CN117904181A - Application of upland cotton GhANN gene in drought resistance and salt tolerance of upland cotton - Google Patents
Application of upland cotton GhANN gene in drought resistance and salt tolerance of upland cotton Download PDFInfo
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Abstract
The invention discloses application of a upland cotton GhANN gene in drought resistance and salt tolerance of upland cotton, and belongs to the technical field of genetic engineering. The nucleotide sequence of GhANN gene is shown as SEQ ID NO: 1. The invention also discloses application of GhANN gene in improving drought resistance and salt tolerance of upland cotton. The invention discovers that GhANN gene is obviously expressed in salt and drought stress, and silences GhANN gene in upland cotton, and discovers that GhANN gene silencing causes leaf wilting of upland cotton, reduction of antioxidant enzyme activity, increase of MDA content and reduction of expression of related genes of adversity stress after drought and salt stress treatment, namely, silences GhANN gene weakens drought resistance and salt tolerance of cotton, which proves that GhANN gene positively regulates and controls upland cotton to respond to drought and salt stress. The invention provides important gene resources for the upland cotton to cope with abiotic stress.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a upland cotton GhANN4 gene 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 fibers in the textile industry. Wherein upland cotton (Gossypium hirsutum L.) is used as one of the cultivars of cotton crops, and occupies 95% of the annual cotton yield worldwide, and has wide application prospect. Cotton planting and production is mainly performed in arid and semiarid regions, cotton exhibiting higher drought tolerance than rice, wheat and corn. However, moisture and salt-alkali limitations still have a significant impact on cotton fiber yield and lint quality, a major non-biological factor affecting cotton production worldwide. Although the main goal of cotton breeders has been to increase productivity and fiber quality for many years, changes in climatic factors and extreme weather frequency events, such as drought and salt and alkali, pose a significant threat to cotton production.
The Annexin gene family belongs to a polygene protein family, members of the Annexin gene family are commonly existing in plants and play an important role in plant growth and development and response to adversity stress, but the excavation and screening utilization of drought resistance and salt resistance gene resources in cotton Annexin family members are not seen up to the present.
Disclosure of Invention
The invention aims to provide application of a upland cotton GhANN-4 gene in drought resistance and salt tolerance of upland cotton, so as to solve the problems in the prior art, the upland cotton GhANN-4 gene positively regulates and controls cotton to respond to drought and salt stress, and GhANN-4 gene is up-regulated in upland cotton to improve the drought resistance and salt tolerance of upland cotton.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides an application of a upland cotton GhANN gene in regulation and control of drought resistance and/or salt tolerance of upland cotton, wherein the nucleotide sequence of the upland cotton GhANN gene is shown as SEQ ID NO: 1.
The invention also provides application of the protein coded by the upland cotton GhANN gene in regulation and control of drought resistance and/or salt tolerance of upland cotton, and the nucleotide sequence of the upland cotton GhANN gene is shown as SEQ ID NO: 1.
The invention also provides application of the recombinant vector in regulation and control of drought resistance and/or salt tolerance of upland cotton, and the recombinant vector comprises the upland cotton GhANN gene.
The invention also provides application of the recombinant strain in regulating drought resistance and/or salt tolerance of upland cotton, and the recombinant strain comprises the recombinant vector.
Further, drought and/or salt tolerance of upland cotton is improved by up-regulating the expression level of the upland cotton GhANN gene in upland cotton.
The invention also provides a method for improving drought resistance and/or salt tolerance of upland cotton, which comprises the step of up-regulating the expression level of the gene GhANN of upland cotton in upland cotton.
Further, the up-regulating the expression level of the upland cotton GhANN gene in upland cotton comprises the steps of: introducing the upland cotton gene or the recombinant vector or the recombinant bacterium into a upland cotton plant.
