CN115725615A - Upland cotton GhABC1K14-A09 gene and application thereof in drought resistance and salt tolerance of upland cotton - Google Patents

Abstract

The invention discloses a upland cotton GhABC1K14-A09 gene and application thereof in drought resistance and salt tolerance of upland cotton, belonging to the technical field of genetic engineering. The nucleotide sequence of the gene is shown as SEQ ID NO:7, the invention also discloses application of the upland cotton GhABC1K14-A09 gene in improving drought resistance and salt tolerance of plants. The invention utilizes VIGS technology to silence target genes in cotton, and after drought and salt stress treatment, finds that the target genes can cause activities of antioxidase (CAT, POD and SOD), content reduction of soluble sugar and chlorophyll and content increase of MDA (multidrug resistance), expression reduction of stress-related genes such as GhSOS1, ghNHX1 and GhCBL3, and the like, namely, the silencing of the target genes causes resistance reduction of the cotton, and proves that the genes positively regulate and control the cotton to respond to drought and salt stress.

Description

Upland cotton GhABC1K14-A09 gene and application thereof in drought resistance and salt tolerance of upland cotton

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a upland cotton GhABC1K14-A09 gene and application thereof in drought resistance and salt tolerance of upland cotton.

Background

Cotton (Gossypium spp.) is one of the most important commercial crops in the world and is also an important source of natural fiber in the textile industry. Wherein, the upland cotton (Gossypium hirsutum L.) is used as one of the cultivated species of cotton crops, accounts for 95 percent of the annual yield of the cotton in the world, and has wide application prospect. Cotton planting and production is carried out mainly in arid and semi-arid regions, and cotton exhibits higher drought tolerance than rice, wheat and corn. However, moisture and saline-alkali limitations still have a great influence on cotton fiber yield and lint quality, and are major abiotic factors affecting cotton production worldwide. Although the major goals of cotton breeders have been to improve productivity and fiber quality for many years, changes in climatic factors and extreme weather frequency events, such as drought and salinity, pose a significant threat to cotton production.

The ABC1K (Activity of bc1 complex kinase) gene family belongs to an atypical protein kinase family, and the members of the ABC1K gene family are ubiquitous in prokaryotes and eukaryotes and play an important role in plant growth and development and stress response, but the discovery and screening utilization of drought-resistant and salt-tolerant gene resources in the ABC1K family members of cotton is not seen so far.

Disclosure of Invention

The invention aims to provide a upland cotton GhABC1K14-A09 gene and application thereof in upland cotton drought resistance and salt tolerance, so as to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a Gossypium hirsutum GhABC1K14-A09 gene, the nucleotide sequence of which is shown in SEQ ID NO: shown at 7.

The invention also provides a primer for amplifying the upland cotton GhABC1K14-A09 gene, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 1-2.

The invention also provides a recombinant vector which comprises the upland cotton GhABC1K14-A09 gene.

The invention also provides a recombinant bacterium, which comprises the recombinant vector.

The invention also provides a method for amplifying the upland cotton GhABC1K14-A09 gene, which takes cDNA of cotton as a template and utilizes the primer to carry out PCR amplification to obtain the upland cotton GhABC1K14-A09 gene.

The invention also provides application of the upland cotton GhABC1K14-A09 gene or the primer or the recombinant vector or the recombinant bacterium in drought resistance and/or salt tolerance of plants.

Further, the drought resistance and/or salt tolerance of the upland cotton can be improved by up-regulating the GhABC1K14-A09 gene level of the upland cotton in plants.

Further, the plant is a cotton plant of the family Malvaceae.

Further, the plant comprises upland cotton.

