CN115044563B - Application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants - Google Patents
Application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants Download PDFInfo
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- CN115044563B CN115044563B CN202210717614.6A CN202210717614A CN115044563B CN 115044563 B CN115044563 B CN 115044563B CN 202210717614 A CN202210717614 A CN 202210717614A CN 115044563 B CN115044563 B CN 115044563B
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Abstract
The invention provides application of a cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants, and relates to the technical field of plant molecular biology. The cotton gene is GhSINA12 gene, and the experimental study of the invention shows that when upland cotton is subjected to drought stress, the GhSINA12 gene obviously up-regulates expression. Meanwhile, the cotton GhSINA12 transgenic line has positive regulation and control effect in drought stress response. The gene silencing technology induced by virus is utilized to silence and express cotton GhSINA12 gene, which shows that the drought tolerance of GhSINA12 silencing line in drought stress is obviously reduced. The invention shows that the cotton GhSINA12 gene can respond to drought stress, improves drought resistance of plants to a certain extent, and can regulate and control drought resistance of plants by relying on an ABA passage. The invention provides a new method for regulating and controlling drought stress response of target plants, and cultivating and screening drought-resistant plants.
Description
Technical Field
The invention relates to the technical field of plant molecular biology, in particular to application of a cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants.
Background
The trend of global climate gradually towards more drought and more lack of precipitation (ARNETH et al, 2019) drought has become a major factor leading to crop yield reduction, and is one of the major environmental stresses suffered in plant growth and development, and the cultivation of high-quality drought-resistant plants is urgent.
When a plant is subjected to drought stress, the change of photosynthesis of the plant characterizes the degree of the plant subjected to the drought stress. When the water is insufficient, the expansion degree of the air holes is limited, and CO 2 The decrease in absorption efficiency directly leads to a decrease in photosynthesis (FLEXAS et al, 2004). Further, studies have shown that chlorophyll is also one of the important factors in drought stress resistance, and that when plants are subjected to drought stress, the chlorophyll content in the plantsAnd consequently decrease, thereby affecting photosynthesis (FAROOQ et al, 2009). In addition, active oxygen metabolism is also critical in drought stress resistance of plants. Reactive Oxygen Species (ROS) are present when O 2 Substances which are not completely reduced when utilized by aerobic organisms, ROS are in a state of dynamic equilibrium in plants under normal growth conditions, but when the plants are stressed, the imbalance of ROS pathway causes ROS to increase, but when ROS increase to a certain extent, toxic effects are generated on the plants, so that some components in plant cells are damaged (ROUT and SHA,2001:FAROOQ, etc., 2009). Active oxygen scavenging relies on antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and Catalase (CAT) to reduce plant injury under drought stress (MILLER et al, 2010). Secondly, plant hormones are also involved in various processes of drought stress resistance of plants, and major hormones such as abscisic acid (ABA), cytokinin (CK), gibberellin (GA), auxin (IAA), ethylene (ETH) and the like (WILKINSON et al 2012), wherein ABA is a major hormone that responds to abiotic stress of plants.
Agricultural drought has become an important factor limiting cotton yield and quality. The growth and development of cotton, such as plant height, leaf area index, fiber quality, canopy and root development, are severely restricted by drought stress (LOKA et al, 2011). Several common physiological strategies for drought stress resistance have evolved in cotton, such as the GaMYB85 gene of cotton to enhance plant drought tolerance by decreasing stomatal density, the GhWRKY41 gene of cotton to positively regulate drought resistance in plants, and to enhance drought resistance by regulating stomatal closure and expression of antioxidant related genes (BUTT et al, 2017). The research shows that the overexpression of the AtLOS5 gene in cotton improves the abscisic acid level of transgenic cotton and drought resistance of plants, the accumulation of endogenous ABA and proline in medium cotton institute 35 (Z35) and transgenic cotton under normal conditions is similar, but drought stress greatly increases the content of endogenous ABA and proline in Z35 and transgenic cotton leaves, transgenic cotton accumulates 25% more endogenous ABA than Z35 cotton, and the result shows that the AtLOS5 transgenic cotton obtains better drought resistance phenotype by improving the ABA content and ABA-induced physiological regulation (YUE and the like, 2012).
ubiquitin-26S protease system (UPS) is a ubiquitous protein modification mechanism, mainly mediates posttranslational modification of protein and degradation of substrate protein, and plays an irreplaceable role in various aspects of plant growth and development, including physiological processes of plant growth, stress and the like. Enzymes involved in the ubiquitination process, mainly including three enzymes, E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme and E3 ubiquitin ligase, E3 ligase plays an important role in many cellular processes. In plants, members of the part SINA (Seven in absentia) family are part of the E3 ubiquitin ligase complex and the SINA family is highly conserved. Currently, SINA E3 ubiquitin ligases have been found in a variety of plants, wherein 10, 5 and 6 SINA E3 ubiquitin ligases have been identified in Populus euphratica, arabidopsis thaliana and rice, respectively, and play a very important role in various aspects of plant life activities.
The molecular biology means is utilized to find drought-resistant genes, define drought-resistant mechanisms of cotton, and create drought-resistant cotton varieties, which is an effective way for solving the problem of drought stress. In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants, and provides a novel method for regulating and controlling drought stress response of target plants and cultivating and screening drought resistant plants.
In one aspect, the invention provides an application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants, wherein the cotton gene is GhSINA12 gene.
After the cotton is subjected to simulated drought treatment, in 24 upland cotton SINA genes, the real-time fluorescence quantitative (qRT-PCR) analysis shows that the expression quantity of the GhSINA12 gene is obviously induced under the treatment of PEG6000 (the induction expression of the GhSINA12 is most obvious under the condition of 15% of PEG6000 treatment at the time of 6h and 12 h); meanwhile, in natural drought, the transcription level of GhSINA12 gene is obviously increased. Subcellular localization experiments were performed on GhSINA12 and localization of GhSINA12 to the nucleus was demonstrated by DAPI staining.
In vitro ubiquitination experiments are carried out by prokaryotic expression and purification of the GhSINA12 protein fused with MBP tag, ubiquitin activating enzyme E1 and ubiquitin binding enzyme E2 (UbcH 5 b) are utilized, and are incubated with protein to be detected (MBP-GhSINA 12, MBP), SDS-PAGE analysis is carried out, and detection is carried out through Biotin antibody anti-Biotin, so that the GhSINA12 has E3 ubiquitin binding enzyme activity.
In the present invention, the CDS sequence of the GhSINA12 (GH_A13G0056) gene may be downloaded from Cotton genome database Cotton FGD (https:// cottonfgd.
In another aspect, the invention provides an application of a biological material containing the GhSINA12 gene or a protein encoding the GhSINA12 gene in improving drought resistance of plants.
The RT-PCR and qRT-PCR are used for detecting the change of the expression level of the GhSINA12 gene in the T3 generation overexpression line of the arabidopsis, and the result shows that the exogenous gene GhSINA12 has different degrees of expression in the transgenic arabidopsis. Arabidopsis thaliana T transformed with GhSINA12 gene 3 The generation and wild type Arabidopsis seeds were sown on MS medium containing different concentrations of mannitol, resulting in over-expressed lines with root lengths significantly longer than the WT line. The GhSINA12 transgenic line has positive regulation and control effect in drought stress response.
