CN117904178B - Application of GhBRXL4.3 gene in regulating salt tolerance and/or low temperature tolerance in upland cotton - Google Patents

Abstract

The invention discloses an application of GhBRXL4.3 gene in regulation and control of salt tolerance and/or low temperature resistance of upland cotton, and belongs to the technical field of genetic engineering. The nucleotide sequence of the GhBRXL4.3 gene is shown in SEQ ID NO:1, the invention utilizes VIGS technology to silence target gene in cotton, after salt and low temperature stress treatment, the target gene is found to cause antioxidant enzyme (SOD, POD and CAT) activity, soluble sugar and chlorophyll content reduction and MDA content increase, ghSOS1, ghSOS2, ghNHX1, ghCIPK, ghHDT4D, ghCBF1 and GhPP C and other adversity stress related gene expression reduction is found, namely, the silencing target gene leads to cotton resistance reduction, and the gene is proved to positively regulate cotton to respond to salt and low temperature stress.

Description

Application of GhBRXL4.3 gene in regulating salt tolerance and/or low temperature tolerance in upland cotton

Technical Field

The invention relates to the technical field of genetic engineering, and in particular to the application of the GhBRXL4.3 gene in regulating the salt tolerance and/or low temperature tolerance of upland cotton.

Background technique

Cotton is an annual herbaceous plant of the Malvaceae family. It not only provides natural textile fibers, but also provides rich seed oil and protein. It is one of the important economic crops in the world. Therefore, cotton cultivation has important socioeconomic significance on a global scale. Despite the economic importance of cotton, various environmental factors, including biotic and abiotic stresses, pose a great threat to cotton production. Due to global climate change, major abiotic stresses such as drought, salinity, extreme temperature, waterlogging, heavy metals, and hypoxia will hinder plant growth and development, affect crop yield and quality, and affect agricultural sustainable development. In order to maximize survival efficiency, plants have developed a variety of defense mechanisms and strategies to cope with various adverse conditions. In the defense mechanism of plants, many stress response genes help plants tolerate the adverse effects of various environmental factors by regulating transcriptome levels.

BREVIS RADIX (BRX) is a highly conserved plant-specific gene family that exists in all higher plants for which data are available. It is involved in the longitudinal and radial expansion of hypocotyls and roots, the development of embryos and leaves, and the asymmetric division of stomatal cells. The BRX gene family plays an important role in plant growth and development and in response to adverse stresses. However, there has been no research on the mining and screening of salt-tolerance and low-temperature-tolerance gene resources in cotton BRX family members.

Summary of the invention

The purpose of the present invention is to provide an application of the GhBRXL4.3 gene in regulating the salt tolerance and/or low temperature tolerance of upland cotton to solve the problems existing in the above-mentioned prior art. The GhBRXL4.3 gene positively regulates the response of cotton to salt and low temperature stress, and up-regulating expression in cotton can improve its salt tolerance and low temperature tolerance.

To achieve the above object, the present invention provides the following solutions:

The present invention provides application of a GhBRXL4.3 gene or a fragment thereof in regulating salt tolerance and/or low temperature tolerance of upland cotton. The nucleotide sequence of the GhBRXL4.3 gene is shown in SEQ ID NO: 1.

The present invention also provides the use of the protein encoded by the GhBRXL4.3 gene or a fragment thereof in regulating the salt tolerance and/or low temperature tolerance of upland cotton.

The present invention also provides the use of a recombinant vector comprising the GhBRXL4.3 gene or a fragment thereof in regulating the salt tolerance and/or low temperature tolerance of upland cotton.

The present invention also provides the use of a host bacteria containing the recombinant vector in regulating the salt tolerance and/or low temperature tolerance of upland cotton.

Preferably, the salt tolerance and/or low temperature tolerance of the upland cotton is improved by up-regulating the expression level of the GhBRXL4.3 gene or a fragment thereof in the upland cotton.

The present invention also provides a method for constructing a transgenic upland cotton with salt tolerance and/or low temperature tolerance, comprising up-regulating the expression level of the GhBRXL4.3 gene or a fragment thereof in the upland cotton to improve the salt tolerance and/or low temperature tolerance of the upland cotton.

Preferably, the nucleotide sequence of the GhBRXL4.3 gene is as shown in SEQ ID NO:1.