The invention discloses the following technical effects:

the expression patterns of the upland cotton ABC1K gene (GhABC 1K) family under four abiotic stresses of high temperature, low temperature, drought and salt are analyzed, and the GhABC1K family member can respond to the drought and salt stress and simultaneously discover high expression of GhABC1K14-A09 under the drought and salt stress. In order to further determine the molecular mechanism of GhABC1K14-A09 gene response to drought and salt stress, a VIGS technology is used for silencing target genes in cotton plants, and after drought and salt stress treatment, 4 target genes are found to cause the activities of antioxidase (CAT, POD and SOD), the content of soluble sugar and chlorophyll and the content of MDA to be increased, and the expression of adversity stress related genes GhSOS1, ghNHX1, ghCBL3, ghCIPK6, ghSOS2, ghHDT4D, ghEREB A, ghDREB2C, ghWRKY and GhRD29A is reduced, so that the silencing of the GhABC1K14-A09 gene causes the cotton resistance to be reduced, and further proves that the GhABC1K14-A09 gene positively regulates the cotton response to drought and salt stress, and provides important genes for upland salt stress resistance breeding of upland cotton.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a vector map of the vectors pGM-T and TRV 2;

FIG. 2 shows the PCR amplification of target gene, PCR of target gene and silent vector construct bacterial liquid and double digestion results; a, PCR amplification of a target gene; b, amplifying target gene VIGS fragments; PCR of target gene VIGS fragment and silent vector construct bacterial liquid; d, double enzyme digestion of the target gene and the silent vector construct; 14 represents GhABC1K14-A09; v and M represent TRV2 vector and Marker (D2000), respectively;

FIG. 3 is a TRV2 GhPDS positive control phenotype;

FIG. 4 shows the result of detecting the silencing efficiency of the target gene in TRV2: ghABC1K14-A09 plant;

FIG. 5 is a phenotypic analysis of upland cotton plants under drought and salt stress;

FIG. 6 shows the detection result of MDA content in target gene-silenced plants;

FIG. 7 shows the detection of the activity levels of antioxidase SOD, POD and CAT in plants with silent target genes;

FIG. 8 shows the result of detecting the content of soluble sugar in a plant with target gene silencing;

FIG. 9 shows the chlorophyll content detection result of target gene silencing plants;

FIG. 10 shows the expression of stress-related genes in silenced plants.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The expression patterns of a gossypium hirsutum ABC1K gene (GhABC 1K) family under four abiotic stresses of high temperature, low temperature, drought and salt are analyzed by the inventor, and the member of the GhABC1K family can respond to the drought and salt stress, and meanwhile, ghABC1K14-A09 is found to show high expression in the drought and salt stress. In order to further define the molecular mechanism of GhABC1K14-A09 gene responding to drought and salt stress, the GhABC1K14-A09 gene is cloned, a VIGS silencing vector of the gene is constructed, and the biological functions under the drought and salt stress are researched. The experiments were as follows:

1. test materials and methods

1.1 Experimental materials

The experimental material used was cotton 113 of the upland cotton variety, the seeds of which were provided by the cotton research institute of the Chinese academy of agricultural sciences.

1.2 vectors and competent cells

pGM-T cloning vector (VT 302-02, containing TOP10 E.coli competent cells) and GV3101 Agrobacterium competent cells were purchased from Tiangen Biochemical technology Co., ltd and Shanghai Weidi Biotechnology Co., ltd, respectively, and VIGS vector systems for gene silencing (TRV 1, TRV2 and TRV 2-GhPLDS) were given by the Cotton institute of Chinese academy of agricultural sciences, and the vector maps are shown in FIG. 1.

1.3 Experimental methods

1.3.1 primer design

NCBI Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /) was used to design cloning primers for the GhABC1K14-A09 gene, qRT-PCR primers, and primers for VIGS silencing vector construction, the Primer sequences (SEQ ID NOS: 1-6) are shown in Table 1:

TABLE 1 primer sequences

Primer name	Primer sequence (5 '→ 3')	Purpose(s) to
			GhABC1K14-A09-F	GAACTATCTTCCTCTCTCTCTCT	Cloning of genes
GhABC1K14-A09-R	TCAACATCTGCTATACAAGC	Cloning of genes
			GhABC1K14-A09-F	TCAACGTCGTCAAGTCAGGG	qRT-PCR
GhABC1K14-A09-R	CGTGCTAACACACGAGACGA	qRT-PCR
			GhABC1K14-A09-F	CGGAATTCAGCTTCGGTTGGTTGAGGTT	VIGS
GhABC1K14-A09-R	CCGCTCGAGCCATGGCTGACCAAGTTCCT	VIGS

Note: underlined fonts indicate restriction sites, with GAATTC and CTCGAG indicating EcoRI and XhoI restriction sites.