A VIGS experiment is carried out on TM-1 cotton, the GhSINA12 gene is silenced and expressed through a virus-mediated gene silencing technology, the drought resistance of GhSINA12 gene silencing plants is obviously reduced after about 8 days of natural drought treatment, a wilting phenotype appears in advance on TRV 00 plants, and the simulation result of PEG6000 drought treatment is the same as that of natural drought stress. In addition, SOD, POD, CAT of the TRV 00 strain is obviously higher than that of the TRV GhSINA12 strain, and MDA is obviously smaller than that of the TRV GhSINA12 strain, which shows that after GhSINA12 gene silencing, drought resistance of cotton plants is obviously reduced.
After the GhSINA12 gene is silenced, the expression level of genes related to drought stress, such as transcription factor BHLH1, kinase MAPKKK17, calbindin CML45, 1-aminocyclopropane-1-carboxylic acid synthase ACS1, auxin response protein SAUR36 and the like, is obviously reduced.
In one embodiment, the biological material is an expression vector, cloning vector, engineering bacterium, or recombinant cell.
In one embodiment, the plant is a dicotyledonous or monocotyledonous plant, preferably a dicotyledonous plant;
more preferably, the dicotyledonous plant comprises cotton or Arabidopsis thaliana. The cotton is preferably upland cotton.
In one embodiment, the use comprises upregulating expression of the GhSINA12 gene in the plant of interest to increase drought resistance in the plant.
In another aspect, the invention provides a method of increasing drought resistance in a plant, the method comprising increasing the expression level of the GhSINA12 gene in a plant of interest or increasing the activity or content of a protein encoding the GhSINA12 gene.
In one embodiment, the method comprises introducing the cotton GhSINA12 gene into a plant of interest to obtain a transgenic plant with increased drought resistance.
In one embodiment, the method comprises agrobacterium-mediated transformation of a plant expression vector comprising a GhSINA12 gene into a plant.
In one embodiment, the plant expression vector drives over-expression of the GhSINA12 gene by a constitutive or inducible promoter; preferably, the constitutive promoter is a 35S promoter.
In one embodiment, the GhSINA12 gene may be introduced into a plant cell, tissue or organ by a plant expression vector.
In the invention, transcriptome sequencing (RNA-seq) analysis is carried out on drought stress lines after GhSINA12 gene silencing, GO enrichment analysis and KEGG analysis are respectively carried out on up-regulated genes and down-regulated genes, wherein the enrichment degree of MAPK signal channels and plant hormone signal transduction channels is very high, and genes of abscisic acid (ABA) channels are simultaneously contained in the two channels. Part of the genome transcriptome data was then validated by qRT-PCR, which indicated that GhSINA12 might respond to cotton drought stress through ABA pathway.
In another aspect, the invention provides application of the GhSINA12 gene in identifying and/or screening drought-resistant cotton varieties, genomic DNA of cotton to be identified is used as a template, fluorescent quantitative PCR amplification is performed by using specific PCR primers of the GhSINA12 gene, and drought-resistant properties of the cotton to be identified are determined according to amplification results.
The beneficial effects are that:
the experiment shows that the GhSINA12 gene has better drought resistance than wild plants in the transgenic plants up-regulated by the gene, and the invention provides valuable gene resources for cultivating new varieties of plants with drought resistance.
The invention provides a method for regulating drought stress resistance/improving drought resistance of plants, which comprises regulating and controlling the expression quantity of GhSINA12 genes in plants, and influencing the drought resistance effect of the plants by directly regulating and controlling the expression of the genes, and has high efficiency.
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 description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the qRT-PCR analysis of cotton treated by PEG6000 for different time periods (wherein A is phenotype of 0, 1, 3, 6, 12 and 24 hours of water-cultured cotton treated by PEG 6000; B is qRT-PCR of GhSINA gene treated by PEG6000 for 0, 1, 3, 6, 12 and 24 hours);
FIG. 2 shows the expression analysis of GhSINA12 gene (wherein A is the expression level of GhSINA12 in 6h with different PEG6000 concentrations; B is the expression level of GhSINA12 in 12h with different PEG6000 concentrations; C is the expression level of GhSINA12 after 7 days of natural drought treatment);
FIG. 3 shows the structure of SINA E3 ubiquitin ligase in cotton (GhSINA 12 gene member has two highly conserved domains: RING-finger domain from N-terminal and one SINA domain from C-terminal);
FIG. 4 shows PCR products of GhSINA12 gene;
FIG. 5 shows subcellular localization of GhSINA12 protein and E3 ubiquitin ligase activity (subcellular localization of GhSINA12, scale bar = 10 μm; ghSINA12 in vitro E3 ubiquitin ligase activity assay);
FIG. 6 shows heterologous expression of GhSINA12 gene in Arabidopsis thaliana (A is the transcript level of GhSINA12 in wild-type and over-expressed GhSINA12 plants as determined by RT-PCR, action as control; B is the transcript level of GhSINA12 in wild-type and over-expressed GhSINA12 plants as determined by qRT-PCR, action as internal control);
FIG. 7 is an observation of drought resistance of Arabidopsis over-expressing GhSINA12 gene (A is phenotype of WT and GhSINA12 transgenic lines grown for 14d on 0mM, 150mM mannitol and 300mM mannitol medium; B is root length data statistics of WT and GhSINA12 transgenic lines grown for 14d on 0mM, 150mM mannitol and 300mM mannitol medium);
FIG. 8 shows TRV 00 and TRV GhSINA12 gene silencing efficiency analysis (A is soil-cultured cotton TRV GhCLA1 plant albino phenotype, B is qRT-PCR for detecting soil-cultured cotton silencing efficiency, ghHIS3 is used as an internal reference control, C is water-cultured cotton TRV GhCLA1 plant albino phenotype, qRT-PCR for detecting water-cultured cotton silencing efficiency, ghHIS3 is used as an internal reference control);
FIG. 9 shows the drought resistance observation of GhSINA12 gene-silenced cotton (A, natural drought treatment of cotton, phenotype difference about 8 days, about 10 days, detection of MDA, POD, CAT, SOD physiological index in drought stressed cotton leaves, C, simulated PEG6000 drought treatment of cotton for about 2.5 hours, and D, detection of MDA, POD, CAT, SOD physiological index in drought stressed cotton leaves);
FIG. 10 is a graph showing MAPK and gene transcript level verification associated with hormone signaling pathway;
FIG. 11 shows the variation of the transcription level of drought stress pathway and related genes (A is the enrichment of drought stress related pathway, B is the analysis of the expression level of drought stress pathway and pathway gene, and C is the transcriptome verification of two pathway genes).
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Materials and methods
Cotton plants were the upland cotton genetic standard line TM-1, tobacco plants were nicotiana benthamiana (Nicotiana benthamiana), and arabidopsis plants were arabidopsis thaliana of the Columbia type (Columbia), which are commercially available.
3. Experimental method and experimental results
1. Extraction of plant RNA and cDNA Synthesis
1.1 extraction of RNA and quality and purity detection
Samples of different tissue sites of TM-1, including roots, stems and leaves, were taken separately. Total RNA from different tissue sites was extracted using RNA prep Pure kit (TIANGEN, beijing). The extraction steps are as follows: during the proper period of cotton growth, the sample is stored at-80 ℃, the sample is taken out and placed in liquid nitrogen when in use, and then is transferred to an ultralow temperature tissue grinding instrument to be ground for 1min (the sample which is not fully ground is ground for 30s again until the sample is ground into powder); extracting RNA by using an RNA prep Pure kit (operating according to the use instruction of the kit) to obtain an RNA solution; RNA quality was measured on a 1.6% agarose gel and RNA purity was measured on a Nanodrop2000 as follows: and (3) RNA quality detection: 1.6g agarose was weighed, the agarose was melted in 100mL 1 XTAE buffer and 8. Mu.L Super was added (US/>INC) nucleic acid gel dye gel preparation. Mixing the extracted RNA with loading buffer, and adding into the sample loading hole. 125V, electrophoresis for 20min, and then transferred to a UV gel imager for observation. All samples were taken under UVThe two non-dispersive strips are adopted, the brightness of the large strip (5 Kb) is about 2 times of the brightness of the small strip (2 Kb), which indicates that the quality of the extracted RNA is high; and (3) detecting the purity of RNA: 1. Mu.L of sterilized ddH was pipetted with a pipette 2 O is used as a blank, the blank strip almost coincides with the abscissa under the detection of a Nanodrop2000 instrument, then 1 mu L of the RNA solution collected in the step (12) is sucked for detection one by one, the concentration of RNA is marked, and the OD of the RNA sample is analyzed 260/280 Value, OD of all RNA samples 260/280 The values are all about 2.0, which indicates that the purity of the extracted RNA is high.
