20. Mu.l of the mixture was placed in a PCR apparatus at 50℃for 15min and 85℃for 5sec. The product can be used immediately for qPCR reactions or stored at-20 ℃ and used within half a year.

2.2.4 Fluorescent quantitative PCR

(1) Specific primers of GhXTH gene were designed by Oligo 7 software, and cotton GhActin gene was used as reference gene.

TABLE 4 Gene sequence

(2) Fluorescent quantitative PCR

This was done using the Cwbio (China) UltraSYBR Mixture (Low ROX) kit and the Applied Biosystems 7500 instrument. The specific process is as follows:

1) Diluting the cDNA stock solution by 5 times;

2) Configuration of the reaction system (on ice operation):

Table 5 System

Reagent(s)	Dosage of
		2×UltraSYBRMixture	10.0μl
PCRForwardPrimer(10μM)	0.4μl
		PCRReversePrimer(10μM)	0.4μl
CDNA template (diluted working solution)	2.0μl
		DH 2 O (sterile distilled water)	upto20μl

Mixing the prepared system uniformly, centrifuging until no bubble exists, and then carrying out fluorescence quantitative PCR by Applied Biosystems 7500: the PCR procedure was set up according to the two-step method: pre-denaturation: 95 ℃ for 2min;95 ℃ for 5s; at 60 ℃,34s (fluorescence signal is collected in this step), 40 cycles are set in these two steps; and finally, analyzing a dissolution curve: 95 ℃ for 15s;60 ℃ for 20s;95℃for 15s. The data were processed using Microsoft Excel 2019 software to calculate gene expression levels and Origin 2022 software was plotted.

2.2.5GhXTH22 quantitative analysis

The relative expression amounts of GhXTH to different cold treatment times of two materials of cotton cold sensitive variety Hei 21 and cotton cold resistant variety H559 were calculated by using a method of 2 ^-△△Ct. As can be seen from FIG. 1, after stress treatment for 0H, 1H, 3H, 6H, 12H and 24H at 5594℃in cotton cold sensitive variety YM21 and cotton cold resistant variety H, the expression level of GhXTH in H559 leaves increased significantly with the increase of the treatment time compared with YM21, and the expression level reached the highest at 12H treatment, indicating that the gene may be related to cotton cold resistance.

Cloning of the 3GhXTH2 Gene and construction of the plant expression vector of PBI121-GhXTH22

2.3.1GhXTH22 Gene cloning primer design

The full length of the CDS sequence GhXTH was ligated to the PBI121 vector to construct a 35S promoter vector. Primers containing appropriate cleavage sites were designed at the start codon and stop codon, respectively. The cleavage sites used for the PBI121 vector were XbaI and SacI.

GhXTH22 the primer sequences of the cleavage site are as follows:

TABLE 6 primer sequences

2.3.2PCR cloning GhXTH Gene

(1) PCR reaction system

According to PRIMESTAR GXL DNA polymerase instructions, the PCR reaction system is as follows:

Table 7 System

Reagent name	Dosage of reagent
		5×PrimeSTARGXLBuffer	10μl
dNTPMixture	4μl
		Primer F (10. Mu.M)	2μl
Primer R (10. Mu.M)	2μl
		TM-1cDNA	2μl
ddH2O	Upto50μl

(2) PCR reaction procedure:

(3) Detection of PCR products

Mu.l of the PCR product was taken, 3. Mu.l of 6X Loading Buffer was added, mixed well, spotted on a 1% agarose gel and the size of the band was checked by electrophoresis to determine if it was about 873 bp.

(4) PCR product purification

Using the product purification kit (Vazyme, DC 301), the procedure was as follows:

1) Rapidly cutting gel containing target DNA fragment under ultraviolet lamp, weighing gel, wherein 100mg gel is equivalent to 100 μl volume, and taking the gel volume as one gel volume;

2) An equal volume of Buffer GDP was added. Water bath at 50-55 deg.c until the gel is dissolved completely;

3) The adsorption column was placed in a collection tube, and 700. Mu.l or less of the sol was transferred to the adsorption column, and centrifuged at 12,000Xg for 30-60sec.

4) The filtrate was discarded and the column was placed in a collection tube. Add 300 μl Buffer GDP to the column. Standing for 1min. Centrifuge at 12,000Xg for 30-60sec.

5) The filtrate was discarded and the column was placed in a collection tube. 700 μl Buffer GW (absolute ethanol added) was added to the column. Centrifuge at 12,000Xg for 30-60sec.

6) And (5) repeating the step 5.

7) The filtrate was discarded and the column was placed in a collection tube. Centrifuge at 12,000Xg for 2min.