1.3.2 Cotton planting, total RNA extraction, cDNA Synthesis and qRT-PCR

1.3.2.1 Cotton planting

a. Placing equal amount of nutrient soil (matrix: vermiculite = 1:1) in each flowerpot, placing in a large pot containing tap water, and soaking until water is absorbed to the surface of the flowerpot for cotton seed germination;

b. planting seeds of the medium cotton 113 in the flowerpot, and culturing in a climatic incubator (16 h of illumination, 8h of darkness, 28 ℃ of temperature and 70 percent of humidity) in order to ensure consistent seedling emergence of the seeds, wherein the planting depth of the seeds is kept at 1.5 cm;

c. when the cotton grows to the four-leaf stage, selecting tender leaves for sampling, quickly freezing the sampled product in liquid nitrogen, and storing at-80 ℃ for later use.

1.3.2.2 extraction of Cotton Total RNA

The extraction was carried out according to the instruction of RNAprep pure polysaccharide polyphenol plant total RNA extraction kit (cat No. DP441, purchased from Tiangen Biochemical technology Co., ltd.).

1.3.2.3 reverse transcription of Cotton RNA (first Strand cDNA Synthesis)

Reverse transcription of the total RNA extracted above was performed according to the following procedure:

a. thawing RNA taken from-80 ℃ on ice; 5 XFastKing-RT SuperMix reagent and RNase-free ddH taken from-20 ℃ were combined ₂ O was melted at room temperature and then quickly placed on ice, which was gently shaken and mixed well before use.

b. The reaction system was prepared as follows (table 2):

composition of matter	Amount of use (mu L)
		5×FastKing-RT SuperMix	4
Total RNA	2
		RNase-free ddH 2 O	Make up to 20. Mu.L

c. The reverse transcription reaction was carried out according to the following reaction procedure (table 3):

reaction temperature (. Degree. C.)	Reaction time (min)
		42	15
95	3

d. After the reaction is finished, the concentration and purity of the cDNA are detected, and the cDNA is stored at the temperature of minus 20 ℃ for later use.

1.4 construction of target Gene amplification and silencing vector

1.4.1 PCR amplification of target genes

The GhABC1K14-A09 gene was amplified using a 2 XPPro Taq premix (dye-containing) kit (product No. AG11109, available from Ai Kerui Co., ltd.) using medium cotton 113cDNA as a template, as follows (Table 4):

after the operation was completed, the mixture was gently shaken, mixed, centrifuged for a short time, and reacted according to the following procedure (table 5):

the full length of the GhABC1K14-A09 gene is amplified from upland cotton by a PCR method, and the nucleotide sequence of the full length gene is shown as SEQ ID NO:7, and (c):

the nucleotide sequence of GhABC1K14-A09 is shown in SEQ ID NO:7, and (c):