1.2 Synthesis of plant cDNA:
with Prime Script TM RT reagent Kit with gDNA Eraser (Takara, china) cDNA was synthesized. Respectively taking 1.0 mug RNA of different tissues as templates, firstly removing genome DNA contained in a sample by using gDNA Eraser, and then synthesizing cDNA by PrimeScript RT Enzyme Mix I reverse transcription, wherein the specific steps are as follows:
(1) Preparing a system for removing genome DNA (prepared according to a system in table 1); (2) incubation at 42℃for 5min; (3) immediately and rapidly transferring to ice for cooling; (4) Preparing a reverse transcription system (prepared according to the system of Table 2 on ice); (5) the following procedure was performed in a PCR apparatus: 16min at 37 ℃; 5s at 85 ℃;4 ℃ Pause; (6) Immediately transfer to ice and use sterilized ddH for cDNA 2 O is diluted 4-5 times. (7) storing at-20 ℃.
TABLE 1 removal of genomic DNA System
TABLE 2 reverse transcription System
2. Real-time fluorescent quantitative (qRT-PCR) experiment
The primer-Blast tool in NCBI website was used to design GhSINA primer, and the specific primer of GhSINA was designed, the primer sequence is shown in Table 3 below.
TABLE 3 real-time fluorescent quantitative primers
qRT-PCR was performed using Light Cycler 480II system (Roche, germany) with ChamQ Universal SYBR qPCR Master Mix (Nanjing, norpran) as reagent and GhHIS3 as reference gene. Each sample was subjected to 3 technical replicates. With 2 -ΔCT qRT-PCR data were analyzed by the method. qRT-PCR experimental procedure referring to the specification, the following is specific:
(1) qRT-PCR system is shown in Table 4:
TABLE 4 fluorescent quantitative PCR reaction System
(2) qRT-PCR reaction process: (1) pre-denaturation at 95 ℃ for 30s, one cycle; (2) denaturation at 95℃for 10s, annealing at 60℃for 30s,40 cycles; (3) 15s at 95 ℃,60 s at 60 ℃, 15s at 95 ℃,1 cycle; (4) fluorescence scanning and collecting fluorescent signals.
3. Gene cloning
Amplification of 1GhSINA12 Gene:
specific primers were designed using the full length CDS sequence of GhSINA12 (gh_a13g0056) as shown in table 5 below:
TABLE 5 Gene full-length cloning primers
The cDNA mixed dilution of the extracted rhizome and leaf is used as a template, and TKS (TaKaRa, china) enzyme is used for amplifying the target gene. The above mixed solution was placed in a 200. Mu.L PCR tube on ice according to the system shown in Table 6, and after slight centrifugation, the mixture was placed in a PCR reaction apparatus, and the PCR procedure was as follows: (1) pre-denaturation at 94℃for 3min; (2) denaturation at 98℃for 10s; (3) annealing at 55 ℃ for 15s; (4) extending at 68℃for 60s; (5) circulating the steps (2), (3) and (4) for 30 times; (6) extending at 68 ℃ for 7min; (7) constant temperature at 4 ℃.
TABLE 6 GhSINA12 clone PCR System
And (3) recycling PCR product glue: the PCR amplified product of GhSINA12 was subjected to gel recovery using FastPure Gel DNA Extraction Mini Kit (Vazyme), and specific procedures were performed according to the instructions of the kit, and the recovered DNA was stored at-20℃for subsequent experiments after measuring the concentration.
GhSINA12 was ligated to pEASY-Blunt Zero vector: (1) The pEASY-Blunt Zero vector and the target gene recovery product were mixed according to a ratio of 1:4, mixing the mixture on ice, wherein the optimal reaction system is 3-5 mu L, and diluting the target gene recovery product if the concentration of the target gene recovery product is too high; (2) controlling the temperature by a PCR instrument, and reacting for 15min at 25 ℃.
Transformation of E.coli: (1) Melting 100 μl of competent on ice, adding T carrier with target fragment under aseptic condition, mixing, and standing on ice for 25min; (2) Heat shock is conducted for 45s at the temperature of 42 ℃, the centrifuge tube is transferred to ice, and the centrifuge tube is kept stand for 2min without shaking; (3) Adding 600 mu L of LB liquid medium without antibiotics into a centrifuge tube in an ultra-clean bench, and carrying out shaking culture for 1.5h at 37 ℃ and 200rpm to revive thalli; (4) In an ultra clean bench, 50. Mu.L of resuscitator solution is coated on a blue and white spot screening culture medium, and the plate is placed in an incubator at 37 ℃ for culturing for about 12 hours to select monoclonal.
Colony PCR detection: (1) Picking large and full monoclonals in a sterilized ultra-clean bench, placing in 400 MuLLB culture solution containing kanamycin, shaking table at 37 ℃ at 200rpm for 3 hours; (2) Using EasyTaq (full gold, china) enzyme, and using M13_F on pEASY-Blunt Zero vector and GhSINA12_R as primer pair, detection was carried out with the following detection system (Table 7):
TABLE 7 PCR detection system of GhSINA12
(3) Sucking 2 mu L of bacterial liquid on an ultra-clean workbench, uniformly mixing on ice according to the system, and placing in a PCR instrument; 4) The PCR procedure was as follows: (1) pre-denaturation at 94℃for 6min; (2) denaturation at 98℃for 10s; (3) annealing at 55 ℃ for 15s; (4) extending at 72 ℃ for 60s and 31 cycles; (5) final extension at 72℃for 6min; (6) constant temperature of 4 ℃; (5) preparing 1.6% agarose gel and spotting; (6) 120V electrophoresis for 16min, and observing and analyzing; (7) The correct bacterial solution of 50. Mu.L of the target band was sucked by a pipette and sent to the company for sequencing, sequencing returned sequences were aligned by ClustalX, and the correct bacterial solution was used for subsequent experiments.
Plasmid extraction: the FastPure Plasmid Mini Kit (Vazyme, china) plasmid was used, and specific procedures were performed according to the instructions of the kit. The concentration of the recovered plasmid was measured and stored at-20℃for further use.
4. Structural analysis
(1) The GhSINA12 protein sequence was entered into the site predicted domain using the SMART site (http:// SMART. Embl-heidelberg. De /).
(2) Cotton SINA genes were mapped to chromosomes using TBtools Gene Location Visualize from GTF/GFF.
(3) The SINA genes of upland cotton, arabidopsis and rice are constructed by using MEGAX to carry out evolutionary tree construction. Arabidopsis and rice SINA genes are used to website phytozome (https:// phytozome-next. Jgi. Doe. Gov).