The present invention discloses the following technical effects:

The present invention analyzes the expression pattern of the BRX gene (GhBRX) family of upland cotton under four abiotic stresses of salt, drought, high temperature and low temperature, and finds that GhBRX family members can respond to salt and low temperature stress, and also finds that GhBRXL4.3 is highly expressed under salt and low temperature stress. In order to further clarify the molecular mechanism of GhBRXL4.3 gene response to salt and low temperature stress, the target genes were silenced in cotton plants using VIGS technology. After salt and low temperature stress treatment, it was found that the four target genes would cause a decrease in antioxidant enzyme (CAT, POD and SOD) activity, soluble sugar and chlorophyll content and an increase in MDA content, and reduce the expression of stress-related genes GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhHDT4D, GhCBF1 and GhPP2C, indicating that silencing the GhBRXL4.3 gene leads to reduced cotton resistance, further proving that the GhBRXL4.3 gene positively regulates cotton's response to salt and low temperature stress, providing important gene resources for salt and low temperature tolerance breeding of upland cotton.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a map of vector pEASY-T5 Zero (a) and TRV2 vector (b);

Figure 2 is the results of PCR amplification of the target gene, PCR of the target gene silencing vector construct and double enzyme digestion; a: PCR amplification results of the target gene, lane 1 is the PCR amplification product, lane 2 is Marker (D2000); b: PCR detection results of the target gene silencing vector construct, lanes 1-5 are all target gene silencing vector construct bacterial solution, lane 6 is Marker (D2000); c: Double enzyme digestion results of the target gene and silencing vector construct, lanes 1-4 are all double enzyme digestion products, lane 5 is Marker (D2000);

Fig. 3 Phenotypes of control and silenced plants; a is the phenotype of TRV:GhCLA positive control; b is the phenotype of TRV:GhCLA, WT, TRV:00, TRV:GhBRXL4.3 before VIGS stress;

FIG4 is the test results of target gene silencing efficiency in TRV:GhBRXL4.3 plants under salt stress and low temperature stress;

FIG5 is a phenotypic analysis of upland cotton plants under salt and low temperature stress;

FIG6 is the MDA content detection result of target gene silenced plants;

FIG7 is the detection of the activity levels of antioxidant enzymes SOD, POD and CAT in target gene silenced plants;

FIG8 is the test result of soluble sugar content in target gene silenced plants;

FIG9 is the result of chlorophyll content detection in target gene silenced plants;

FIG10 shows the expression of stress-related genes GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhHDT4D, GhCBF1 and GhPP2C in silenced plants.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The present invention analyzes the expression pattern of the BRX gene (GhBRX) family of upland cotton under four abiotic stresses of salt, drought, high temperature and low temperature, and finds that GhBRX family members can respond to salt and low temperature stress, and also finds that GhBRXL4.3 is highly expressed under salt and low temperature stress. In order to further clarify the molecular mechanism of GhBRXL4.3 gene response to salt and low temperature stress, the target gene was silenced in cotton plants using VIGS technology. After salt and low temperature stress treatment, it was found that GhBRXL4.3 would cause a decrease in antioxidant enzyme (CAT, POD and SOD) activity, soluble sugar and chlorophyll content and an increase in MDA content, and reduce the expression of stress-related genes GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhHDT4D, GhCBF1 and GhPP2C, indicating that silencing the GhBRXL4.3 gene leads to reduced cotton resistance, which further proves that the GhBRXL4.3 gene positively regulates cotton's response to salt and low temperature stress, providing important gene resources for salt and low temperature tolerance breeding of upland cotton.

The above technical solution is further described below with reference to specific embodiments.

The main experimental materials and reagents involved in the following examples are:

(1) Test materials

The seeds of upland cotton Xinshi K25 (xinshiK25) were provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

Strains and vectors: GV3101 Agrobacterium competent cells and pEASY-T5 Zero cloning vector (CT501-01, containing DH5α Escherichia coli competent cells) were purchased from Shanghai Weidi Biotechnology Co., Ltd. and Quanshijin Biotechnology Co., Ltd., respectively. The VIGS vector system (TRV1, TRV2 and TRV:GhCLA) used for gene silencing was donated by the Transgenic Research Group of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The map of the pEASY-T5 Zero vector is shown in Figure 1.

(2) Test reagents

The test reagents used are shown in Table 1.

