CN107723301A - A kind of cotton anti-drought gene and its expression vector and application - Google Patents

Abstract

The invention discloses a cotton drought-resistant gene GhAPX2, the base sequence of which is shown in SEQ ID NO:1. The inventor cloned the ascorbic acid peroxidase gene GhAPX2 for the first time, and verified the use of the GhAPX2 gene in enhancing the drought resistance of cotton, providing important theoretical support for cotton drought-resistant breeding. In addition, using the method for enhancing the drought resistance of cotton of the present invention can effectively enhance the drought resistance of the target cotton, is simple and convenient, has good repeatability, has no effect on the growth and development of cotton, and is suitable for popularization and application.

Description

A kind of cotton drought resistance gene and its expression vector and application

technical field

The present invention relates to the field of genetic engineering. Specifically, the present invention relates to the field of cotton genetic engineering. More specifically, the present invention relates to a cotton drought-resistant gene and its expression vector and application.

Background technique

Since the 20th century, with the rapid increase of the global population, the demand for water resources has increased rapidly, but water resources are limited. At the same time, the rapid development of industrialization has led to the reduction of a large amount of arable land, and the further drought cycle has become shorter and shorter. , the area of damage is also increasing, which poses a serious threat to agricultural production, and the output of crops has dropped significantly due to water shortage. Therefore, how to explore the best way to solve the drought problem is a major test for the sustainable development of agriculture in the world. About 1/3 of the world's arable land currently has insufficient water supply. 36% of the world's total land area is arid and semi-arid, and this proportion is even as high as 43% of the available arable land. In China, arid and semi-arid areas are mainly distributed in Inner Mongolia, Gansu and other northwest inland areas, accounting for about 1/2 of the country's land area. In recent years, the impact of drought on crops ranks first among various abiotic stresses.

Cotton is a plant of Malvaceae Malvaceae, which is an important oilseed and economic crop. As the main source of natural fiber, it has a wide distribution in the world, and more than 80 countries are planting cotton. Cotton is divided into 4 subgenera and 51 species in plant taxonomy, including 46 diploid cotton species and 5 tetraploid cotton species. Diploid cotton species include Asian cotton (G. arboreum) and grass cotton (G.herbaceum L), tetraploid cotton includes sea island cotton (G.barbadense) and upland cotton (G.hirsutum) [9]. Among them, almost all of the world's cotton production comes from upland cotton, which accounts for more than 90%, while sea-island cotton only provides about 5% of the production. Therefore, as a country with a large population, the consumption of upland cotton is particularly huge. It is an indispensable and important strategic material and plays an irreplaceable role in the fields of national life, medical security, and science and technology.

Xinjiang is currently the largest base of cotton production in my country, accounting for about 50% of the country's total cotton production. However, the problem of water shortage has become increasingly severe, restricting the agricultural development of this region. To alleviate the emergency situation of water shortage in Xinjiang, it is necessary to invest heavily in the development of water-saving irrigation technology for farmland. At the same time, it is imperative to cultivate and create new drought-resistant varieties and improve the drought-resistant level of crops. Therefore, excavating the existing cotton germplasm resources with excellent drought resistance and using them as research objects for cross-breeding or molecular breeding is a shortcut to solve the drought hazard, which makes it of great significance to carry out research on the drought-resistance of cotton and the genetic mechanism of drought-resistance.

Contents of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. Therefore, an object of the present invention is to provide a cotton ascorbic acid peroxide (APX2) gene with drought resistance function.

The present invention is accomplished based on the inventor's following findings: plant ascorbic acid peroxidase (APX) is an important enzyme class for scavenging oxygen free radicals (ROS) in plants, and plant cytoplasmic APX (cAPX) is extremely active in the APX subfamily. The highest degree of environmental tolerance is the key enzyme in the survival of plants in adversity. So far, the discovered APX family consists of four subfamilies: cytoplasmic APX (cAPX), whose main enzyme is APX1; chloroplast APX, including stroma-type (sAPX) and thylakoid-type (tAPX); mitochondrial APX (mitAPX); Body APX (mAPX). Many studies have shown that this enzyme plays an important role in the process of plant stress resistance. At present, the existence of APX enzyme has been found in various plants, such as spinach, jujube, woad, sugarcane, litchi and so on. According to previous studies, there have been many reports on the stress resistance of cytoplasmic APX1 gene in rice, jujube, pea, and cotton, but there are few studies on APX2 gene, which is also a cytoplasmic ascorbic acid peroxide.