5. Subcellular localization
(1) The homologous primer Design software CE Design (http:// www.vazyme.com) was downloaded by the accession number nupran, the CDS sequence of GhSINA12 of upland cotton (with the stop codon removed) and the complete vector sequence of pCambia2300 were input into the CE Design, and two restriction sites BamHI and EcoRI were selected to automatically generate primers as shown in Table 8.
TABLE 8 subcellular localization amplification primers
(2) Amplifying a target gene with a carrier fragment by using the plasmid extracted in the step 3 as a template, wherein the system is as follows:
TABLE 9 amplification PCR System
(3) The amplification procedure is the same as the gene amplification procedure.
Purifying a target gene: the product was recovered using FastPure Gel DNA Extraction Mini Kit (Vazyme) and the specific procedure was followed according to the instructions of the kit.
And (3) enzyme cutting of a carrier:
(1) The plasmid of the pCambia2300 (CAMBIA) vector was subjected to a double cleavage reaction using the restriction endonucleases BamHI and EcoRI to form a linearized cleavage site, the double cleavage system being as follows:
TABLE 10 double enzyme digestion System
(2) Uniformly mixing the solution on ice according to the system, centrifuging 4000g for 1min, and placing in a water bath at 37 ℃ for 6h;
(3) Sucking 5. Mu.L of the solution in (2), mixing 1. Mu.L of 10×Loading Buffer, using pCambia2300 (CAMBIA) plasmid as a control, performing electrophoresis on 120V with 1.6% agarose for 19min, detecting enzyme digestion effect, and analyzing;
(4) The solution in (3) was purified according to the recovery method of step 3.
Ligation of the target Gene with vector: clon from Northenan IncII One Step Cloning Kit the target gene GhSINA12 and the vector pCambia2300 (CAMBIA) are connected by the following connection system:
TABLE 11 connection System
The mixture was prepared on ice according to the system, and centrifuged at 4000g for 30s. Then the mixture is transferred to a PCR instrument at 37 ℃ for reaction for 30min, and the mixture is quickly transferred to ice after the reaction is finished.
Recombinant transformed E.coli and colony detection: coli was transformed with the GhSINA12 and pCambia2300 (CAMBIA) recombinant according to step 3.
Recombinant material grain acquisition: the upstream universal primer HP158_F of the pCambia2300 vector and the downstream primer of the gene are used as a colony PCR detection primer pair, colony PCR detection is carried out according to the method in the cloning experiment of the step 3 gene, the correct detection strip is sent to a company for sequencing, and the recombinant product plasmid is extracted according to the method in the step 3 after the sequencing is correct.
Agrobacterium transformation: (1) Melting GV3101 competent ice, adding 1 μg of plasmid of GhSINA12 and pCambia2300 recombinant product under aseptic condition, gently mixing, and standing on ice for 25min; (2) liquid nitrogen for 5min; (3) water bath at 37 ℃ for 5min, and the water surface can not shake; (4) immediately transferring to ice and standing for 4min; (5) Adding 500 mu L of prepared sterile LB solution into an ultra-clean workbench, and carrying out shake culture for 3h at the temperature of 28 ℃ and the speed of 200rpm to revive thalli; (6) 4000rmp, centrifuging for 5min, collecting about 120 mu L of bacterial liquid, coating LB plates with double-antibody (kanamycin and rifampicin) resistance, and culturing for 48h in a constant temperature incubator at 28 ℃; and (7) selecting a monoclonal, detecting colony PCR and preserving bacteria.
Transient transformation of tobacco: the seeds of Benshi tobacco (Nicotiana benthamiana) are scattered on nutrient soil and vermiculite according to the proportion of 1: and (3) transferring the mixed nutrient soil in the proportion to an illumination incubator for culture. The tobacco seedlings growing well for about 15 days are moved to a new pot (2 seedlings in one pot) with old soil, the plastic film is removed after the transparent plastic film is coated for one week, and subcellular localization is carried out when the tobacco grows to 4-6 leaves, and the concrete steps are as follows: (1) Taking out the recombinant plasmid GhSINA12-pCambia2300 agrobacterium solution and the empty vector pCambia2300 agrobacterium solution from-80 ℃ in the first two days of transient transformation of tobacco, respectively sucking 100 mu L in a super clean bench, transferring to 600 mu L double-resistance (kana and rifampin) LB solution, and performing shake culture for 12h for activation at 28 ℃ and 200 rpm; (2) Under aseptic conditions, the activated bacterial solutions are respectively transferred into 150mL conical flasks filled with 50mL LB solution (kanamycin and rifampicin double resistance), and the solution is cultured at 28 ℃ at 200rpm until the solution turns orange yellow; (3) Transferring to a 50mL centrifuge tube, centrifuging for 9min at 4800rmp, and discarding the supernatant; (4) preparing 100mL of heavy suspension with proper volume; (5) Re-suspending the bacterial cells, and adjusting the OD600 value of the re-suspended bacterial liquid to be 1.0; (6) Standing at room temperature for 2-3h, and picking tobacco with flat leaf surfaces for injection; (7) After 72h of injection, the injected leaves were cut and washed clean with ddH2O, and subcellular localization observation experiments were performed using a laser confocal microscope.
6. In vitro ubiquitination experiment
And (3) glue preparation: SDS-PAGE gels (7.25% gels) were prepared at appropriate concentrations according to the molecular weight of the protein samples.
Electrophoresis: adding an electrophoresis liquid into the electrophoresis tank; the voltage is regulated to 80V, the glue is run, and when the sample strip just enters the lower layer of separation glue, the voltage is regulated to 120V (constant voltage);
the sample system was added as follows:
TABLE 12 in vitro ubiquitination sample loading System
When the sample strip runs to a position 0.5cm away from the lower edge of the rubber plate, turning off the power supply to perform electric transfer; the PVDF membrane is soaked in pure methanol for about 30s-1min in advance, and then soaked in the membrane transferring liquid for standby.
After the glue is taken out, balancing for 15min in the film transfer liquid; opening a gel clamp, placing the gel clamp in a clean tray, pouring 3/4 volume of transfer membrane liquid, sequentially placing one piece of fiber support pad net, three pieces of pre-cut chromatographic filter paper, gel, PVDF membrane, three pieces of pre-cut chromatographic filter paper and a second piece of fiber support pad net (bubbles among materials of each layer are necessarily removed, and the membrane and the gel are particularly in close contact); closing a gel clamp, placing the gel clamp in an electrotransfer tank, and pouring transfer membrane liquid; placing the electrophoresis tank in ice water, connecting a power supply, and setting 200mA for 2 hours;
blocking and developing: after the transfer is finished, the clamping box is put back into a tray filled with film transferring liquid, the clamping box is opened, the PVDF film is taken out, the gel contact surface is upwards, the PVDF film is put into a culture dish filled with TBST, and the film is washed for 5min at room temperature; transfer to TBST solution containing 3% BSA, block overnight at 4' C (or 1h at ambient temperature); washing the membrane with 20mLTBST for 5min, repeating for three times; pouring 10mL of primary anti-dilution Buffer (Blocking buffer+1:2000 antibody) on a shaker for 2-3h at about 55 rpm; washing the membrane with 20mLTBST for 10min, repeating for three times, and about 80 rpm; pouring 10mL of dilution buffer containing secondary antibody (1:2000), and gently shaking at room temperature for 1h; washing the membrane with 20mLTBST for 8min, repeating the washing with 20mTBS three times; placing the washed film in an ultrahigh-sensitivity chemiluminescent imaging machine, and dripping a proper amount of mixed color developing solution on the film to develop color.