The invention discloses the following technical effects:
The invention adopts gene family analysis to mine candidate genes related to growth and development, and discovers that GhANN gene plays an important role in cotton abiotic stress. In order to further define the regulation mechanism of GhANN gene in abiotic stress, the target gene is cloned, a VIGS silencing vector of the target gene is constructed, and the abiotic function is researched, so that after drought or salt stress treatment, ghANN gene silencing leads to more obvious leaf wilting of upland cotton, the activity of antioxidant enzymes (CAT, POD and SOD) in the leaf is reduced, the MDA content is increased, and the expression level of the gene related to the stress of a silencing plant is reduced. The silencing GhANN gene weakens drought resistance and salt tolerance of upland cotton, so that the GhANN gene is proved to positively regulate and control the response of upland cotton to drought and salt stress, and important gene resources are provided for the upland cotton to cope with abiotic stress.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are 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 other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a map of pEASY-T5Zero vector and a map of TRV vector;
FIG. 2 shows the results of PCR amplification and double digestion of the desired gene and silencing vector construct; a, target gene PCR amplification result; b, a target gene VIGS fragment and a silencing vector construct bacterial liquid PCR amplification result; c, carrying out double enzyme digestion on the target gene and the silencing vector construct to obtain a PCR detection result of target gene bacterial liquid; wherein, marker: DNA molecular weight standard Marker2000 (bp); 1-4: ghANN4-1,2,3,4;1-3: ghANN4, 4-1,2,3;
FIG. 3 shows the TRV GhCLA positive control phenotype;
FIG. 4 shows the results of the detection of silencing efficiency of a gene of interest in a TRV GhANN4 plant;
FIG. 5 is a phenotypic analysis of upland cotton plants under drought and salt stress of TRV 00 and TRV GhANN; wherein, CK: a stress-free control group; PEG stress: drought stress; naCl stress: salt stress;
FIG. 6 shows the activity of the target gene silencing plant antioxidant enzymes (SOD, POD and CAT) and the MDA content detection; a: an index under drought stress; b: index under salt stress;
FIG. 7 is a diagram showing the expression of stress-related genes in a silent plant; a: drought stress related gene expression; b: salt stress related gene expression;
FIG. 8 is a sequencing drawing of cloning product of upland cotton GhANN 4.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 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 invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 application of upland cotton GhANN4 Gene in drought resistance and salt tolerance of upland cotton
1. Experimental materials and reagents
1.1 Experimental materials
The material used in the invention is upland cotton variety Xinshi K18, and seeds are provided by shihe seed agricultural science institute.
1.2 Experimental reagents
The reagents used in the invention are shown in Table 1:
Table 1 reagents used in the experiments
1.3 Strain and vector
GV3101 Agrobacterium competent cells and pEASY-T5Zero cloning vector (CT 501-01, DH 5. Alpha. Containing E.coli competent cells) were purchased from Shanghai Weidi Biotechnology Inc. and full gold Biotechnology Inc., respectively, and the VIGS vector system for gene silencing (including TRV1, TRV2 and TRV-GhCLA) was given away by the cotton institute transgenic subject group of China academy of agriculture, and the pEASY-T5Zero vector map is shown in FIG. 1.
1.4 Medium and solution preparation
A. the formulation of the medium is shown in Table 2:
table 2 culture medium formulation
Note that: the culture medium is sterilized at 121deg.C for 20min
B.100mL 50 xTAE buffer: 24.2g Tris+3.72gNa 2EDTA·2H2 o+5.71mL glacial acetic acid;
50mg/mL rifampicin (Rif) and 20mg/mL Acetosyringone (AS): respectively dissolving 5g of Rif powder and 2g of AS powder in 100 mM DS SO solution, respectively filtering and sterilizing with 0.22 μm filter membrane, packaging, and preserving at-20deg.C;
d.0.5M morpholinoethanesulfonic acid (MES): 10.65g MES powder is dissolved in 50mL ddH 2 O, the pH value is adjusted to 5.6 by NaOH, the volume is fixed to 100mL, and the mixture is filtered and sterilized by a filter membrane with the thickness of 0.22 mu m, packaged and stored at the temperature of 4 ℃;
e.1m MgCl 2: 9.521g of MgCl 2 powder (heat of amplification) was dissolved in 100mL of ddH 2 O, sterilized by filtration through a 0.22 μm filter and stored at 4 ℃;
f.500mL of heavy suspension: 10mL 0.5M MES+1mL 20mg/mLAS +5mL of 1M MgCl 2, sterile ddH 2 O up to 500mL.