According to the previous research, the inventors used protein two-dimensional electrophoresis technology to detect a significant increase in the expression of APX enzyme in the differential expression of cotton root proteins under drought stress at the seedling stage of cotton. After identification by mass spectrometry, it was confirmed that the enzyme belonged to the cytoplasmic ascorbic acid hyperactivity of upland cotton. Oxidases, the gene belongs to cytoplasmic APX, which is named GhAPX2, and its base sequence is shown in SEQ ID NO:1.

The inventors also found that the APX enzyme activity can actively respond to the water deficit environment through the water control test of the cotton material at the flowering and boll stage, and measured the photosynthetic physiological indicators under drought stress, and its sensitivity to drought is higher than that of proline and malondialdehyde. APX enzymes are considered to play an important role in the drought resistance of cotton.

In another aspect of the present invention, the cotton drought resistance gene plant expression vector pCAMBIA1304-GhAPX2 is obtained by digesting the fragment shown in SEQ ID NO: 1 into the vector pCAMBIA1304.

Transforming the expression vector into a cotton variety without the GhAPX2 gene through the method of Agrobacterium infection can improve the ability of the cotton variety to resist drought.

The cotton with the drought resistance gene GhAPX2 is used as the male parent or female parent to cross the female parent or male parent without the drought resistance gene; the offspring are screened for drought resistance, and new cotton varieties with drought resistance characteristics can be obtained.

According to the embodiments of the present invention, the expression of GhAPX2 gene can enhance the drought resistance performance of cotton.

The inventor cloned the ascorbic acid peroxidase gene GhAPX2 for the first time, and verified the use of the GhAPX2 gene in enhancing the drought resistance of cotton, providing important theoretical support for cotton drought-resistant breeding. In addition, using the method for enhancing the drought resistance of cotton of the present invention can effectively enhance the drought resistance of the target cotton, is simple and convenient, has good repeatability, has no effect on the growth and development of cotton, and is suitable for popularization and application.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 shows the RNA agarose electrophoresis figure of 36 in Xinlu in one embodiment of the present invention;

Fig. 2 shows the partial positive clone electrophoresis in one embodiment of the present invention;

Figure 3 shows the nucleic acid sequence and deduced amino acid sequence of the coding region of GhAPX2 in one embodiment of the present invention;

Fig. 4 shows the ProtScale prediction APX2 hydrophobicity detection figure in one embodiment of the present invention;

Figure 5 shows a graph of the prediction results of the transmembrane region in one embodiment of the present invention;

Figure 6a shows the expression pattern diagram of transgenic positive plants at the stage of 3 true leaves in one embodiment of the present invention;

Figure 6b shows the expression pattern diagram of non-transgenic plants at the stage of 3 true leaves in one embodiment of the present invention;

Figure 7a shows a pattern of cotton fiber expression in transgenic positive plants in one embodiment of the present invention;

Figure 7b shows the pattern of cotton fiber expression in non-transgenic plants in an embodiment of the present invention.

detailed description

The following specific implementation is to describe the present invention in detail, and the embodiments described below are exemplary, and are only used to explain the present invention, but should not be construed as limiting the present invention.

Unless otherwise stated below, the unspecified reagents and instruments involved in the following examples can all come from conventional commercially available products.

Example 1

Cloning and Bioinformatics Analysis of Cotton Drought Resistance Gene GhAPX2

1. Materials

The strong drought-resistant variety "Xinluzhong 36" was used as the test material (all provided by the Crop Genetics and Breeding Laboratory of Xinjiang Agricultural University). When 4-5 true leaves are cultivated at the seedling stage by hydroponics, cut the leaves and put them into sealed bags, mark them well, freeze them with liquid nitrogen for 10 minutes, and store them in a -80°C refrigerator for later use.