7. Genetic transformation of Arabidopsis thaliana
Planting arabidopsis thaliana:
columbia (Columbia) Arabidopsis thaliana grows into four leaves and is transplanted into the nutrient soil.
Genetic transformation of Arabidopsis thaliana:
(1) In the first two days of the full bloom stage of Arabidopsis thaliana, the recombinant plasmid GhSINA12-pCambia2300 (containing a stop codon) Agrobacterium solution was removed from-80 ℃, 100. Mu.L was aspirated in a super clean bench, 600. Mu.L of double-resistant LB solution (Canada and rifampin) was added, and incubated at 28℃and 200rpm for 13h; (2) The 700. Mu.L of the bacterial solution was transferred to a 150mL Erlenmeyer flask containing 50mL of LB solution (kanamycin and rifampicin resistance) in an ultra clean bench, and incubated at 28℃and 200rpm until the solution became orange-yellow; (3) Transferring the culture solution to a 50mL centrifuge tube, centrifuging for 9min at 5000g, and discarding the supernatant; (4) preparing a proper volume of heavy suspension; (5) Re-suspending the thallus with re-suspension, and using re-suspension as blank control to re-suspend the thallus OD 600 The value is adjusted to 1.0; (6) After the OD value is regulated, 0.02% of silwet-77 is added and mixed uniformly; (7) Soaking the arabidopsis inflorescence with the pod cut off in advance in the mixed solution for 1min, marking, placing in a greenhouse, performing darkness treatment, and removing black cloth after 24 h; (8) transformation was repeated once after 5 days of growth.
Screening of Arabidopsis positive seedlings:
After the arabidopsis is ripe, harvesting the seeds to be T 0 Removing impurities, drying in a 37 deg.C incubator for one week to allow the Arabidopsis thaliana to be completely after-ripened, taking out seeds, and collecting GhSINA 12T 0 Sowing the seeds of the generation on MS (containing kana antibiotics) culture medium, vernalizing at 4 ℃ for 2 days, transferring to an incubator, and growing for about 15 days to see that the arabidopsis is positiveSeedling.
Phenotype observation of arabidopsis:
GhsINA12 transgenic Arabidopsis T was performed using a sterile 0.1% agarose solution 3 The generation seeds and wild type Arabidopsis were spotted on MS medium (0 mM, 150mM, 300 mM) containing different concentrations by using a 2.5. Mu.L pipette, and the culture was performed for 2 days of vernalization, and the incubator was cultured for 14 days, so that the phenotype difference between GhSINA12 transgenic Arabidopsis and wild type Arabidopsis roots was observed.
8. Cotton Virus Induced Gene Silencing (VIGS) technique
And (3) constructing a carrier: 80-200bp in GhSINA12 sequence is selected as silencing fragment, and the designed primer is shown in Table 13.
TABLE 13 VIGS amplification primers
The T vector is used as a template to amplify a target gene with a homologous arm of a tobacco mosaic virus vector pTRV2, the system and subcellular localization experiment is carried out, and the PCR procedure is as follows: (1) pre-denaturation at 94℃for 3min; (2) denaturation at 98℃for 10s; (3) annealing at 55 ℃ for 15s; (4) extension at 68℃for 7s; (5) circulating the steps (2), (3) and (4) for 30 times; (6) extending at 68 ℃ for 7min; (7) constant temperature at 4 ℃.
Purifying a target gene: the specific steps are the same as the purification mode of subcellular localization experiment.
Cleavage of pTRV2 vector:
(1) The plasmid of pTRV2 (CAMBIA) vector was subjected to a double cleavage reaction using restriction endonucleases BamHI and EcoRI to form a linearized cleavage site, the double cleavage system being as follows:
TABLE 14 double cleavage System
(2) Uniformly mixing the solution on ice according to the system, centrifuging 4000g for 1min, and placing in a water bath at 37 ℃ for 6h;
(3) Sucking 5. Mu.L of the solution in (2), mixing 1. Mu.L of 10×Loading Buffer, using pCambia2300 (CAMBIA) plasmid as a control, performing electrophoresis on 120V with 1.6% agarose for 19min, detecting enzyme digestion effect, and analyzing;
(4) Purifying the solution in step (3) according to the method for recovering the PCR product gel in step 3.
Ligation of the target Gene with vector: clon from Northenan IncII One Step Cloning Kit the target gene GhSINA12 and the vector pTRV2 are connected by the kit, and the connection system is as follows:
TABLE 15 connection System
The mixture was prepared on ice according to the system, and centrifuged at 4000g for 30s. Then the mixture is transferred to a PCR instrument at 37 ℃ for reaction for 30min, and the mixture is quickly transferred to ice after the reaction is finished.
Recombinant transformed E.coli and colony detection: ghSINA12 and pTRV 2 The recombinant was transformed into E.coli according to step 3.
Recombinant material grain acquisition: by pTRV 2 The upstream universal primer of the vector and the downstream primer GhSINA 12-2R of the gene are used as a colony PCR detection primer pair, the colony PCR detection is carried out according to the method in the cloning experiment of the step 3 gene, the correct detection strip is sent to a company for sequencing, and the recombinant product plasmid is extracted according to the method in the step 3 after the sequencing is correct.
Agrobacterium transformation: (1) Thawing GV3101 on ice, and aseptically adding 1 μg of GhSINA12 and pTRV 2 The recombinant product plasmid is gently mixed and placed on ice for 25min; (2) liquid nitrogen for 5min; (3) water bath at 37 ℃ for 5min, and can not shake; (4) immediately transferring to ice and standing for 4min; (5) Adding 600 mu L of prepared sterile LB solution into an ultra-clean workbench, and carrying out shake culture for 3h at 28 ℃ and 200rpm to revive thalli; (6) 4000g, centrifuging for 5min, collecting about 120 mu L of bacterial liquid, coating LB plates with double-antibody (kanamycin and rifampicin) resistance, and culturing for 48h in a constant temperature incubator at 28 ℃; (7) Selecting monoclonal, colony PCR detection and protectionBacteria.
Agrobacterium transformation of cotton:
selecting seeds of selfing age TM-1, and downwards sowing radicle into the seeds 3;1, coating a transparent film on the mixed nutrient soil and vermiculite, and carrying out agrobacterium injection on seedlings in a period that true leaves are not grown, wherein the specific steps are as follows: (1) Taking out the stored positive bacterial liquid TRV from the refrigerator at the temperature of minus 80 DEG C 2 -GhSINA12, helper plasmid TRV 1 Empty vector TRV 2 -00, cotton control albino gene GhCLA1 bacterial liquid, and transferring the four bacterial liquids into 1mL LB liquid culture medium containing kana and rifampicin dual-resistance antibiotics, 220rmp, and culturing at 28 ℃ for 12h in an oscillating way; (2) Taking out all bacterial liquid, placing the bacterial liquid in an conical flask for continuous expansion culture, adding the bacterial liquid into 50mL of double-resistance culture medium, and carrying out shaking culture for 14h at 220rmp and 28 ℃; (3) Taking out the cultured bacterial liquid, placing the bacterial liquid into a centrifuge tube, centrifuging the bacterial liquid in the centrifuge for 8min at 6000rmp, and collecting bacterial bodies; (4) Adding a proper amount of heavy suspension into the deposited bacterial liquid, vortex oscillating to heavy suspension bacterial body, and OD (optical density) of the bacterial liquid 600 The value is adjusted to 1.3, and the mixture is kept stand for 3 hours at room temperature in a dark place; (5) Standing at room temperature in dark for 3 hr, and standing TRV 2 Ghsina12, empty vector TRV 2 -00, uniformly mixing the bacterial liquid of the GhCLA1 and the bacterial liquid of the auxiliary plasmid TRV1 according to the ratio of 1:1; (6) Using a 1mL sterile syringe, the bacterial solution was aspirated, the cotyledons were scratched with a needle, then injected at the back of the cotyledons, the leaves were filled with the bacterial solution, and water droplets were dropped.