1.5 Major instruments
Visible spectrophotometers, high-speed refrigerated centrifuges, water baths, electrophoresis apparatuses, artificial climate boxes, gel cutting apparatuses, ultra-low temperature refrigerators, fluorescence quantitative apparatuses, gradient PCR apparatuses, sterilization pans, electronic balances, microwave ovens, table-type constant temperature shaking tables, ice-making machines, ultra-clean benches, biochemical incubators, ultra-micro spectrophotometers, ultra-pure water apparatuses and mini vortex mixing apparatuses.
2. Test method
2.1 Primer design
Cloning primers, qRT-PCR primers, and VIGS silencing primers (the size of the VIGS silencing primer product fragment is 300-500 bp) of the GhANN gene (SEQ ID NO. 1) were designed using NCBIPrimer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/primer-BLAST /), and the primer sequences are shown in Table 3:
TABLE 3 list of primers used in the present invention
Note that: the base fragments shown under the underlines in the table are cleavage sites.
2.2 Extraction and reverse transcription of RNA
2.2.1RNA extraction
RNA was extracted according to the instructions of the polysaccharide polyphenol plant total RNA extraction kit (DP 441, tiangen).
2.2.2 Reverse transcription
A. Taking out the packaged RNA from the refrigerator at-80 ℃ and melting on ice, taking out 5X FastKing-RT Supermix reagent and RNase-Free ddH 2 O from the refrigerator at-20 ℃ and melting on ice, and mixing by light shaking;
b. The reaction system is shown in Table 4:
TABLE 4 reverse transcription reaction system
C. The reaction procedure is shown in Table 5:
Table 5 reaction procedure
D. After the reaction, the purity and concentration of cDNA were measured, and the cDNA was packaged and stored at-20 ℃.
2.3 Amplification of fragments of interest and ligation and transformation of cloning vectors
2.3.1 Amplification of fragments of interest
Amplification of the target gene was performed using a cDNA of Sinorhizite K18 as a template and Taq 2X PCRMix with Dye V2 premix (containing dye) kit, and the amplification system was as shown in Table 6:
TABLE 6 amplification System
After the reaction solution is added according to the system, the mixture is gently mixed, centrifuged for a short time, and the reaction is carried out according to the reaction procedure of Table 7:
TABLE 7 amplification reaction procedure
After the completion of the reaction, 1.8% agarose gel electrophoresis was used to determine whether the size of the target gene was consistent with that expected. The results showed that the sequence of the amplified product (SEQ ID NO. 2) was consistent with the expected size.
The full length of the gene of upland cotton GhANN has a nucleotide sequence shown as SEQ ID NO. 1.