2. Methods and results

2.1 Primer design

The mRNA sequence of ascorbic acid peroxidase cAPX2 in upland cotton was used as the reference sequence (accession number gi|211906475 in Gene Bank). The amino acid sequence of cotton APX2 obtained from the previous mass spectrometry analysis obtained the mRNA sequence of this amino acid. The full length of the mRNA sequence is 1123bp, and the CDS of the coding region is located at 506bp-808bp. Using the mRNA sequence as a template, Rrimer Premier 5.0 software was used to design primer sequences according to the principles of primer design, and the designed sequences were sent to Beijing BGI to synthesize primers. The primer sequences are as follows. Combined with the primer synthesis report, set the temperature gradient, and explore the optimal Tm value of the primer to be 55°C.

Forward primer: Pf 5'-CATTTTTTCACACTCTCCGACG-3'

Reverse primer: Pr 5'-GAAAATGCAACAGCTACGAACA-3'

2.2 Total RNA extraction method

The Trizol method was used to extract the RNA in the cotton leaf tissue. The RNA extraction reagent was purchased from Hetiangen Reagent Company. The specific extraction steps were as follows:

(1) Take 0.3 g of leaves, add liquid nitrogen and grind them into white powder in a mortar. Add the white powder to a 1.5ml centrifuge tube;

(2) Add 1ml Trizol reagent to the centrifuge tube, shake vigorously for 15s, then place it in the ice box for 5min, then centrifuge at 12000rpm/4℃ for 15min;

(3) Transfer the supernatant to a new 1.5ml centrifuge tube. Add 300ul chloroform to the centrifuge tube and invert up and down 20 times. Place in an ice box for 5 minutes, then centrifuge at 12000rpm at 4°C for 15 minutes;

(4) Transfer the upper liquid phase to a new centrifuge tube again, add 1 volume of -20°C isopropanol, and gently invert up and down 10 times. Place the centrifuge tube in an ice bath for 30 minutes, then centrifuge at 12,000 rpm at 4°C for 15 minutes;

(5) Discard the supernatant liquid in the centrifuge tube, fully soak with 1ml 75% ethanol, and tap the precipitate. Then centrifuge at 12000 rpm for 3 min at 4°C. Suck out the ethanol in the centrifuge tube with a pipette gun and place it in the ultra-clean workbench to blow for 2 minutes to completely evaporate the alcohol;

(6) Add 30ul RNase-free water into the centrifuge tube, and detect the RNA quality after fully dissolving.

Take 2uL of extracted total RNA, and use 1% agarose gel electrophoresis to detect the result. The concentration of RNA and the ratio of 260nm and 280nm were measured with a German Berger hummingbird micro-spectrophotometer.

Hummingbird-type micro-spectrophotometer measurement results showed that OD ₂₆₀ /OD ₂₈₀ was between 1.9 and 2.1, and OD ₂₆₀ /OD ₂₃₀ was greater than 2.0, indicating that the total RNA extracted from cotton tissue was free from polysaccharide, polyphenol and protein contamination. Then, electrophoresis was performed on the extracted RNA. After the EB solution was dipped into the gel block for 15 minutes, it was irradiated by ultraviolet light. The electrophoresis results are shown in Figure 1. The three subunit bands of RNA are relatively clear and bright, indicating that the quality of RNA extraction is relatively high. Good, suitable for subsequent molecular cloning experiments.

2.3 Reverse transcription (synthesis of the first strand of cDNA)

The reverse transcription kit (Thermo Reagent Company) was used, and the specific operation steps were as follows:

(1) Add an appropriate amount of Total RNA to an RNase-free PCR tube to ensure that the RNA content is 0.1-5ng, then add 1 μl of Oligo(dT)18Primer, and then add Water to 12 μl, and the total volume is now 12 μl;

(2) Add 5×Reaction Buffer 4μl, Ribolock RNase Inhibitor (20U/μl) 1μl, 10mM dNTP Mix 2μl, RevertAid M-MuLv RT (200U/μl) 1μl, and the total reaction volume is 20μl;

(3) Gently blow and mix with a pipette;

(4) 42°C water bath for 60 minutes;

(5) Water bath at 70°C for 5 minutes, then place in an ice box, and move to -80°C for storage.

2.4 PCR reaction setup

According to the PCR reaction kit (Quanshijin Company), using high-fidelity Taq DNA polymerase, the reaction system is shown in Table 3-1. The PCR program was set as follows: pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, 35 cycles, and extension at 72°C for 10min.