Table 1 Reagents used in the experiment

Solution preparation:

a. The formula of the culture medium is shown in Table 2:

Table 2 Culture medium formula

Note: The culture medium needs to be sterilized at 121℃ for 20 minutes

b.100mL 50×TAE buffer: 24.2g Tris+3.72g Na ₂ EDTA·2H ₂ O+5.71mL glacial acetic acid;

c. 50 mg/mL rifampicin (Rif) and 20 mg/mL acetosyringone (AS): Dissolve 5 g of Rif powder and 2 g of acetosyringone powder in 100 mL of DMSO solution, filter and sterilize with a 0.22 filter membrane, package, and store at -20°C;

d. 0.5M morpholineethanesulfonic acid (MES): Dissolve 10.65g MES powder in 50mL ddH ₂ O, adjust the pH to 5.6 with NaOH, then make up to 100mL, filter sterilize with a 0.22 filter membrane, package, and store at 4°C;

e.1M MgCl ₂ : Dissolve 9.521g MgCl ₂ powder in 100mL ddH ₂ O, filter through a 0.22 filter to sterilize, and store at 4°C;

f. 500mL resuspension: 10mL 0.5M MES + 1mL 20mg/mL AS + 5mL 1M MgCl ₂ , add sterile ddH ₂ O to make up to 500mL.

(3) Test equipment

Visible spectrophotometer, high-speed refrigerated centrifuge, water bath, electrophoresis apparatus, artificial climate chamber, gel cutter, ultra-low temperature refrigerator, fluorescence quantitative instrument, gradient PCR instrument, sterilizer, electronic balance, microwave oven, desktop constant temperature shaker, ice maker, clean bench, biochemical incubator, ultra-micro spectrophotometer, ultrapure water instrument and mini vortex mixer.

Example 1 Application of GhBRXL4.3 gene in regulating drought resistance and salt tolerance of upland cotton

1. Primer design

NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design cloning primers, qRT-PCR primers, VIGS silencing primers (VIGS silencing primer product fragment size is 459 bp), and fluorescent quantitative primers for adversity-related genes of the GhBRXL4.3 gene (SEQ ID NO: 1). The primer sequences are shown in Table 3:

Table 3 Primer list

Note: The underlined characters in the table indicate restriction enzyme cutting sites, among which GAATTC and GGTACC indicate EcoRⅠ and KpnI restriction enzyme cutting sites.

2. RNA extraction and reverse transcription

RNA was extracted according to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit.

Reverse transcription (synthesis of first-strand cDNA):

a. Take out the aliquoted RNA from the -80℃ refrigerator and melt it on ice. Take out 5×FastKing-RT SuperMix reagent and RNase-Free ddH ₂ O from the -20℃ refrigerator and melt them on ice. Shake gently to mix.

b. Reaction system see Table 4:

Table 4 Reverse transcription reaction system

c. Reaction procedure is shown in Table 5:

Table 5 Reaction procedure

d. After the reaction is completed, test the purity and concentration of cDNA, package it, and store it at -20℃.

3. Amplification of target fragments and connection and transformation of cloning vectors

3.1 Amplification of target fragment

Using the cDNA of xinshiK25 as a template, the target gene was amplified using the Taq 2×PCR Mix with Dye V2 premix (containing dye) kit. The amplification system is shown in Table 6:

Table 6 Amplification system

After adding the reaction solution according to the above system, shake gently to mix, centrifuge briefly, and react according to the reaction procedure in Table 7:

Table 7 Amplification reaction program

After the reaction was completed, 1.8% agarose gel electrophoresis was used to detect whether the size of the target gene was consistent with the expected one.

3.2 Ligation of target gene and cloning vector pEASY-T5 Zero

a. Take out the pEASY-T5 Zero vector from the -80℃ freezer and thaw it on ice.

b. Calculate the volume of the target fragment to be added (molar ratio of vector to target fragment = 1:5), and add the following components to a sterile 1.5 mL centrifuge tube (the entire operation is completed on ice):

Table 8 Connection system

c. Shake gently to mix, centrifuge briefly, and connect at 25°C for 5 minutes.

3.3 Transformation of DH5α E. coli competent cells

The heat shock method was used to transform into DH5α Escherichia coli competent cells, and the target gene sequence primers (see Table 3) were used to perform PCR verification and sequencing of the bacterial liquid (completed by Shanghai Shenggong Biotechnology Co., Ltd.).