SEQ ID NO.1：
ATGGCCACTCTTACAGTGCCCACGACAGTTCCTTCAGTGTCTGAAGATTGTGAACAACTAAGAAAAGCCTTTTCAGGATGGGGAACTAATGAGGGCTTAATCATAGATATATTGGGTCACAGAAATGCGGAGCAACGAAACTTGATTCGAAAAACCTACGCTGAAACCTATGGAGAGGATCTCCTCAAGGCACTAGACAAGGAACTCTCGAATGACTTTGAGAGGCTGGTTCTGCTTTGGGCTCTTGATCCTGCTGAACGTGATGCCCTTTTGGCTAATGAAGCCACCAAAAGGTGGACTTCAAGCAATCAAGTCCTTATGGAAATAGCCTGCACAAGGTCTGCCAACCAACTGCTTCACGCAAGGCAGGCTTATCATGCTCGTTATAAGAAGTCGCTTGAAGAGGACGTTGCTCATCACACGACTGGGGACTTCCATAAGCTCCTCCTACCTCTAGTGAGTTCATACAGATATGAGGGAGAGGAGGTGAACATGACTCTGGCAAAAACAGAGGCGAAGTTGCTTCATGAGAAAATTTCAAACAAAGCTTACAGTGATGACGATGTCATAAGGGTTTTGGCTACAAGAAGCAAGGCACAGATCAATGCAACTCTGAATCACTACAAAAATGAATATGGAAATGACATAAACAAGGACTTGAAGGCTGACCCTAAGGATGAGTTCCTTGCACTACTAAGGTCCACAGTGAAGTGCTTGGTCTATCCGGAAAAGTATTTTGAGAAGGTTCTTCGCCTAGCAATCAATAGACGAGGAACGGATGAAGGAGCTCTTACAAGAGTTGTTTGCACTAGGGCTGAGGTGGATCTAAAGATCATAGCAGATGAGTATCAGCGAAGGAACAGTGTCCCACTGACTCGTGCCATTGTCAAGGACACTCATGGAGACTATGAAAAATTGCTGCTGGTACTTGCAGGACATGTGGAGAATTGA;
Cloning gave a gene sequence silencing fragment of GhANN (SEQ ID NO. 2).
SEQ ID NO.2：
CGGAATTCCACAGAAATGCGGAGCAACGAAACTTGATTCGAAAAACCTACGCTGAAACCTATGGAGAGGATCTCCTCAAGGCACTAGACAAGGAACTCTCGAATGACTTTGAGAGGCTGGTTCTGCTTTGGGCTCTTGATCCTGCTGAACGTGATGCCCTTTTGGCTAATGAAGCCACCAAAAGGTGGACTTCAAGCAATCAAGTCCTTATGGAAATAGCCTGCACAAGGTCTGCCAACCAACTGCTTCACGCAAGGCAGGCTTATCATGCTCGTTATAAGAAGTCGCTTGAAGAGGACGTTGCTCATCACACGACTGGGGGTACCCC.
2.3.2 Ligation of the Gene of interest to the cloning vector pEASY-T5Zero
A. the pEASY-T5Zero vector was removed from the-80℃refrigerator and thawed on ice.
B. The volume of the added target fragment (molar ratio of carrier to target fragment=1:5) was calculated and the following ingredients (whole procedure completed on ice) were added to a sterile 1.5mL centrifuge tube:
table 8 connection system
C. The mixture was gently mixed, centrifuged briefly, and then connected at 25℃for 5 minutes.
2.3.3 Transformation of DH 5. Alpha. E.coli competent cells
The strain is transformed into DH5 alpha escherichia coli competent cells by a heat shock method, and bacterial liquid PCR verification and sequencing (completed by Shanghai biological engineering Co., ltd.) are carried out by using a target gene sequence primer.
2.4 Construction of silencing vectors
The positive plasmid which is successfully sequenced in the DH5 alpha escherichia coli competent cell transformed by 2.3.3 is taken as a template, a silencing fragment is amplified by adding primers of restriction enzymes EcoR I and Kpn I cleavage sites and protective bases, and a GhANN4 fragment is inserted into a TRV2 silencing vector by a double cleavage method to construct the TRV2: ghANN4 silencing vector, wherein the specific cleavage system is as follows: stopping the reaction after adding 10X Loading Buffer at 37 ℃ for 3 hours, recovering the target gene fragment PCR product by gel, recovering the large fragment by enzyme digestion of the carrier, connecting the target fragment with a silencing carrier, converting the connecting product into competent cells of escherichia coli, carrying out bacterial liquid PCR and double enzyme digestion identification, and after the completion, sequencing positive plasmids (Shanghai process), transferring the positive plasmids into the competent cells of agrobacterium GV3101, wherein the operation steps are as follows:
a. GV3101 competent cells were removed from the-80℃ultra-low temperature refrigerator, thawed on ice, split into two tubes, 2. Mu.L of the successfully sequenced plasmid was aspirated, and added to centrifuge tubes for transformation.
