MKK3 Experiment for Protein-Protein Interaction
To confirm the related protein with MKK3, the Yeast Two-Hybrid (Y2H) assays would be performed (Teige, Scheikl, Reiser, Ruis, & Ammerer, 2001). The Y2H experiment is used to identify physical interactions between proteins via downstream activation of the reporting gene. The reporter gene generally influences the measuring of the transcription, but the most common measure involves auxotrophic selection, which reflects the ability of the yeast to thrive in nutrient-controlled environments.
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The quantitative Yeast Two-Hybrid assays would be based on the techniques described by Teige et al. (2001).
Arabidopsis thaliana Col-0 would be used to provide a genetic basis for the study. Seeds were sprouted on 0.5× Murashige and Skoog (MS) medium (Sigma-Aldrich) while the plant was matured at a temperature a range of between 24°C and 26°C, the RH of 30%, and cool-light of ∼50 μmol·m−2·s−1 in the long day involving 16 hours of light and eight hours of darkness conditions (Teige et al., 2001). Arabidopsis protoplasts were obtained based on the method provided by (Cardinale et al., 2000).
Yeast Two-Hybrid Assays
The strain of yeast two-hybrid L40 would be used in the experiment alongside the pBTM116 as the vectors for the LexA-BD the integration of MKKs, as well as pGAD424 (Clontech) for the Gal4-AD synthesis of the MPKs (the method by provided by Vojtek et al. (1993) would be used for the vector). For a given MAPK pathway, MKKs would be used as triggers of certain MAPKs. Tests would be conducted to determine potential downstream MAPK targets of MKKs using well-defined techniques for a pair-wise yeast two-hybrid evaluation involving 14 various MPKS, which account for all the four groups of Arabidopsis MAPKs (Dóczi et al., 2007).
The two plasmids would be necessary for the assays. They include prey plasmid and bait plasmid. The prey plasmid usually codes for the activation domain of the copying factor bonded to specific proteins of interests, known as the prey. In addition, it codes for a selection marker, such as a biosynthesis gene, to ensure that the yeast can develop in a nutrient-controlled medium. The bait plasmid codes for the DNA-binding domain for copying factors attached to a given protein of interest, bait, during the analysis. It also codes a selection marker biosynthesis that ensures the yeast can thrive in a nutrient-regulated medium.
The yeast strain L40 genetic background mentioned above, and further descriptions of other yeast strains by Teige et al. (2001) are provided. That is, all other yeast strains bear the genetic background of W303–1A (MATa ade2–1 can1–100 his3–11 leu2–3 trp 1–1 ura 3–1). The microhomology polymerase chain reaction (PCR) method would be used to eliminate the coding area of the HOG1 gene in the W303–1A type strain varieties. The PCR analysis of chromosomal DNA would confirm the deletion (Teige et al., 2001).
Teige et al. (2001) further described the method applied in yeast two-hybrid. The HOG1 encoding area would be cloned as BamHI element into the direction pBTM116. Replication is used to create C-terminally hemagglutinin (HA)-codded Hog1 with various elements of the HA epitope into the pVR50 plasmid. The required genetic material would be acquired through PCR from the genomic yeast deoxyribonucleic acids.
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The facilitating parts would be cloned as HindIII-BamHI part into YCp33, the yeast vector. Coding areas would be augmented independently and replicated into a vector, while the deoxyribonucleic acids (DNA) alignment/sequencing is used to verify the construct. Proteins are normally bonded with C-terminal with hexa-fractions of HA epitome or an increased green luminous protein duplicate replica.
PCR is used to produce the kinase sedentary mutant to initiate an active silent analytical Bg/ll site. The PCR is further used to develop phosphorylation site mutants with diagnostic Kpnl site and Kspl site. The double mutant was also obtained using the same process. Constructs are integrated with 800-bp promoter (endogenous/produced from within) and released to YCp33 to determine yeast manifestation. The constructs used for two-hybrid analysis would be acquired from the screen. Alternatively, a subcloning would be conducted.
The yeast two-hybrid screen also involves other growth assays. A two-hybrid screen would be conducted with HOG1p as DNA-joining inducement, replicated into pBTM116. Testing would be performed on L40 involving element cDNA collection. Transformants will be evaluated for histidine prototrophy on the least medium with two mM 3- amino triazole. Histidine-positive replicas would be additionally analyzed for LacZ correspondent genetic factor activation in the filter-lift analyses. Library plasmids will be acquired from the introduced foreign genetic material into E. coli strain HB101 of DNA yeast while the choosing M9 medium is not containing any leucine.
The quantitative two-hybrid assays would be performed with cell excerpts with two mM o-nitrophenyl β-D galactopyranoside used as the substrate while interaction analysis would be evaluated as distinct β-galactosidase activity in units/milligram of protein.
Various yeast strains would be analyzed to determine their capacity to subdue the harmful phenotype of forming part of the overactive HOG trail. Shortly, N-terminally trimmed SSK2 would be assessed under the influence of GAL promoter. Any remaining strains would be checked using spotting serial dilutions to determine their survival noted through culture development on galactose and glucose plates.