PCR reaction system

2.5 purpose strip glue recovery

(1) Column balance step: Use a pipette to inhale 500 μl of balance solution BL and add it to the adsorption column CA2, centrifuge at 12,000 rpm for 1 min, discard the liquid in the collection tube, and put CA2 back into the tube;

(2) Cut off the target band from the gel block after electrophoresis under ultraviolet light, and put it into a clean centrifuge tube prepared in advance and marked;

(3) Add 500 μl PN to the centrifuge tube containing the glue block, place it in a 50°C water bath for about 10 minutes, and gently turn the centrifuge tube several times during this period to ensure that the glue block is fully dissolved;

(4) Add the gel block solution to the adsorption column CA2, place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 60 sec, discard the liquid in the collection tube, and put the adsorption column back into the collection tube;

(5) Add a certain volume of absolute ethanol to PW in advance according to the instruction manual, then add 600 μl PW to CA2, centrifuge at 12,000 rpm for 60 sec, discard the liquid in the collection tube, and put CA2 back into the collection tube;

(6) Repeat step 5 once;

(7) Put CA2 back into the collection tube and centrifuge at 12,000rpm for 2min to remove as much PW liquid as possible. Then put CA2 on clean paper at room temperature for 5 minutes, and thoroughly dry the filter layer in CA2 to prevent the residual rinse solution from affecting subsequent experiments;

(8) Put CA2 into a new clean centrifuge tube, add 30 μl of EB dropwise to the middle of the adsorption membrane, leave it at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

2.6 Connection conversion

(1) Add 4 μl of DNA gel to the centrifuge tube to recover the product, add 1 μl of pEASY-T1 vector, mix gently, and keep at 25°C for 20 minutes (in PCR);

(2) Take out Escherichia coli competent Trans T1 from the -80°C refrigerator, melt it in an ice bath, take 50 μl and add it to the above 5 μl system, mix well, and bathe in ice for 30 minutes;

(3) Heat shock in a water bath at 42°C for 45 seconds, then quickly ice bath for 2 minutes (do not shake);

(4) Add 800 μl LB liquid medium (without antibiotics) to a 2.0ml centrifuge tube in the ultra-clean workbench, add 55 μl competent system, and incubate on a shaking table at 37°C and 200 rpm for 1 hour to recover the bacteria;

(5) 4000rpm at room temperature for 2min, discard part of the supernatant, leave 250μl, pipette evenly;

(6) Heat the LB solid medium, cool it at room temperature at about 50°C, add 1/1000 volume of Kanna antibiotics, shake well and pour the plate;

(7) After the LB solid medium is solidified, draw 150 μl of competent cells to smear on the plate (keep away from the alcohol lamp and smear quickly).

2.7 Picking a single colony to expand culture and bacterial liquid PCR

Usually the test is done in the morning. Take out 8 centrifuge tubes, add 1 μl of Kanna antibiotics to each centrifuge tube, then add 1ml of LB liquid medium, pick 8 single colonies with a 2.5 μl pipette tip on each petri dish, shake the gun repeatedly, and generally see tiny colonies. particles. After culturing on a shaker at 37° C. and 200 rpm for 4 to 5 hours, PCR amplification was performed using the bacterial solution as a template. The PCR reaction system and conditions of the bacterial solution were the same as those for the above-mentioned PCR. After the bacterial liquid was sampled, it was returned to the shaker for overnight cultivation. After PCR, agarose gel electrophoresis was performed to screen positive clones. The next morning, the positive clones were subpackaged, preserved, and sent to Beijing BGI for sequencing.

According to the method from 2.3 to 2.7, use RNA as template, reverse transcribe to obtain cDNA of two materials respectively; then use cDNA as template to carry out PCR amplification, cut and recover the amplified target cDNA and use it as template to pass through pEASY-T1 vector Connect and transform Escherichia coli competent, plate culture, pick a single colony and culture, and use bacterial liquid PCR to identify and screen positive clones. Among them, the electrophoresis results of 36 positive clones in Xinluzhong are shown in Figure 2. Using DL 2000 DNA Marker as a marker, it was found that the cloned target fragment was around 1000 bp, which was 969 bp. The length of the fragment met the requirements of primer design, and the fragment was further sequenced .