B. After the above steps were completed, 350. Mu.L of LB liquid medium (without antibiotics) was added, shaking culture was performed for 2 hours (28 ℃,200 rpm), the bacterial liquid was uniformly spread on the solid medium (Kan + and Rif antibiotics were added), and the culture was performed for 2 days in the dark at 28 ℃.
C. After the culture is completed, picking the single colony into 5mL LB liquid culture medium (adding Kan + and Rif antibiotics), and culturing for 16h according to the condition of shaking culture in the step b;
d. After the cultivation is completed, the bacterial liquid is preserved by using 50% glycerol (bacterial liquid: glycerol=1:1), and is preserved at 80 ℃ for standby; the bacterial liquid PCR was performed to confirm that the vector was positive.
2.5 Upland cotton VIGS silencing target Gene
VIGS silencing of new stone K18 seedlings was performed as follows:
a. Planting new stone K18 seeds, soaking the seeds until the seeds grow to seventh day and the cotyledons are fully unfolded until nutrient soil in the flowerpot absorbs water to the surface, stopping soaking, and standing for later use.
B. Kan + and Rif were added to LB liquid medium for use, wherein the final concentrations of Kan + and Rif were 50. Mu.g/mL and 25. Mu.g/mL, respectively. The VIGS vector system and the objective gene bacterial liquid taken out from-80 ℃ were thawed on ice, at 28 ℃, activated at 200rpm for 16h (bacterial liquid: LB liquid medium=1:10). And after the activation is finished, the propagation is carried out according to the same proportion.
C. after the bacterial liquid is propagated, centrifuging for 10min at a rotation speed of 5000rpm, pouring out supernatant, retaining bacterial cells, suspending the bacterial cells by using a spectrophotometry heavy suspension, and adjusting the OD 600 to 0.8.
D. After resuspension was completed, the cells were left in the dark for 3 hours, and after resuscitating, TRV1 was mixed with 1:1 of a cell mass suspension containing TRV2:00 (blank control group), TRV2: ghCLA (positive control group) and TRV2: ghANN4 (experimental group), respectively, and thoroughly mixed.
E. On the seventh day of the growth of upland cotton seedlings, they were soaked in water according to the method of step a. VIGS injections were performed on the eighth day of upland cotton seedling growth, with specific manipulations: and d, cutting the back of the cotyledon by using a 1mL syringe needle (the wound is not too large and the needle point is just large), injecting the mixed bacterial liquid in the step d into the upland cotton cotyledon, and filling the whole cotyledon with the bacterial liquid as much as possible.
F. after injection, in order to achieve better infection effect, the seeds are wrapped by a plastic bag, and are cultivated under normal growth conditions after being placed in darkness at 25 ℃ for 24 hours.
2.6 Identification of silenced plants
And after the positive control upland cotton seedlings whiten, adopting experimental groups and blank group upland cotton young leaves to carry out fluorescent quantitative experiments, and detecting the silencing efficiency.
2.7 Drought and salt stress treatments of Gene-silenced cotton plants
After the injected positive cotton seedlings are whitened, blank control and experimental groups are respectively subjected to salt and drought stress treatment by using 400mmol/LNaCl and 15% PEG for carrying out phenotype observation on cotton seedlings grown to four weeks of age.
2.8 Determination of physiological index of Gene-silenced cotton leaf
And detecting the activities of antioxidant enzymes (SOD, POD and CAT) and MDA content of cotton leaves of a blank control group and an experimental group respectively according to a conventional method so as to analyze the response of a target gene silencing plant to drought and salt stress.