9. Measurement of enzyme Activity index
MDA activity index determination:
when the cotton VIGS is stressed by adversity, the plants are sampled and measured by using a MDA kit for testing the soxhlet test, and the specific steps are operated according to the use instruction of the kit, and the calculation formula is as follows:
MDA content (nmol/g fresh weight) = [ ΔA ∈×d) ×v2×109 ]/(W×v1/(V) =32.3×ΔA/(W);
v- -total volume of sample extract, 1mL; v1- -adding the sample volume of the reaction system, and 0.2mL; v2- -total reaction liquid volume of sample extract and working liquid, 5×10 -4 L is; d- -cuvette optical path, 0.5cm; epsilon- - - -MDA molar extinction coefficient 155X 10 3 L/mol/cm; w is the sample mass, g;
protein concentration determination:
when cotton VIGS is stressed by adversity, plants are sampled and assayed by the Vazyme kit BCA Protein Quantification Kit, and the specific steps are operated according to the use instructions of the kit and are calculated as follows:
drawing a standard curve by taking the protein content (mug) as an abscissa and the light absorption value as an ordinate; according to the measured light absorption value, the protein content of the sample can be calculated on a standard curve; protein concentration was calculated: dividing the detected protein content by the sample volume of 20 mu L, and multiplying by the corresponding dilution times to obtain the actual concentration of the sample to be detected.
CAT activity index measurement:
when the cotton VIGS is stressed by adversity, the plants are sampled and tested by using a Suzhou Granism CAT kit, and the specific steps are operated according to the use instruction of the kit, and the calculation formula is as follows:
CAT(μmoL/min/mg prot)＝[(ΔA+0.0137)÷0.1412]÷(V1×Cpr)÷T；
V- -adding volume of the extracting solution, 1mL; v1- -adding sample volume, 0.01mL; t-reaction time, 5min; cpr- -sample protein concentration, mg/mL;
POD activity index measurement:
when the cotton VIGS is stressed by adversity, the plants are sampled and measured by using a POD kit for testing, and the specific steps are operated according to the use instruction of the kit, and the calculation formula is as follows:
POD (Δod470/min/g fresh weight) =Δa/V (w×v1/V)/(1)/t=100×Δa/Cpr;
v- -adding volume of the extracting solution, 1mL; v1- -adding sample volume, 0.01mL; t-reaction time, 1min; cpr- -sample protein concentration, mg/mL;
measuring SOD activity indexes:
when the cotton VIGS is stressed by adversity, the plants are sampled and measured by using a soxhlet test SOD kit, and the specific steps are operated according to the use instruction of the kit, and the calculation formula is as follows:
percent inhibition = [ (a blank 1-a blank 2) - (a sample tube-a sample control) ]/(a blank 1-a blank 2) ×100%;
SOD activity (U/mg prot) = [ percent inhibition ++1-percent inhibition ++v2 ] ++v1×cpr ++10×percent inhibition ++1-percent inhibition ++cpr×d;
v- -adding volume of the extracting solution, 1mL; v1- -sample volume added to the reaction system, 0.02mL; v2- -total volume of reaction system, 0.2mL; d, sample dilution multiple, namely 1 when undiluted; cpr- -sample protein concentration, mg/mL.
10. Transcriptome sequencing analysis
Plant sample: firstly, carrying out a VIGS experiment on cotton, carrying out drought treatment on a cotton silencing group and a control group after the GhSINA12 gene is confirmed to be silenced, and sampling cotton tissues after plants are subjected to drought stress, wherein each group of samples is not less than 0.4g. Sequencing was then performed by extracting 6 sets of RNAs and constructing a library by using Illumina NovaSeq 6000 platform.
Differential expression gene volcanic diagram: comparing the differentially expressed genes between the two sets of data by constructing a dataset by using DESeq2 software; then, DESeq difference analysis was performed on the data based on the negative binomial (Negative Binomial) distribution. GO enrichment analysis of differentially expressed genes: enrichment analysis is carried out through the GO engineering program of the TBtools software, after the Enrichment analysis is finished, visualization of Enrichment analysis results is carried out through the Enrichment Grapher program in the software, and then pictures are adjusted. KEGG enrichment analysis of differentially expressed genes: enrichment analysis is carried out through KEGG Enrichment Analysis program of TBtools software to obtain enrichment analysis results, visualization is carried out through Enrichment Bar Plot small program in the software, finally, a visualized picture is obtained, and the picture is stored.
Experimental results:
1. variation of expression level of GhSINA gene under drought stress
1.1 screening of GhSINA Gene
To screen for the GhSINA gene that may be associated with drought stress, cotton was first subjected to simulated drought treatment, TM-1 cotton was hydroponic, and when a second true leaf was grown, drought treatment was performed with 15% PEG6000, while 6 time points (0, 1, 3, 6, 12, 24 h) were sampled (FIG. 1A). Specific primers were designed for 24 upland cotton SINA genes identified by previous studies (Table 3). According to real-time fluorescence quantitative (qRT-PCR) analysis, the expression level of the GhSINA12 gene is obviously induced under the treatment of PEG6000, the transcription level of the GhSINA12 is rapidly increased to about 2 times of the control level when the PEG6000 is treated for 1h, the induced expression of the GhSINA12 is most obvious at 12h, and the GhSINA12 begins to rapidly decrease after 24h (B in fig. 1).
1.2 analysis of expression of GhSINA12 Gene
Since GhSINA12 was most significantly induced under the conditions of 15% of PEG6000 treatment at 6h and 12h, in order to understand under which conditions the transcription level of the gene was the highest, the PEG6000 treatments at different concentrations were 10%, 20% and 30% respectively on TM-1 cotton, and it was found by qRT-PCR experiments that the transcription level of GhSINA12 was the highest when the PEG6000 treatments were 10% both at 6h and 12h periods, and at the same concentration, the transcription level at 12h was higher than that at the same concentration (A and B in FIG. 2). We also performed natural drought treatment, soil culture of cotton, removal of water basin of cotton in a period of two leaves and one heart, water-deficient drought treatment as drought group, control group watering on time, and qRT-PCR analysis of two groups of leaf sampling treatment when leaf wilting phenotype appears in drought group, found that transcription level of GhSINA12 gene was significantly increased in natural drought (FIG. 2C).
1.3 sequence analysis of GhSINA12 Gene
Domain analysis of the coding sequence of GhSINA12 revealed that GhSINA12 also had SINA and RING domains (fig. 3); according to the specific position information of 24 upland cotton SINA genes on upland cotton chromosomes, the chromosome distribution of SINA is analyzed, wherein the 24 upland cotton SINA genes are co-located on 16 chromosomes, and the different chromosomes contain different SINA numbers. The target gene GhSINA12 is positioned on the 13 th chromosome of the A subfamily.
Cloning and functional verification of 1.4GhSINA12 Gene
cDNA of roots, stems and leaves of a cotton genetic standard line TM-1 is mixed and used as a gene cloning template, a CDS full-length sequence of the GhSINA12 gene is obtained through PCR amplification, as shown in a figure (4), water is used as a negative control, and a PCR detection band of the GhSINA12 is positioned at about 1000bp and is consistent with the fragment size of the GhSINA12 gene. Subcellular localization experiments are carried out on GhSINA12, and the images are observed and collected under a laser confocal microscope. Localization of GhSINA12 to the nucleus was demonstrated by DAPI staining (a in fig. 5). As a result, ghSINA12 was observed on the nucleus, and a coincidence with the DAPI channel image was observed from the combined image, indicating that GhSINA12 was localized in the nucleus.