4. Construction of silencing vector

The positive plasmid sequenced successfully in the above "3.3 Transformation of DH5α E. coli Competent Cells" was used as a template, and the silencing fragment was amplified by adding restriction enzyme EcoR I and Kpn I restriction sites and primers with protective bases (see Table 3), and the GhBRXL4.3 fragment was inserted into the TRV2 silencing vector by double enzyme digestion to construct the TRV:GhBRXL4.3 silencing vector. The specific enzyme digestion system is shown in Table 9 below:

Table 9 Enzyme Digestion System

At 37°C, after 3 hours, 10× Loading Buffer was added to stop the reaction, the target gene fragment PCR product was recovered by gel, the large fragment was digested by vector, the target fragment was connected with the silencing vector, the ligation product was transformed into Escherichia coli competent cells, and the bacterial liquid PCR and double enzyme digestion were performed for identification. After completion, the positive plasmid was sequenced (Shanghai Biotech), and then transferred into Agrobacterium GV3101 competent cells. The specific operations are as follows:

a. Take out the GV3101 competent cells from the -80℃ ultra-low temperature freezer, thaw them on ice, divide them into two tubes, take 2μL of the successfully sequenced plasmid, add it to the centrifuge tube, and transform it.

b. After completing the above steps, add 350 μL of LB liquid culture medium (without antibiotics), shake and culture for 2 hours (28°C, 200 rpm), evenly spread the bacterial liquid on the solid culture medium (with Kan ⁺ and Rif antibiotics added), and culture in the dark at 28°C for 2 days.

c. After the culture is completed, a single colony is picked into 5 mL of LB liquid medium (with Kan ⁺ and Rif antibiotics added) and cultured for 16 hours according to the shaking culture conditions in step b;

d. After the culture is completed, the bacterial solution is preserved with 50% glycerol (bacterial solution: glycerol = 1:1) and stored at -80°C for future use; PCR is performed on the bacterial solution to confirm that the vector is positive.

5. VIGS silencing target genes in upland cotton

VIGS silencing was performed on Xinshi K25 seedlings. The specific method is as follows:

a. Plant the seeds of Xinshi K25. When they grow to the seventh day and the cotyledons are fully expanded, soak them in water until the nutrient soil in the flowerpot absorbs the water to the surface. Then stop soaking and set them aside for later use.

b. Add Kan ⁺ and Rif to LB liquid medium for later use, where the final concentrations of Kan ⁺ and Rif are 50 μg/mL and 25 μg/mL respectively. Thaw the VIGS vector system and target gene bacterial solution taken out from -80℃ on ice, and activate at 28℃, 200rpm for 12-16h (bacterial solution: LB liquid medium = 1:10). After activation, expand the culture in the same ratio.

c. After the bacterial liquid is expanded, centrifuge at 5000 rpm for 10 minutes, pour off the supernatant, retain the bacteria, and use a spectrophotometer to suspend the bacteria with a resuspension solution. The _OD600 should be between 1.8-2.0.

d. After resuspension, place in the dark for 3 hours to allow the bacteria to recover, then mix TRV1 with the bacterial resuspension containing TRV:00 (blank control group), TRV:GhCLA (positive control), and TRV:GhBRXL4.3 (experimental group) in a 1:1 ratio and mix them thoroughly.

e. On the seventh day of the growth of the cotton seedlings, soak them in water according to the method of step a. On the eighth day of the growth of the cotton seedlings, perform VIGS injection on them. The specific operation is as follows: use a 1mL syringe needle to cut the back of the cotyledon (note that the wound should not be too large, the size of a needle tip is sufficient), and inject the mixed bacterial solution of step d into the cotton cotyledon, so that the bacterial solution fills the entire cotyledon as much as possible.

f. After the injection is completed, in order to achieve a better infection effect, wrap it in a plastic bag, place it in the dark at 25℃ for 24 hours, and then culture it under normal growth conditions.

g. After the positive control cotton seedlings turned white, the young cotton leaves of WT (untreated), blank control group and experimental group were taken for fluorescence quantitative experiment to detect their silencing efficiency.

5.1 Salt and low temperature stress treatment of target gene silenced cotton plants

After the injected positive cotton seedlings showed albinism, the cotton seedlings grown to four weeks old in the blank control group and the experimental group were treated with salt and low temperature with 200mmol/L NaCl and 12℃, respectively, and the phenotypic observation was carried out.

5.2 Determination of physiological indexes of target gene silenced cotton leaves

The antioxidant enzyme activities (SOD, POD and CAT), MDA content, soluble sugar content and chlorophyll content of cotton leaves in the blank control group and the experimental group were detected according to conventional methods to analyze the response of the target gene silenced plants to salt and low temperature stress.