>14-A09

ATGGACGCAGCAGCGCCGCCGTGGCTCGTCTACTGCGGTGTCGATCCCGTCCGTTTCTCTTCCCCGCGCTCTAACAGAGTATCTATTCGTACTCGAACCAGACCGGTTCTCGCTGTAGCAACCGACCCTAAACCTACTCGTAAGACTCCGTCTCAGTCCTCTCCTTCAAACAACAACGTCAACGGCTCCTCCAAGTCCTCCTTATCTAAAAAGTCCGTGAACGGAGTTCCTACGAGGATGGGAGACGTTTCACAGGAAATAAAAAGAGTGAGAGCACAAATGGAAGAAAATGAAGATTTGGCTATACTAATGAGAGGACTTCGTGGCCAAAATTTACGGGATTCACAGTTTGCTGATGACAATATTCAGCTTCGGTTGGTTGAGGTTGATGAAAGCAGTGAGTTCTTACCTTTGGCATATGATCCGGCTAGCATCTCAGCATACTGGGGGACACGGCCTCATGCTGTTGCAACTCGTATCATACAGTTACTATCTGTTGCTGGGGGTTTCCTCTCACGCTTGGCTATGGATGTGGTAAACAAGAAGGTCAAAGAGCATGAAGTGGCTAGAGCTATTGAATTAAGGGAAATTGTTACCTCTTTGGGCCCAGCGTATATAAAGCTTGGGCAAGCATTGAGCATTCGACCGGATATACTTTCTCCTGTTGCCATGATGGAGCTGCAAAAGCTTTGTGATAAGGTTCCTTCATTTCCAGATGACATAGCAATGGCTCTTATTGAAGAGGAACTTGGTCAGCCATGGCAAGAAATCTACTCTGAACTTTCCTCTTCGCCAATAGCTGCTGCATCTCTTGGACAAGTGTATAAAGGACGCTTGAAAGAGAATGGAGATCTGGTTGCTGTCAAAGTGCAGAGGCCTTTTGTTCTTGAGACAGTGACTGTTGATTTGTTCATCATAAGAAACTTGGGTTTGGTGCTCCGAAAGTTTCCTCAGATCTCCATAGATGTGGTTGGATTGGTTGATGAATGGGCTGCACGATTCTTTGAGGAGCTAGATTATATTAACGAGGGTGAAAATGGACAACTCTTTTCTGAAATGATGCGTAAGGACCTTCCACAGGTTGTTATACCAAGGACTTATCAGAAATACACATCAAGGAAGGTTCTTACTACAGAGTGGATCGAAGGAGAGAAGCTATCACAAAGTACAGAAAGTGATGTTGGTGAATTGGTCAATGTTGGAGTGATATGCTACCTAAAGCAGTTGCTTGATACCGGGTTTTTCCATGCTGACCCACATCCTGGGAATTTGATCCGTACTCCTGATGGAAAGCTAGCCATACTCGACTTTGGTCTCGTGACAAAATTAACTGATGACCAGAAGTATGGAATGATTGAAGCAATTGCTCATCTTATCCACCGGGACTATCCAGAAATAGTTAAGGATTTTGTTAAACTTGATTTCATTCCTGAGGGGGTTAATTTAGAGCCAATCTTGCCCGTTCTGGCAAAGGTTTTTGATCAAGCACTTGAGGGTGGGGGTGCAAAAAACATCAACTTTCAGGACCTGGCATCAGATTTGGCGCAGATAACATTTGATTATCCATTTAGGATACCTCCTTATTTTGCTCTTATAATTAGGGCAATTGGGGTGTTGGAAGGAATAGCTTTAGTGGGGAATCCTGATTTTGCTATCGTGGATGAGGCCTATCCTTATATTGCACAGAGACTGCTCACTGATGAATCCCCGCGGCTAAGGAATGCTTTGCGTTATACAATATACGGGAAATCAGGTGTTTTTGATGCTGAAAGATTCATTGATGTGATGCAAGCCTTTGAGAATTTCATAACTGCAGCAAAAAGTGGAGGTGGGGAGAATTTGAATGGGGATATGGCAGAACTAGGTCTTCTACAAAGGCAAGCAGATATTTCCTTCCCTAGATTTCTACCAAGTGAATCTCAATCAAAGCAACCTGTTCAAACAAGAGCGGCCTTAGGATTTCTACTGTCTGAGAAGGGAAATTTTTTCCGAGAATTTCTTCTTGATGAGATTGTGAAGGGCATTGATGCACTTAGCAGGGAACAGTTGGTTCAAATAATGTCAGTGTTGGGAGTAAGAAATGCTGCCCCAGTGTTTAGCTTGGTTCCAACAGTTGGACCCTTCAAACCAGCAGGACTTTTGCCTTCAATAACCGAGGAAGACAGAGTTATACTGAATAATGTCCAAAAAATTCTCGAATTCTTAACAGCGGGAAGTGCAATATCAACGTCGTCAAGTCAGGGTGTAAATGTTGCTCAGGTTATCCAGGAATTGCTTCCAGTGTTGCCAGGCATATCTGCCAGGGTTCTTCCTGAGTTGATTAGCCGGTTATCGTCTCGTGTGTTAGCACGTTTAATCCGGGATACATTTTTGTAA。