2.8 Bioinformatics analysis

Homology alignment and multiple sequence comparison were performed by searching GeneBank and using software such as DNAMAN. A series of online software and programs were used to analyze the gene sequence of GhAPX2 and its encoded protein sequence.

A phylogenetic tree was constructed using MEGA 5.05 software, and the transmembrane structure of the amino acid sequence encoded by the cotton ascorbate peroxidase gene was analyzed using online software (http://www.cbs.dtu.dk/services/TMHMM).

The sequencing results were compared with the NCBI database and found that the gene clone bands were correct. The open reading frame (ORF) of the cloned GhAPX2 and its encoded amino acid sequence are shown in FIG. 3 . Use ProtParam to predict ( http://web.expasy.org/protparam/ ) the physicochemical properties of the protein encoded by GhAPX2 online. The results showed that the protein encoded by the GhAPX2 gene has a molecular formula of C ₁₂₃₅ H ₁₉₁₃ N ₃₃₁ O ₃₆₅ S ₇ , a total number of atoms of 3851, a molecular weight of 27462.1, and contains various amino acids such as Ala, Arg, Asn, Cys, Leu, Asp, Gly, and Thr. Among them, the content of Leu is the most abundant, which is 11.2%, followed by Ala, which is 10%. The predicted instability index is 35.32, which is a stable protein. The overall average hydrophilic index is -0.346, and the isoelectric point is 5.63.

Further application of ProtScale to analyze the hydrophobicity of amino acids showed that the peptide chain of 36APX2 in Xinlu was dominated by hydrophilic amino acid residues, so it was speculated that the encoded protein was a hydrophilic protein (Figure 4).

Using NCBI CDS analysis, the amino acid encoded by GhAPX2 contains a conserved sequence, and it is a member of the plant POD superfamily. In addition, the application of Protein Blast comparison analysis found that the protein has three binding sites, namely the heme binding site, the substrate binding site and the K ⁺ ion binding site (K ⁺ binding site), also contains an ascorbate peroxidase conserved domain consisting of a total of 243 amino acid fragments at positions 5-247.

Transmembrane region prediction:

The possible transmembrane regions of the protein were analyzed using the TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0) online program. The results show that the protein does not have a transmembrane region and belongs to the extramembrane protein, as shown in Figure 5 .

Signal peptide prediction:

SignalP (http://www.cbs.dtu.dk/services/SignalP) online program was used to predict the signal peptide of 36 APX proteins in Xinlu. The results showed that the protein did not contain a signal peptide, and it was presumed to be a non-secreted protein.

Subcellular localization:

The online program SubLock v 1.0 ( http://www.bioinfo.tsinghua.edu.cn/SubLoc/ ) was used to localize the gene, and it was confirmed again that the gene was located in the cytoplasm.

Example 2

Construction of Cotton Drought Resistance Gene Plant Expression Vector pCAMBIA1304-GhAPX2

The carrier plasmid used in this application is pCAMBIA1034, in combination with the existing restriction sites on the carrier plasmid, use DNAMAN software to analyze the sequence of the GhAPX2 gene to find out the available restriction sites, design gene-specific primers at both ends of the ORF of the GhAPX2 gene and The expression vector pCAMBIA1034-GhAPX2 was constructed by adding BglⅡ and SpeⅠ restriction sites at both ends, and verified by sequencing.

The designed forward primer sequence is Pf 5'-CAGATCTGATGACCAAGTGTTACC-3';

Reverse primer sequence Pr 5'-GACTAGTCTTATGCATCAGCAAATCC-3'.

PCR reaction system

Embodiment 3GhAPX2 gene functional verification

1. Materials

The embryogenic callus of Xinhai 14 sea-island cotton, which was obtained from hypocotyl-induced callus through somatic embryogenesis, was used as the test material and transformed recipient in this example.

YEB (liquid/solid) medium: used for the cultivation of Agrobacterium tumefaciens; tryptone 5g/L, yeast extract 1g/L, sucrose 5g/L, MgSO ₄ 7H ₂ O 0.5g/L, agar Powder 15g/L (solid), pH=7.0.

MS co-cultivation medium: glucose 30g/L, coagulant 1.8g/L, MgCl 0.75g/L, macroelement I 100ml/L, trace element II 5ml/L, iron salt 5ml/L, _B5 organic matter 5ml/L, pH = 5.8.