8) The column was placed in a 1.5ml centrifuge tube, 20-30. Mu.l of sterilized water was added to the center of the column, and the column was left for 2min. Centrifuge at 12,000Xg for 1min. The column was discarded and the DNA was stored at-20 ℃.

Construction of 2.3.3PBI121-GhXTH plant expression vector

(1) Double enzyme digestion and glue recovery of PBI121 plasmid

The PBI121 plasmid was digested with XbaI and SacI, and the digested product of the PBI121 vector was purified by agarose gel electrophoresis. The enzyme digestion reaction system is as follows:

Table 8 System

Reagent name	Dosage of reagent
		XbaI	1μl
SacI	1μl
		CutSmart	5μl
PBI121 plasmid	1μg
		ddH2O	Upto50μl

(2) Ligation of PCR gel recovery product and restriction enzyme digestion PBI121 plasmid

Use of Vazyme homologous recombinase reagentsIIOne Step Cloning Kit carrying out a ligation reaction:

Table 9 System

Reagent name	Dosage of reagent
		5XCEⅡBuffer	2μl
ExnaseⅡ	1μl
		PBI121 double enzyme cutting carrier	25～100ng
PCR fragment	10～100ng
		ddH2O	Upto10μl

After the system is completed, the components are blown and evenly mixed, and the reaction is carried out for 30min at 37 ℃.

(3) Ligation product transformation of E.coli

1) Adding 100ul of escherichia coli DH5 alpha competent ligation reaction system, adding ligation reaction product into the ligation reaction system, and ice-bathing for 25min;

2) Heat shock in 42 ℃ water bath for 45s;

3) Ice bath for 2min; 700ul of non-resistant LB liquid medium is added, and the mixture is incubated for 1h at 37 ℃ and 200 rpm;

4) Centrifuging at 5000rpm for 1min, leaving about 100ul of supernatant, mixing, and coating on LB plate containing kana resistance;

5) Culturing at 37 deg.C overnight;

(4) Detection and sequencing of Positive clones

1) Selecting a monoclonal from the transformation plate, putting the monoclonal into a liquid LB culture medium containing Kan, and carrying out shaking culture at a constant temperature of 37 ℃ for 8 hours;

2) Colony PCR verifies positive clones and sends the correct monoclonal to Shang Ya biotechnology company for sequencing, 3 replicates per sequence.

(5) Preservation of positive bacterial liquid

And (3) performing PCR verification on bacterial liquid, and adding glycerol into bacterial liquid with correct sequencing until the final concentration is more than 20%, and preserving at-80 ℃. The correctly sequenced plasmid was returned for transformation of Agrobacterium.

(6) Transformation of Agrobacterium

Transformation of Agrobacterium tumefaciens GV3101 competent cells by freeze thawing:

1) The agrobacterium is thawed at-80 ℃ and the ice water is inserted into the ice in a mixed state.

2) Mu.l of competent plasmid DNA was added to 1. Mu.g, and the mixture was stirred by hand to the bottom of the tube, and then allowed to stand on ice for 5min, liquid nitrogen for 5min, and ice-bath for 5min at 37 ℃.

3) Adding 700ul of non-resistant LB liquid medium, and shake culturing at 28deg.C for 2-3 hr

4) 100-150Ul of bacterial liquid is placed on an LB plate containing kana and rifampicin, and is placed in an incubator at 28 ℃ for 2-3 days in an inverted manner.

5) Positive clones are selected, cultured for 48 hours at 28 ℃ on LB liquid culture medium with resistance, and the bacterial liquid is preserved for standby at-80 ℃ after the bacterial liquid with correct strips is verified by PCR and glycerol is added.

2.3.4 Agrobacterium-mediated transformation of Arabidopsis thaliana

(1) Arabidopsis thaliana culture

And planting Columbia wild type Arabidopsis thaliana in a phytotron, growing to a full bloom stage, and cutting off pod which is already fruiting.

(2) Transformation of Arabidopsis inflorescence infection

1) Activating bacterial liquid: inoculating 20 μl of Agrobacterium solution stored at-80deg.C into 1ml LB liquid culture medium (corresponding antibiotics: kanamicin and rifampin are added), culturing at 28deg.C and 180rpm for 14-18 hr;

2) Expanding and shaking: adding 500 μl of activated bacterial liquid into 50ml of LB liquid culture medium containing corresponding antibiotics, culturing at 28deg.C and 180rpm until OD ₆₀₀ value of bacterial liquid is about 0.8-1.2 (about 18-20 h), centrifuging at 5000rpm for 10min, discarding supernatant, and collecting thallus;

3) Preparation of infection transformation medium: 1/2MS halving, 5% sucrose, 0.02% Silwet L-77, pH adjusted to 5.6-5.7 with NaOH, 0.1mM AS (acetosyringone);

4) Resuspension of the thalli with a transformation medium, and adjusting the OD ₆₀₀ to 0.6-0.8;

5) Dip dyeing: placing the arabidopsis inflorescence into a transformation medium for 60s, and culturing for 24h under the condition of weak light or light shading after dip dyeing;

6) Placing the treated arabidopsis thaliana under normal conditions for culture, and carrying out secondary infection by using the same method after one week;

7) After the arabidopsis seeds are ripened, the arabidopsis seeds are harvested to obtain transgenic T ₀ generation seeds.