To determine whether its GhSINA12 has E3 ubiquitin-binding enzyme activity, an in vitro ubiquitination experiment (FIG. 5B) was performed by prokaryotic expression of purified MBP-tagged fusion GhSINA12 protein, using ubiquitin activating enzyme E1, ubiquitin binding enzyme E2 (UbcH 5B), incubation with the protein to be tested (MBP-GhSINA 12, MBP) for 2h 30℃for SDS-PAGE analysis and detection by Biotin antibody anti-Biotin. The results showed that ladder bands could be observed in the presence of E1, E2, E3 and ubiquitin Ub at the same time, and ladder bands could not be displayed in the absence of any of the above, thus demonstrating that GhSINA12 has E3 ubiquitin-binding enzyme activity.
2. Response of GhSINA 12-overexpressed Arabidopsis thaliana to drought stress
2.1 heterologous expression of cotton GhSINA12 in Arabidopsis thaliana
Before the full bloom stage of arabidopsis thaliana, transferring the GhSINA12 gene into arabidopsis thaliana by an inflorescence dip-dyeing method, and harvesting seeds as T 0 Instead, they were planted on MS medium containing kanamycin resistance to screen positive seedlings. Because the overexpression vector of the GhSINA12 gene has kanamycin resistance, the arabidopsis seeds transferred with the GhSINA12 gene can grow normally on MS culture medium containing kanamycin resistance, but wild arabidopsis cannot grow normally. Seed propagation of Arabidopsis thaliana to T 3 At the time of generation, the seeds are subjected to colony positive detection, and the Arabidopsis T is detected by RT-PCR and qRT-PCR 3 The expression level of GhSINA12 gene in the generation of the over-expression line is changed (A and B in FIG. 6),the result shows that the exogenous gene GhSINA12 has different degrees of expression in the transgenic arabidopsis thaliana strain.
2.2 drought resistance observations of Arabidopsis thaliana overexpressing GhSINA12 Gene
Arabidopsis thaliana T transformed with GhSINA12 gene 3 The generation seeds and wild type Arabidopsis thaliana were sown on MS medium containing mannitol at different concentrations of 0mM, 150mM and 300mM, and marked separately, and after vernalization for 48 hours, the medium was placed vertically in an incubator for 14 days, and the phenotype was observed, and the root phenotype of the GhSINA12 strain was quite distinct, wherein the WT and the overexpressing strain were not significantly different in MS medium of 0mM mannitol, but the root length of the overexpressing strain was significantly longer than that of the WT strain in mannitol medium of 150mM and 300mM (A in FIG. 7). Meanwhile, the root length of the plants was observed, and it was found that the root length of the overexpressed lines was significantly longer than that of the WT lines in 150mm and 300mm mannitol MS medium (B in fig. 7). These data indicate that the GhSINA12 gene enhances resistance of Arabidopsis to drought stress.
3. Silencing cotton response to drought stress in GhSINA 12-expressing cotton
3.1 detection of Cotton silencing efficiency
To understand the response of the GhSINA12 gene to drought in cotton, we studied the response of the GhSINA12 gene-silenced strain to drought. The cotton of TM-1 was subjected to VIGS experiments, silencing expression of the GhSINA12 gene by virus-mediated gene silencing techniques, and when the positive control cotton cotyledon whitened very significantly around 10 days after agrobacterium injection, natural drought treatment and 15% PEG6000 simulated drought treatment were performed (fig. 8 a and C). Meanwhile, cotton leaves are sampled, qRT-PCR experiments are carried out, and the silencing efficiency of the GhSINA12 gene in cotton plants is analyzed. The results showed that the silencing efficiency of GhSINA12 in the silencing plants was around 50% (B and D in fig. 8).
3.2 silencing expression of GhSINA12 to reduce drought resistance of Cotton plants
When the cotton whitens obviously, the basin is filled with water every day. After 3 consecutive days, the water pan is removed to perform natural drought treatment on cotton, and about 8 days after the natural drought treatment, the drought resistance of GhSINA12 gene silencing plants is obviously reduced, and the wilting phenotype appears in advance on TRV 00 plants (A in figure 9). And when the whitening of the water-cultured cotton is obvious, carrying out PEG6000 simulated drought treatment, and photographing about two half hours (C in fig. 9), wherein the result is the same as the natural drought stress.
In general, to prevent cells from being adversely affected by excessive ROS, plants form CAT, POD, etc. to eliminate the excessive ROS. In addition, when plants are damaged in adverse conditions, membrane lipid peroxidation often occurs, and the final decomposition product of membrane lipid peroxidation is MDA, and the content of MDA can reflect the degree of damage to the plants in adverse conditions. Therefore, these indicators can reflect drought resistance of plants to some extent. The results of these drought-resistant physiological indicators showed that under both treatments, the SOD, POD, CAT strain TRV:00 was significantly higher than the TRV: ghSINA12 strain, and the MDA was significantly smaller than the TRV: ghSINA12 strain (FIGS. 9B and D). The result shows that after GhSINA12 gene silencing, the drought resistance of cotton plants is obviously reduced. Taken together, it was shown that the resistance of the GhSINA 12-silenced expression lines was significantly reduced in drought stress.
RNA-seq analysis of differentially expressed genes after GhSINA12 Gene silencing
To explore the mechanism of drought resistance attenuation of cotton caused by GhSINA12 gene, we performed RNA-seq analysis on the drought-resistant TRV:00 and TRV: ghSINA12 plants. From these, genes related to drought stress, such as transcription factor BHLH1, kinase MAPKKK17, calbindin CML45, 1-aminocyclopropane-1-carboxylic acid synthase ACS1, auxin response protein SAUR36, etc., were screened out, and we performed qRT-PCR analysis on the above 5 genes to verify that the results are consistent with RNA-seq (FIG. 10).
To more fully understand the response of the GhSINA12 gene after silencing in drought stress, the whole transcriptome data was first analyzed, and 765 genes were up-regulated and 2540 genes were down-regulated among 3305 genes differentially expressed after the GhSINA12 gene silencing. GO enrichment analysis is performed on the whole differential expression genes, and we find that the catalytic activity of the differential expression genes in Molecular Functions (MF) and the metabolic process enrichment degree in Biological Processes (BP) are high, and the catalytic activity and the metabolic processes are closely related to drought stress. And then carrying out KEGG analysis on the up-down regulated genes of the differential expression genes respectively, and finding that the enrichment degree of the mitogen-activated protein kinase (MAPK) signal channel and the plant hormone signal transduction channel is high whether the differential expression genes are up-regulated or down-regulated.