5.3 Fluorescence quantification of stress-related genes in target gene silencing lines

Young leaves of the silenced strain were picked, and RNA was extracted using the RNAprep pure polysaccharide and polyphenol plant total RNA extraction kit (catalog number DP441, purchased from Tiangen Biochemical Technology Co., Ltd.), and reverse transcribed using the FastKing one-step genomic cDNA first-strand synthesis premix kit. Finally, the expression levels of stress-related genes GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhHDT4D, GhCBF1 and GhPP2C were detected using the SuperReal fluorescent quantitative premix reagent enhanced kit (catalog number FP205, purchased from Tiangen Biochemical Technology Co., Ltd.). The specific methods refer to the instructions.

6. Results and Analysis

6.1 Amplification of target gene fragments and construction of silencing vector

Using the cDNA of Xinshi K25 as a template and the primers as shown in Table 3, the gene silencing fragment was cloned using the Taq 2×PCR Mix with Dye V2 premix (containing dye) kit, and the fragment size was detected by agarose gel electrophoresis, and the sequence was determined by sequencing. The result is shown in Figure 2a, and the target fragment was successfully amplified. The gene sequence is shown in SEQ ID NO: 2.

The nucleotide sequence of GhBRXL4.3 is shown in SEQ ID NO: 1:

Note: The cloning primers of GhBRXL4.3 gene are indicated by underline fonts, and the start codon and stop codon of GhBRXL4.3 gene are in bold fonts.

SEQ ID NO.2:

AGGTCTGAAGATGAGAGCTCGAAAATGGAATCGGCAGAAGAAAGCCCTGTAACACCACCATTGACCAAAGAACGCGTACCTCGTAATTTATACCGTCCAGCGGGGATGGGGATGGGCTACTCATCCTCAGATTCACTTGATCAGCACCCAATGCAGGCCAGGCATTATTGTGACTCTGGTCTTACTTCAACCCCAAAAGTTTCTAGCATTAGTGGTGCCAAGACAGAGATATCATCAATGGATGCCTCTATGAGGAGTAGCTCAT CAAGAGAGGCTGATCAGTCCGGGGAGCTATCTATCAGTAACGCCAGTGATCTCGAGACTGAGTGGGTTGAACAAGATGAACCAGGCGTTTACATTACAATCAGAGCCTTGCCAGGAGGCAAAAGGGAGCTTAGGCGAGTTAGATTCAGCCGAGAAATATTTGGAGAAATGCATGCTAGACTGTGGTGGGAAGAG.

Note: The sequence size of the GhBRXL4.3 gene silencing fragment is 459 bp.

The positive plasmid detected correctly was used as a template and the primers were as shown in Table 3. The Taq 2×PCR Mix with Dye V2 premix (containing dye) kit was used to amplify the VIGS fragment, and the fragment size was detected by agarose gel electrophoresis. The result is shown in Figure 2b. After the target fragment of the appropriate size was successfully connected to the pGM-T vector, the positive plasmid was connected to the TRV2 vector by double enzyme digestion, and the positive plasmid was identified by bacterial liquid PCR and double enzyme digestion (the results are shown in Figure 2c), and sequencing was successful.

6.2 Silencing of target genes and detection of silencing efficiency

The TRV:GhBRXL4.3 silencing vector successfully constructed above was used to infect cotton seedlings with fully flattened cotyledons. It was found that on the 7th day after infection, the positive control (TRV:GhCLA) plants began to show albinism, and the albinism was obvious on the 15th day after VIGS silencing (see Figure 3), indicating that the VIGS silencing system was successfully constructed. The results of the silencing efficiency test of the target gene are shown in Figure 4. The results showed that compared with the control group, the expression of GhBRXL4.3 in the TRV:GhBRXL4.3 plants was significantly inhibited, and its silencing efficiency reached 100% (36/36). The expression analysis of the VIGS silenced plants and the control plants indicated that the target gene had been silenced.