2.3.5 Cold tolerance identification of transgenic Arabidopsis plants

(1) The harvested seeds are planted on 1/2MS containing kanamycin after sterilization, vernalization is carried out at 4 ℃ for 2 days, the seeds are transferred into a manual climate test box, positive plants grow normally about 10 days, and negative plant leaves turn yellow and do not grow any more.

(2) Transplanting the positive arabidopsis plants into nutrient soil, extracting DNA after growing for one month, and detecting the positive plants by PCR, wherein the primers used in the detection are as follows:

TABLE 10 primer sequences

Primer name	Primer sequence (5 'to 3')
		35S	SEQ ID No.9：GACGCACAATCCCACTATCC
GhXTH22-R	SEQ ID No.10：TTAAGACATACTGCACTCAGGCG

(3) And breeding to T ₃ generation to obtain homozygous transgenic arabidopsis strain.

(4) The T ₃ generation plant and the wild type plant (WT) are planted in nutrient soil, the arabidopsis seedling grows out of true leaves about 10 days, then the arabidopsis seedling moves into a flowerpot to grow, and the planting and the cultivation are carried out under the same condition, and the expression quantity of the GhXTH gene in the GhXTH22 transgenic plant is found to be obviously higher than that of the wild type plant by using a qRT-PCR technology (figure 2). The results indicate that GhXTH22 was successfully overexpressed in Arabidopsis.

(5) We selected GhXTH transgenic Arabidopsis thaliana and WT with consistent growth conditions for 25-30d for cold tolerance testing. In the control group, the phenotype of WT and transgenic lines was identical, but in the experimental group, after treatment at 4℃for 12-18h and-8℃for 2.5-3h and recovery in a 22℃incubator for 7d, the transgenic lines somewhat recovered green, new leaves developed, continued bolting growth, while the wild type of Arabidopsis was almost completely withered and yellow (FIG. 3). After cold treatment, the survival rates of the three GhXTH22 over-expressed lines were 86.4%, 75.9% and 77.7%, respectively, with the survival rate of the wild-type plants being only 37.0% (fig. 4).

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. Application of GhXTH2 gene in cotton cold stress resistance.
2. 2. The use according to claim 1, wherein the nucleotide sequence of GhXTH gene is shown as SEQ ID No. 1.
3. 3. The use according to claim 1 or 2, wherein the GhXTH gene has the amino acid sequence shown in SEQ ID No. 2.
4. 4. The use according to claim 1, wherein the temperature of the cold stress is-8 ℃ to 4 ℃.
5. 5. The method for obtaining the cold-resistant arabidopsis plant is characterized by comprising the following steps of:

   1) Ligating the GhXTH gene of claim 1 into a PBI121 vector to obtain a plant expression vector;

   2) Transforming the plant expression vector obtained in the step 1) into escherichia coli to obtain escherichia coli transformed bacteria;

   3) Transforming the escherichia coli transformed bacteria obtained in the step 2) into agrobacterium tumefaciens to obtain agrobacterium tumefaciens transformed bacteria;

   4) Infecting the arabidopsis inflorescence with the agrobacterium tumefaciens transformation bacteria obtained in the step 3), and harvesting arabidopsis seeds after maturation;

   5) And 4) planting the arabidopsis seeds obtained in the step 4), vernalizing for 2d at the temperature of-8-4 ℃, and culturing to obtain cold-resistant arabidopsis plants.
6. 6. The method according to claim 5, wherein the upstream primer used in the amplification of GhXTH gene in step 1) has a nucleotide sequence shown in SEQ ID No.3 and the downstream primer has a nucleotide sequence shown in SEQ ID No. 4.
7. 7. The method according to claim 5, wherein the conditions of the step 1) connection include: the temperature is 37 ℃ and the time is 30min;

   the PBI121 vector was digested with XbaI and SacI.
8. 8. The method according to claim 1, wherein the E.coli of step 2) comprises E.coli DH 5. Alpha.
9. 9. The method according to claim 1, wherein the step 3) of transforming the agrobacterium tumefaciens includes agrobacterium tumefaciens GV3101 by freeze thawing.
10. 10. The method of claim 1, wherein the number of infections in step 4) is 2, the interval is 7d and the time per infection is 60s.