1.4.2 ligation of the target Gene to the cloning vector pGM-T

a. pGM-T vector was removed from a-80 ℃ refrigerator and then thawed on ice.

b. Calculate the volume of the added target fragment (molar ratio of carrier to target fragment = 1:5) and add the following ingredients to a sterile 1.5mL centrifuge tube (the whole procedure was done on ice):

c. the mixture was gently shaken and mixed, centrifuged briefly, and then ligated overnight at 16 ℃.

1.4.3 transformation of TOP10 E.coli competent cells

Transforming the strain into TOP10 escherichia coli competent cells by a heat shock method, and carrying out bacterial liquid PCR verification and sequencing by using a target gene sequence primer (completed by Shanghai biological engineering Co., ltd.).

1.4.4 construction of silencing vectors

Taking the positive plasmid with successful sequencing of 1.4.3 as a template, carrying out silent fragment amplification by adding primers of restriction enzyme EcoR I and Xho I enzyme cutting sites and protection bases, and inserting the fragment of GhABC1K14-A09 into a TRV2 silent vector by a double enzyme cutting method to construct a TRV2 GhABC1K14-A09 silent vector, wherein a specific enzyme cutting system is as follows:

and (3) adding 10 Xloading Buffer to stop reaction after 37 ℃ for 3h, recovering a target gene fragment PCR product by glue, recovering an enzyme digestion large fragment by a vector, connecting the target fragment with a silencing vector, transforming the connecting product into an escherichia coli competent cell to perform a blue-white spot screening experiment, performing bacterial liquid PCR and double enzyme digestion identification, sequencing positive plasmids (Shanghai's engineering) after completion, and transferring the positive plasmids into agrobacterium GV3101 competence.

1.5VIGS silencing of genes of interest

VIGS silencing is carried out on medium cotton 113 seedlings by the following specific method:

a. planting medium cotton 113 seeds according to the method of 1.3.2.1, soaking the medium cotton 113 seeds when the medium cotton 113 seeds grow to the seventh day and cotyledons are completely unfolded until the water is absorbed to the surface by the nutrient soil in the flowerpot, stopping soaking, and standing for later use.

b. Adding Kan into LB liquid culture medium ⁺ And Rif for use, wherein Kan ⁺ And Rif at 50. Mu.g/mL and 25. Mu.g/mL, respectively. The VIGS vector and the target gene plasmid taken out from-80 ℃ were thawed on ice, and activated at 28 ℃ and 200rpm for 12-16h (plasmid: LB broth = 1. After activation, propagation is carried out according to the same proportion.

c. After the propagation of the bacteria liquid is finished, centrifuging for 10min at the rotating speed of 5000rpm, pouring out supernatant, reserving thalli, and suspending the thalli by using a heavy suspension liquid by using a wind-solar photometer to obtain OD ₆₀₀ Is 0.8-1.0.

d. After completion of the resuspension, the cells were allowed to stand in the dark for 3 hours, and after the cells were recovered, TRV1 was mixed with a cell resuspension solution 1:1 containing TRV2 (blank control), TRV2: ghPDS (positive control), TRV2: ghABC1K14-A09 (experimental group), respectively, and sufficiently mixed.

e. On the seventh day of cotton seedling growth, it was soaked according to the method of step a. The eighth day of growth of cotton seedlings, the VIGS injection was performed by: d, cutting the back of the cotyledon by using a 1mL syringe needle (the wound is not easy to be too large, and the size is just the same as that of the needle point), and injecting the mixed bacterial liquid obtained in the step d into the cotton cotyledon to fill the whole cotyledon as much as possible.

f. After injection, in order to achieve better infection, the cells were wrapped with a plastic bag, left at 25 ℃ in the dark for 24 hours, and then cultured under normal growth conditions.

g. And after the positive control cotton seedlings are whitened, carrying out fluorescence quantitative experiment detection on the silencing efficiency by adopting cotton young leaves of the experimental group and the blank group.