6.3 Phenotypic analysis of target gene silenced plants under salt and low temperature stress

Four-week-old VIGS silenced lines, including target gene silenced TRV:GhBRXL4.3 and control TRV:00 plants, were selected for salt and low temperature stress treatment. The results are shown in Figure 5. It was found that after 12 days of treatment with 200mmol/L NaCl and 12℃ low temperature, the cotyledons of the plants injected with TRV:00 bacterial solution and target gene silenced bacterial solution had completely fallen off, and the true leaves were wilted and yellowed compared with the control group. It was also found that the true leaves of TRV:GhBRXL4.3 silenced plants were severely dehydrated compared with TRV:00 plants, and the yellowing and wilting were also more serious, indicating that the GhBRXL4.3 gene is involved in the salt and low temperature tolerance of upland cotton.

6.4 Effects of salt and low temperature stress on MDA content in leaves of target gene silenced cotton

The MDA content of the GhBRXL4.3 gene silenced plants and empty vector plants after stress was detected and analyzed. The results are shown in Figure 6. The results showed that the MDA content of the GhBRXL4.3 silenced plants under salt and low temperature stress increased significantly, indicating that the silencing of the GhBRXL4.3 gene caused a decrease in cotton resistance.

6.5 Effects of salt and low temperature stress on antioxidant enzyme activities in leaves of target gene silenced cotton

The activities of antioxidant enzymes (SOD, POD and CAT) of the silenced plants were detected, and the results are shown in Figure 7. Compared with the TRV:00 control plants, the activities of CAT, SOD and POD in the GhBRXL4.3 silenced plants showed varying degrees of decrease after 8 days of salt and low temperature stress.

6.6 Effects of salt and low temperature stress on soluble sugar content in leaves of target gene silenced cotton

The results are shown in FIG8 . Compared with the soluble sugar content in the leaves of the control TRV:00, silencing of the GhBRXL4.3 gene caused a decrease in the soluble sugar content, indicating that silencing the GhBRXL4.3 gene would increase the sensitivity of cotton to salt and low temperature stress.

6.7 Effects of salt and low temperature stress on chlorophyll content in leaves of target gene silenced cotton

When plants are subjected to high-intensity abiotic stress, the leaves of the plants will show obvious yellowing and wilting. The detection of chlorophyll content is one of the important indicators for detecting plant stress tolerance. By testing the chlorophyll content of cotton after stress, it was found that the chlorophyll content of the GhBRXL4.3 gene-silenced plants was significantly lower than that of the control plants (see Figure 9), indicating that the silencing of the GhBRXL4.3 gene leads to a weakening of the salt and low temperature tolerance of cotton plants.

6.8 Effects of salt and low temperature stress on the expression of stress genes in silent strains

The results are shown in Figure 10. The expression of GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhHDT4D, GhCBF1 and GhPP2C genes in TRV:GhBRXL4.3 plants was significantly reduced compared with that in TRV:00, indicating that silencing the GhBRXL4.3 gene will affect the expression of genes related to adverse stress.

In summary, upland cotton in which GhBRXL4.3 was silenced by VIGS was more sensitive to salt and low temperature stress than the control, indicating that cotton resistance was reduced. Therefore, it was proved that the above gene positively regulated cotton's response to salt and low temperature stress.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (5)

1. The application of the GhBRXL4.3 gene in regulation and control of salt tolerance and/or low temperature resistance of upland cotton is characterized in that the nucleotide sequence of the GhBRXL4.3 gene is shown in SEQ ID NO:1 is shown in the specification; silencing the GhBRXL4.3 gene and improving the sensitivity of upland cotton to salt and/or low temperature.
2. 2. Use of a protein encoded by the ghbrxl4.3 gene of claim 1 for modulating salt tolerance and/or low temperature tolerance of upland cotton, wherein the ghbrxl4.3 gene is silenced to increase salt and/or low temperature sensitivity of upland cotton.
3. 3. Use of a recombinant vector comprising the ghbrxl4.3 gene of claim 1 for regulating and controlling salt tolerance and/or low temperature tolerance of upland cotton, wherein silencing the ghbrxl4.3 gene increases sensitivity of upland cotton to salt and/or low temperature.
4. 4. Use of a host bacterium comprising the recombinant vector of claim 3 for regulating salt tolerance and/or low temperature tolerance of upland cotton, wherein the ghbrxl4.3 gene is silenced to increase salt and/or low temperature sensitivity of upland cotton.
5. 5. A construction method of transgenic upland cotton with improved salt and/or low temperature sensitivity, which is characterized by comprising the steps of reducing the expression level of GhBRXL4.3 gene in upland cotton and improving the salt and/or low temperature sensitivity of upland cotton; the nucleotide sequence of the GhBRXL4.3 gene is shown in SEQ ID NO: 1.