MS ₀ basic culture medium: glucose 30g/L, macroelement I 100ml/L, trace element II 5ml/L, iron salt 5ml/L, _B5 organic matter 5ml/L, pH=5.8.

2. Method

Taking the embryogenic callus of Xinhai 14 sea-island cotton as an example, the plant expression vector pCAMBIA1304-GhAPX2 of the present invention prepared in Example 2 was used for plant gene transformation, and the specific steps were as follows:

1. Eukaryotic expression vector pCAMBIA1304-GhAPX2 transformed into Agrobacterium LBA4404

After pCAMBIA1304-GhAPX2 was transformed into LBA4404, only transformed Agrobacterium could grow on the selection medium, while non-transformed LBA4404 could not survive due to lack of Kna resistance.

Therefore, the inventors transformed the recombinant plasmid pCAMBIA1304-GhAPX2 prepared in Example 2 into Agrobacterium LBA4404 competent cells, which were qualified for later use.

2. Preparation of Agrobacterium infection solution

The above-mentioned plant expression vector pCAMBIA1304-GhAPX2 that has been transformed into the Agrobacterium engineering strain LBA4404 was streak-inoculated on the YEB solid medium containing Kna (50mg/L), cultured at 28°C for 24-48h, and single clones were picked to contain Kan. (50mg/L) liquid YEB medium, 28°C, 200rpm, 24h culture to the logarithmic phase, centrifuge at 4°C, 5000r/min for 10min to collect the cells, then wash with MS ₀ basic culture medium and resuspend the cells, adjust When the OD ₆₀₀ value reaches 0.5, the Agrobacterium infection solution is obtained and set aside.

3. Agrobacterium infection of cotton embryogenic callus

The embryogenic callus explants of Xinhai 14 were inoculated on MS ₃ proliferation medium and pre-cultured for about 10 days. The vigorously growing embryogenic callus explants were picked and inoculated into sterilized culture bottles, and the pCAMBIA1304-GhAPX2 Agrobacterium infection solution prepared above was added to infect for 15 minutes and shake several times. Pour off the bacterial solution, wash 3-5 times with sterile water, place the callus on sterile filter paper to absorb the bacterial solution on the surface, insert it into MS co-cultivation medium, and culture in dark for 24 hours. The resistant callus was screened, further differentiated into seedlings, and transgenic plants were obtained.

3. Verification of drought resistance of transgenic plants

Fluorescent quantitative PCR reaction to measure the expression of GhAPX2 gene

RT-PCR special kit (Thermo Reagent Company) was used. Add the reaction components to the RT-PCR special plate. The concentration and volume of each component are as follows (20 μl):

Fluorescence quantitative reaction system

Fluorescence quantitative reaction internal reference gene selection

The selection of internal reference genes refers to the screening of real-time quantitative PCR internal control genes in the process of flower fiber development and somatic embryogenesis by Tu Lili et al. ^[96] , and GhUBQ7, which can be stably and continuously expressed during the fiber development of upland cotton, was selected as a candidate internal reference gene. The primer sequences are as follows, and the primers were synthesized by BGI.

Forward primer: Pf 5'-GAAGGCATTCCACCTGACCAAC-3'

Reverse primer: Pr 5'-CTTGACCTTTCTTCTTCTTGTGCTTG-3'

4.2.4 Expression pattern test of GhAPX2 gene at seedling stage

The test was carried out indoors. Choose plump seeds with complete embryos and plant them in the germination box. After the cotyledons are fully unfolded, select plants with consistent growth and move them into plastic pots with 1/2 Hoagland nutrient solution for hydroponic seedlings. Set control (normal watering), stress (11% PEG-6000 simulated drought stress for 48h) two groups of treatments, repeated 3 times. When the cotton leaf age was 3 true leaves, the roots, stems and leaves of the two groups of transgenic positive plants and non-transgenic plants were collected, and they were quickly frozen in liquid nitrogen and stored at -80°C for later use.

It can be seen from Figures 6a and 6b that GhAPX2 was expressed in all cotton tissues at the 3-leaf stage, and whether it was the stress group or the control group, the expression level of this gene was the highest in leaves, followed by roots, and the least in stems. The expression level in medium was significantly higher than that in roots and stems. In addition, the gene expression level in the leaves of the transgenic positive plants increased significantly after stress treatment, which was 4.37 times that of the control group; the leaves of the non-transgenic plants in the stress group were only 1.64 times that of the control group.