1.5.1 treatment of drought and salt stress in Cotton plants with silencing of target genes

After whitening of the injected positive cotton seedlings occurred, the blank control and experimental group-grown cotton seedlings of four weeks old were subjected to salt and drought treatments with 200mmol/L NaCl and 15% PEG, respectively, for phenotypic observation.

1.5.2 determination of physiological indexes of target gene silencing cotton leaves

The activities of antioxidant enzymes (SOD, POD and CAT), MDA content, soluble sugar content and chlorophyll content of cotton leaves of a blank control group and a test group are respectively detected according to a conventional method so as to analyze the reaction of target gene silencing plants to drought and salt stress.

1.5.3 fluorescent quantitation of adversity stress-related genes in target Gene-silenced lines

Picking young leaves of a silent strain, extracting RNA according to an RNAprep pure polysaccharide polyphenol plant total RNA extraction kit (the product number is DP441 and purchased from Tiangen Biochemical technology Co., ltd.), performing reverse transcription by a FastKing one-step method except a genomic cDNA first-strand synthesis premixed kit (the product number is KR118 and purchased from Tiangen Biochemical technology Co., ltd.), and finally performing expression level detection on stress-related genes such as GhSOS1, ghNHX1, ghCBL3, ghSOS2, ghCIPK6, ghHDT4D, ghDREB C, ghDREB A, ghWRKY and GhRD29A by a SuperReal fluorescent quantitative premixed reagent enhanced kit (the product number is FP205 and purchased from Tiangen Biochemical technology Co., ltd.), wherein the specific method refers to the instruction.

2 results and analysis

2.1 amplification of target Gene fragment and construction of silencing vector

The medium cotton 113cDNA was used as template, primers were as in Table 1, and 2 XPro Taq premix (Ai Kerui) was used for full-length amplification of the gene, and the fragment size was checked by agarose gel electrophoresis and sequenced to determine the sequence (FIG. 2 a). Taking the positive plasmid with the correct detection as a template, performing VIGS fragment amplification by using a 2 XPro Taq premix (Ai Kerui) as a primer as shown in Table 1, detecting the size of the fragment by agarose gel electrophoresis (figure 2 b), successfully connecting a target fragment with a proper size with a pGM-T vector, connecting the positive plasmid with a TRV2 vector by a double enzyme digestion method, identifying the positive plasmid by bacterial liquid PCR (figure 2 c) and double enzyme digestion (figure 2 d), and successfully sequencing.

2.2 silencing of target genes and detection of silencing efficiency

The TRV2: ghABC1K14-A09 silencing vector constructed in the construction 2.1 is used for infecting cotton seedlings with completely flattened cotyledons, and the whitening of positive control (TRV 2: ghPDS) plants starts to appear on the 7 th day after infection, and the whitening is obvious on the 15 th day after VIGS silencing (figure 3), which indicates that the construction of the VIGS silencing system is successful. The silencing efficiency detection of the target gene (figure 4) shows that the expression of GhABC1K14-A09 in TRV2: ghABC1K14-A09 plants is obviously inhibited compared with a control group, the silencing efficiency reaches 100 percent (36/36), and the analysis of expression quantity of VIGS silencing plants and control plants shows that the target gene is silenced.

2.3 phenotypic analysis of plants with drought and salt stress on target Gene silencing

Selecting four-week-old VIGS silencing strains, and carrying out drought and salt stress treatment on target gene silencing TRV2: ghABC1K14-A09 and control TRV2:00 plants. As a result, it was found (FIG. 5) that 12 days after PEG and 200mmol/LNaCl treatment, the treated group showed complete cotyledons abscission and wilting and yellowing of the true leaves of the plants injected with the TRV2:00 bacterial solution and the target gene-silencing bacterial solution, and the TRV2: ghABC1K14-A09 silencing plants were found to lose water and have more severe yellowing and wilting than the true leaves of the TRV2:00 strain, indicating that the GhABC1K14-A09 gene participates in the drought and salt tolerance reaction of upland cotton.