2.9 Fluorescent quantitative detection of adversity stress related Gene in Gene-silenced Cotton plants
Young leaves of the gene silencing line are picked before and after stress treatment, RNA is extracted according to RNAprep pure polysaccharide polyphenol plant total RNA extraction kit (product number DP441, purchased from Tian Gen Biochemical technologies Co., ltd.), reverse transcription is performed by a FastKing one-step method to remove a first strand of genomic cDNA synthesis premix kit (product number KR118, purchased from Tian Gen Biochemical technologies Co., ltd.), and finally expression level detection of stress-related genes is performed by a SuperReal fluorescent quantitative premix reagent enhancer kit (product number FP205, purchased from Tian Gen Biochemical technologies Co., ltd.) for specific methods.
Wherein in drought stress treatment, the stress related genes detected by the TRV GhANN4 silent plants are GhCBL3, ghDREB2A, ghDREB2C, ghPP2C, ghRD-2 and GhRD29A.
In the salt stress treatment, the stress related genes detected by the TRV GhANN4 silent plants are GhCIPK6, ghNHX1, ghRD20-1, ghSOS1, ghSOS2 and GhSNRK2.6.
3. Results and analysis
3.1 Construction of the Gene fragment of interest and silencing vector
The primer was designed with NCBI blast-primer, cDNA of New Zeolite K18 was used as a template, and Taq 2XPCRMix with Dye V2 enzyme of Eboltaaceae was used to amplify the target fragment, and a universal DNA purification and recovery kit (Tiangen) was used to recover the target fragment. The recovered target fragment was ligated with pEASY-T5 Zero vector (shown in FIG. 1) to select plasmids. The target fragment (A in FIG. 2) was ligated with the TRV156 vector by a double digestion method and cultured, and positive plasmids were obtained by bacterial liquid PCR (B in FIG. 2), double digestion (C in FIG. 2) and sequencing (FIG. 8). The target fragment ligated to the vector was obtained by sequencing without base deletion and substitution, and thus sequencing was successful. The positive plasmid was transformed into Agrobacterium by freeze thawing for cultivation.
3.2 Detection of silencing efficiency of the Gene of interest and phenotypic analysis
Screening out candidate genes for the growth and development of upland cotton based on gene family data analysis. Therefore, the VIGS technology is utilized to research the effect of the target gene in drought resistance and salt stress regulation of upland cotton. The gene GhANN of the present invention was selected for the VIGS test. Silencing the desired gene in upland cotton new stone K18 by epidermis injection under cotyledons revealed that the positive control (TRV: ghCLA) plants began to whiten on day 8 after infection, and that the positive control whitened significantly on day 12 after silencing, indicating that the Agrobacterium infection of upland cotton was successful (as shown in FIG. 3). The silencing efficiency of the target gene was detected to show that GhANN expression in the TRV GhANN4 plants was significantly inhibited compared to the control group (as shown in FIG. 4). Analysis of the expression levels of VIGS-silenced plants and control plants indicated that the gene of interest had been silenced.
3.3 Phenotypic analysis of Gene-silenced plants of interest by drought and salt stress
Four weeks old VIGS silenced lines were selected, including target gene silencing TRV GhANN, and control TRV 00 plants were subjected to drought and salt stress treatments. As shown in FIG. 5, after 35 days of treatment with 15% PEG and 400mmol/LNaCl, the cotyledons of the treated plants had fallen off completely, the true leaves appeared to wilt and yellow, and it was found that the TRV GhANN 4-silenced plants had severely dehydrated, and the yellow and wilt were also more severe than the TRV 00-line true leaves, indicating that the GhANN gene was involved in drought-resistant and salt-tolerant reactions of upland cotton.
3.4 Effect of drought and salt stress on the antioxidant enzyme Activity of Gene-silenced cotton leaves of interest
The antioxidant enzyme (SOD, POD and CAT) activities of the silenced plants were examined, and as shown in FIG. 6, CAT, SOD and POD activities of GhANN4 silenced plants showed a decrease in various degrees after drought stress for 30 days, compared with untreated plants. After 30 days of salt stress, ghANN4 silenced plants showed a different decrease in SOD and POD activity than untreated plants.