Both pathways simultaneously contain changes in the expression level of ABA pathway genes (fig. 11 a), wherein the heat map analysis of the pathways involved in regulating drought response mechanisms in MAPK and phytohormone signaling pathways, by heat map and MAPK pathway map analysis, found that under drought stress, when the GhSINA12 gene was silenced, the transcription level of the plant abscisic acid receptor (PYR/PYL) related gene in the ABA signaling pathway was down-regulated, thereby up-regulating the transcription level of the 2C type protein phosphatase (PP 2C) related gene, and inhibiting sucrose non-fermentation related protein kinase 2 (SnRK 2) by dephosphorylation, which indirectly resulted in down-regulating the transcription level of the MAPK kinase 17_18 (mapkk17_18) related gene, by decreasing phosphorylation to decrease MAPK kinase 3 (MKK 3), decreasing the related gene of split activated protein kinase (mpk7_14), and finally allowing plants to have reduced stress adaptation, and in the case of the ABA pathway was under conditions, by regulating SnRK 2B to influence on the transcription factor (SnRK 11B). Finally, transcriptome verification (C in FIG. 11) of qRT-PCR results of related genes in the two paths is carried out to accord with transcriptome results.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> scientific research center for Yangtze river of cotton
Application of <120> cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants
<130> PA22015711
<160> 50
<170> PatentIn version 3.3
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<212> DNA
<213> artificial sequence
<400> 19
ctaactgtat ttcattgttt tggtca 26
<210> 20
<211> 21
<212> DNA
<213> artificial sequence
<400> 20
tctcttatgc ttctcggggt g 21
<210> 21
<211> 19
<212> DNA
<213> artificial sequence
<400> 21
tgcttctctt gagcttccc 19
<210> 22
<211> 22
<212> DNA
<213> artificial sequence
<400> 22
aacattgata ctcgtgtttt gg 22
<210> 23
<211> 22
<212> DNA
<213> artificial sequence
<400> 23
tttaggtgga aatcttggag ca 22
<210> 24
<211> 25
<212> DNA
<213> artificial sequence
<400> 24
cttacaaatc gaacatacag tgtgg 25
<210> 25
<211> 20
<212> DNA
<213> artificial sequence
<400> 25
tcgtcaagag ctgtcacgtc 20
<210> 26
<211> 20
<212> DNA
<213> artificial sequence
<400> 26
cgctgacttc acgaagggta 20
<210> 27
<211> 20
<212> DNA
<213> artificial sequence
<400> 27
agtggaagcg aaccatggag 20
<210> 28
<211> 20
<212> DNA
<213> artificial sequence
<400> 28
ggagttcgca tcggcaattc 20
<210> 29
<211> 20
<212> DNA
<213> artificial sequence
<400> 29
atctcggcga tgatgtgacc 20
<210> 30
<211> 20
<212> DNA
<213> artificial sequence
<400> 30
tcagccaacc gggacttaac 20
<210> 31
<211> 20
<212> DNA
<213> artificial sequence
<400> 31
cgggcatcca tttgtctcgt 20
<210> 32
<211> 20
<212> DNA
<213> artificial sequence
<400> 32
ctggtggatt gcaccttgga 20
<210> 33
<211> 20
<212> DNA
<213> artificial sequence
<400> 33
gttctggtcc ggggtatgtc 20
<210> 34
<211> 20
<212> DNA
<213> artificial sequence
<400> 34
ccatgtacac cgcaacatgc 20
<210> 35
<211> 20
<212> DNA
<213> artificial sequence
<400> 35
atcaggggaa cgcctttgag 20
<210> 36
<211> 20
<212> DNA
<213> artificial sequence
<400> 36
catctcaaca acggcgcatc 20
<210> 37
<211> 20
<212> DNA
<213> artificial sequence
<400> 37
atcaccaaac tcaaactata 20
<210> 38
<211> 17
<212> DNA
<213> artificial sequence
<400> 38
cctaaagctg tcataac 17
<210> 39
<211> 20
<212> DNA
<213> artificial sequence
<400> 39
attagcttcg gcctagacgc 20
<210> 40
<211> 20
<212> DNA
<213> artificial sequence
<400> 40
gcctgctttg gacgatgaac 20
<210> 41
<211> 20
<212> DNA
<213> artificial sequence
<400> 41
tggacttgtg gcgagtgatt 20
<210> 42
<211> 20
<212> DNA
<213> artificial sequence
<400> 42
gcaaagcaaa ccctgaacca 20
<210> 43
<211> 22
<212> DNA
<213> artificial sequence
<400> 43
tcaagactga tttgcgtttc ca 22
<210> 44
<211> 20
<212> DNA
<213> artificial sequence
<400> 44
gcgcaaaggt tggtgtcttc 20
<210> 45
<211> 21
<212> DNA
<213> artificial sequence
<400> 45
atggcacctg gtggtggtat c 21
<210> 46
<211> 22
<212> DNA
<213> artificial sequence
<400> 46
tcattgttct ttccaaatcc gg 22
<210> 47
<211> 42
<212> DNA
<213> artificial sequence
<400> 47
acgggggact cttgaggatc catggcacct ggtggtggta tc 42
<210> 48
<211> 41
<212> DNA
<213> artificial sequence
<400> 48
ccgggtaccg agctcgaatt cttgttcttt ccaaatccgg c 41
<210> 49
<211> 42
<212> DNA
<213> artificial sequence
<400> 49
gtgagtaagg ttaccgaatt catggcacct ggtggtggta tc 42
<210> 50
<211> 44
<212> DNA
<213> artificial sequence
<400> 50
cgtgagctcg gtaccggatc cggacactgg taaatcggag gata 44
Claims (10)
1. The application of the cotton SINA E3 ubiquitin ligase gene in improving the drought resistance of plants is characterized in that the cotton SINA E3 ubiquitin ligase gene isGhSINA12Genes of the order ofGhSINA12The gene is GH_A13G0056 gene in cotton genome database;
the plant is cotton or Arabidopsis thaliana.
2. Comprising a composition as defined in claim 1GhSINA12Biological material of genes or saidGhSINA12Application of gene coded protein in improving drought resistance of plants;
the plant is cotton or Arabidopsis thaliana.
3. The use according to claim 2, wherein the biological material is a cloning vector or a recombinant cell.
4. The use according to any one of claims 1-3, wherein said use comprises up-regulating said plant of interestGhSINA12The expression of the gene can improve drought resistance of plants.
5. A method for improving drought resistance in a plant, comprising increasing the drought resistance of a plant of interest as set forth in claim 1GhSINA12The expression level of the gene is increased or the expression level of the gene is increasedGhSINA12Activity or content of the protein encoded by the gene;
the plant is cotton or Arabidopsis thaliana.
6. The method of claim 5, comprising inserting cottonGhSINA12The gene is introduced into target plant to obtain transgenic plant with raised drought resistance.
7. The method of claim 6, comprising the steps ofGhSINA12The plant expression vector of the gene is transformed into the plant through agrobacterium mediation.
8. The method of claim 7, wherein the plant expression vector drives overexpression of the GhSINA12 gene by a constitutive or inducible promoter.
9. The method of claim 8, wherein the constitutive promoter is a 35S promoter.
10. The method of claim 1GhSINA12The application of the gene in identifying and/or screening drought-enduring cotton varieties is characterized in that the cotton genomic DNA to be identified is used as a template, and the gene is utilized GhSINA12The specific PCR primer of the gene is subjected to fluorescent quantitative PCR amplification, and the drought resistance of the cotton to be identified is determined according to the amplification result.
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|---|---|---|---|
| CN202210717614.6A CN115044563B (en) | 2022-06-23 | 2022-06-23 | Application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants |
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| CN202210717614.6A CN115044563B (en) | 2022-06-23 | 2022-06-23 | Application of cotton SINA E3 ubiquitin ligase gene in improving drought resistance of plants |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| SINA E3 Ubiquitin Ligases: Versatile Moderators of Plant Growth and Stress Response;Chongyang Zhang 等;《Molecular Plant 》;第610-612页 * |
| 棉花GhSINA12基因在干旱胁迫反应中的功能研究;王悦力;《万方》;全文 * |
| 植物SINA E3泛素连接酶功能的研究进展;汤晓丽 等;《生物技术通报》;第10-17页 * |
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