2.4 Effect of drought and salt stress on the MDA content of target Gene silenced Cotton leaves

MDA content detection analysis is carried out on the stressed GhABC1K14-A09 gene silencing plant and the empty carrier plant (figure 6), the MDA content of the GhABC1K14-A09 silencing plant under the stress of PEG and salt is obviously increased, and the fact that the cotton resistance is reduced due to the silencing of the GhABC1K14-A09 gene is shown.

2.5 Effect of drought and salt stress on the Activity of antioxidant enzymes in target Gene silencing Cotton leaves

The antioxidant enzyme (SOD, POD and CAT) activities of the silenced plants were tested, and as shown in FIG. 7, the CAT, SOD and POD activities of GhABC1K14-A09 silenced plants showed different decreases after 10 days of drought and salt stress compared with the TRV2:00 control plants.

2.6 Effect of drought and salt stress on silencing soluble sugar content of Cotton leaves in target Gene

Silencing of the GhABC1K14-a09 gene resulted in a decrease in soluble sugar content compared to soluble sugar content in control TRV2:00 leaves and was found to be more reduced under salt stress than under PEG stress (fig. 8), indicating that silencing of the GhABC1K14-a09 gene increased cotton sensitivity to drought and salt stress and was more sensitive to salt stress responses.

2.7 Effect of drought and salt stress on chlorophyll content in target Gene silenced Cotton leaves

When plants are subjected to high-intensity abiotic stress, obvious yellowing and wilting phenomena occur on plant leaves. The detection of the chlorophyll content is one of important indexes for detecting the stress tolerance of plants. The chlorophyll content of cotton leaves subjected to stress is detected, and the chlorophyll content of GhABC1K14-A09 gene-silenced plants is remarkably lower than that of control plants (figure 9), which shows that the GhABC1K14-A09 gene is silenced, and the drought tolerance and the salt tolerance of cotton plants are weakened.

2.8 expression of stress genes in silencing lines by drought and salt stress

The expression of GhSOS1, ghNHX1, ghCBL3, ghCIPK6, ghSOS2, ghHDT4D, ghEREB2A, ghDREB2C, ghWRKY and GhRD29A genes in TRV2: ghABC1K14-A09 plants is obviously reduced relative to that in TRV2:00 (figure 10), and the expression of adversity stress related genes can be influenced by silencing the GhABC1K14-A09 genes.

Taken together, upland cotton silenced GhABC1K14-A09 by VIGS is more sensitive to drought and salt stress than controls, indicating decreased cotton resistance, thus demonstrating that the above genes positively regulate cotton response to drought and salt stress responses.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. The upland cotton GhABC1K14-A09 gene is characterized in that the nucleotide sequence is shown as SEQ ID NO: shown at 7.

2. A primer for amplifying the upland cotton GhABC1K14-A09 gene of claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO: 1-2.

3. A recombinant vector comprising the Gossypium hirsutum GhABC1K14-A09 gene of claim 1.

4. A recombinant bacterium comprising the recombinant vector according to claim 3.

5. The method for amplifying the GhABC1K14-A09 gene of the upland cotton of claim 1, wherein the GhABC1K14-A09 gene of the upland cotton is obtained by performing PCR amplification using cDNA of cotton as a template and the primers of claim 2.

6. Use of the gossypium hirsutum GhABC1K14-A09 gene of claim 1, the primer of claim 2, the recombinant vector of claim 3 or the recombinant bacterium of claim 4 for drought and/or salt tolerance of plants.

7. The use of claim 6, wherein drought and/or salt tolerance of said plant is increased by up-regulating the level of the gossypium hirsutum GhABC1K14-A09 gene in said plant.

8. Use according to any one of claims 6 to 7, wherein the plant is a cotton plant of the family Malvaceae.

9. Use according to claim 8, wherein the plant comprises upland cotton.

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