3.5 Effect of drought and salt stress on MDA content of target Gene-silenced Cotton leaf
MDA content detection analysis is carried out on the GhANN gene silencing plant after stress and the empty vector plant, and found (as shown in figure 6), the content of MDA in the GhANN silencing plant under drought stress is obviously increased compared with that in untreated plants, which indicates that the silencing of GhANN gene causes the reduction of cotton resistance.
3.6 Effect of drought and salt stress on expression of Gene-silenced Cotton stress-related Gene
Compared with the control plant, the expression of the stress related gene of the TRV GhANN4 plant is obviously changed before and after drought stress treatment.
As shown in FIG. 7A, the expression levels of GhCBL, ghDREB2A, ghDREB2C, ghPP C were significantly lower in the silenced plants TRV GhANN4 than in the control plants before and after treatment as compared to TRV 00. The expression level of GhRD-2 in the silenced plant TRV GhANN4 was higher than that of the control plant before treatment, but the expression level of GhRD20-2 was significantly lower than that of the control plant after drought stress treatment. The expression level of GhRD A in the silenced plant TRV GhANN4 was higher than that in the control plant, both before and after the treatment.
Compared with the control plant, the expression of the stress related gene of the TRV GhANN4 plant is obviously changed before and after the salt stress treatment.
As shown in FIG. 7B, the expression levels of GhCIPK, ghRD20-1 were significantly lower in the silenced plants TRV GhANN4 than in the control plants, both before and after treatment, as compared to TRV 00. The expression level of GhSOS1 in the silenced plant TRV GhANN4 was higher than that of the control plant before the treatment, but the expression level of GhSOS1 was significantly lower than that of the control plant after the salt stress treatment. Expression of GhNHX1, ghSOS2 and ghsnrk2.6 was significantly up-regulated in control plants, and significantly down-regulated in silent plants after salt stress treatment.
The results indicate that GhANN4 may have a positive regulatory effect on cotton against drought and salt stress.
In conclusion, upland cotton with VIGS silence GhANN4 is more sensitive to drought and salt stress than a control group, so that the drought resistance and salt tolerance of cotton are reduced, and therefore, the fact that GhANN4 gene positively regulates cotton to respond to drought and salt stress is proved, and the drought resistance and salt tolerance of upland cotton can be enhanced by over-expressing GhANN gene in upland cotton. The invention provides important gene resources for high stress resistance breeding of upland cotton against abiotic stress.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. An application of a upland cotton GhANN gene in regulating drought resistance and/or salt tolerance of upland cotton, which is characterized in that the nucleotide sequence of the upland cotton GhANN gene is shown as SEQ ID NO: 1.
2. The application of protein coded by upland cotton GhANN-4 gene in regulation and control of drought resistance and/or salt tolerance of upland cotton is characterized in that the nucleotide sequence of the upland cotton GhANN-gene is shown as SEQ ID NO: 1.
3. Use of a recombinant vector for regulating drought and/or salt tolerance of upland cotton, wherein the recombinant vector comprises the upland cotton GhANN gene according to claim 1.
4. Use of a recombinant bacterium for controlling drought and/or salt tolerance of upland cotton, wherein the recombinant bacterium comprises the recombinant vector of claim 3.
5. The use according to any one of claims 1 to 4, wherein the upland cotton drought and/or salt tolerance is increased by up-regulating the expression level of the upland cotton GhANN gene in upland cotton.
6. A method for improving drought and/or salt tolerance of upland cotton, comprising the step of up-regulating the expression level of the upland cotton GhANN gene in upland cotton.
7. The method of claim 6, wherein the method of up-regulating the expression level of the gossypium hirsutum GhANN gene in gossypium hirsutum comprises the steps of: introducing the upland cotton gene of claim 1 or the recombinant vector of claim 3 or the recombinant bacterium of claim 4 into a upland cotton plant.
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