4.2.5 Experimental design of expression pattern of GhAPX2 gene at flowering and boll stage

The experiment was carried out in Daejeon. Two treatments of normal irrigation and dry stress were set up for the field resource materials. During the flowering and boll stage (July 5th), the control group was normally irrigated, and the stress group was treated with water control. The transgenic positive plants and non-transgenic plants under the two treatments Select cotton buds that have just bloomed on July 5, tie them up with wool to mark them, and record them as DPA 0 at this time, so that the start time of drought and water control is consistent with the flowering time of buds, and then control water for 0 days and 5 days respectively. , 10 days (also DPA 0, DPA 5, DPA 10) take the cotton bolls of two treatments (Note: July 5th is the water control time, but the control and stress groups are all sampled before watering; July 15th is the stress group Rehydration time (both groups of treatments were also sampled before watering), shaved off the bracts and sepals of the bell, and kept the ovule part in a ziplock bag for storage in liquid nitrogen. room for RNA extraction.

ABI 7500Fast instrument was used for RT-PCR amplification, connected to the instrument through a computer, and the computer was equipped with corresponding software. The software would automatically collect the fluorescence signal of each sample during the RT-PCR process, and examine the influence of the linear relationship and slope of the standard curve. After the reaction, the relative expression can be calculated by the Ct value of each sample [97]. Calculated as follows. 4-6 Use SPSS software to analyze the relative expression of the same material at the same true leaf stage using the LSD method for significant difference analysis.

Relative expression of target gene f(x)=2-ΔΔCt

Seedling stage expression mode uses this formula: ΔΔCt=(Ct target gene-Ct internal reference gene) different tissues under different treatment modes-(Ct target gene-Ct internal reference gene) root control treatment

The expression pattern in the flowering fiber uses this formula: ΔΔCt=(Ct target gene-Ct internal reference gene) different DPA fibers-(Ct target gene-Ct internal reference gene) ODPA fiber

It can be seen from Figures 7a and 7b that at 0 DPA, there was no significant difference in the expression level of this gene in the ovules of the control and stress groups, and the expression level gradually increased at the 5DPA and 10DPA stages with the passage of flowering time. Figure 4 also shows that drought treatment can significantly increase the expression level of GhAPX2. On the whole, under normal irrigation conditions, the relative expression levels of the two cotton varieties were similar, with little difference. However, after drought stress treatment, compared with the control, the expression levels of the transgenic positive plants increased significantly. At 5 DPA, the expression levels of the stress group It was 2.31 times that of the control group, and at 10 DPA, the stress group was 3.56 times that of the control group, while the stress groups of non-transgenic plants were 1.65 times and 1.67 times that of the control group, respectively.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , material or feature is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (9)

1. a kind of cotton anti-drought gene GhAPX2, its base sequence such as SEQ ID NO：Shown in 1.

2. a kind of cotton drought resisting gene plant expression vector pCAMBIA1304-GhAPX2, the expression vector is by by SEQ ID NO：Fragment digestion shown in 1 is obtained to carrier pCAMBIA1304.

Purposes of the 3.GhAPX2 genes in plant drought resistance is strengthened.

A kind of 4. method for strengthening plant drought resistance, it is characterised in that GhAPX2 genes are transferred into plant.

5. according to the method for claim 4, it is characterised in that the plant is cotton.

6. a kind of method for cultivating transgenic drought-resistant cotton, including step：

Clone anti-drought gene；

Build anti-drought gene plant expression vector；

Anti-drought gene plant expression vector is transferred to cotton using Agrobacterium infestation method.

7. according to the method for claim 6, it is characterised in that the anti-drought gene is cotton anti-drought gene GhAPX2.

8. according to the method for claim 6, it is characterised in that the anti-drought gene plant expression vector is pCAMBIA1304-GhAPX2。

9. a kind of method for cultivating drought resisting cotton, including step：

Enter the cotton with anti-drought gene GhAPX2 as male parent or maternal female parent or male parent with without the anti-drought gene Row hybridization；

Screening of drought resistance is carried out to filial generation, obtains the cotton variety with Characteristics of Drought.