The Western blot analysis would be used to assess phosphorylation on HA-bonded Rck2p. The genetic factor would be conveyed in the most appropriate plasmids with various strains or strain framework. Vortexing using glass beads in lysis butter would be used to destroy cells enhanced by protease inhibitors.
Samples would be subjected to dephosphorylation for 15 minutes at 37°C using the treated λ phosphatase. Distinctive elements of phosphatase treatment would be evaluated using additional vanadate. Cell extracts with 40 μg of aggregate protein would be taken from SDS/7.5% polyacrylamide ointment and marked on the nitrocellulose membranes to identify the protein gene adopting the Western blot revealing mechanism or Pierce.
Vitro Kinase Assays
For in vitro kinase assays, Teige et al. (2001) showed that C-terminally HA epitope-bonded HOG1p was immunoprecipitated from extracts of yeast cells containing HA antibody destined to Pan mouse IgG Dynabeads for two hours at 40C prior to and after the activation of HOG1p using salt shock for five minutes (0.4 M NaCl). Every precipitate would be cleaned twice using a lysis buffer and then once using two kinase buffer.
The cleaned beads would then be divided into the same amounts and mixed with other substrates to initiate kinase reaction. The reaction will take place for 15 minutes at normal room temperature, and it would be terminated by introducing a loading buffer of 15 μl 5 × SDS. The p38 inhibitor SB 203580 would be used to analyze the distinctive elements of HOG1 kinase activity. Autoradiography would be added to identify phosphorylation after SDS/PAGE.
Further, a method presented by Dóczi et al. (2007) would also be utilized for the study. The recombinant GST- MPKs (1 μg) would be nurtured in 15 μL of kinase reaction buffer containing immunoprecipitated myc epitope–bonded wild-type or with active alleles of MKK3 obtained from protoplasts. Reactions involving kinase would be conducted under room temperature for about 30 minutes. They would be stopped using SDS loading buffer after 30 minutes and heated or two minutes at 95°C. The products would be analyzed using SDS/PAGE autoradiography and Coomassie Brilliant Blue R 250 staining (Dóczi et al., 2007).
Two-dimensional gel electrophoresis will be used to conduct EF-2 alteration. The polyclonal antisera would be used to react with EF-2 to initiate immunodetection. All protein excerpts will be set prior to and 15 minutes following the osmotic tremor. The aggregate protein of about 50 μg should be isolated on pH 3–10 nonlinear gradients using a stepwise gradient. Successively, strips would be balanced using the mixture of 50 mM Tris⋅HCl, pH 8.8/3% SDS/5 mM DTT, and introduced to SDS/PAGE and Western blotting (Dóczi et al., 2007).
Leucine Incorporation Assays
The 3H-labeled leucine shall be included in the trichloroacetic acid-precipitable component. The plasmid YCp111 with the LEU2 gene would be used to change enhance the development of various straining, including the wild-type and HOG1 Δ strains (Teige et al., 2001). The artificial medium, leucine would be used to grow cells to an OD of 0.5, made more pure by centrifugation, and suspended again using 10 ml of medium.
About 3H-leucine (75 μ Ci, 140 Ci/mmol) will be put to every specimen five minutes prior to osmotic challenge. The process will run parallel with stress process. Cells would be gathered at specific times, including 0, 5, 15, 30, 45, and 60 minutes. The collected cells would then be suspended again in 200-μl lysis buffer and complemented with 10 μg/ml cycloheximide, and later iced up in liquid N2.
The glass globules will be used to break cells, and the aggregated protein will be precipitated using frozen acetone. Protein cubes would be cleaned twice with about 25 percent CCl3COOH and once with 70% acetone, and later determined with 1-ml CH4N2O buffer for clear calculating.
To determine amino acid endorsement, cell, normally the wild type and other strains were grown using the steps defined. A portion of the culture shall be treated with osmotic shock containing 0.4 M NaCl and trailed with instantaneous application of 3H-leucine. These cells would be amassed between five and 12 minutes following the addition of leucine, cleaned once with cold medium, and taken through centrifugation again. A clear concoction will be used to dissolve cell pellets while the radioactivity shall be gauged using liquid sparkling. The uptake of leucine following salt shock will be analyzed as percentage variation of application with no accounting for stress. The assessment noted once 12 minutes are over following the measurement will be used as the reference 100% for every strain.
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Salt stress, specifically, sodium stress, is known to trigger salt overly sensitive (SOS) system in plants, which lead to sodium repossession from the cytosol. It has also been observed that exposure to salt stress often result in transient rise in the level of free cytosolic calcium as perceived by the salt overly sensitive system (Yoo, Cho, Tena, Xiong, & Sheen, 2008).
Transient Express Assays
The plan expression vector pRT100 would be used to clone the reading frames of MPKs and MKKs, and they will be joined at their C-terminal end either to a c-myc epitope or a three HA epitope (MKKs) (Dóczi et al., 2007).
The assays involving Arabidopsis protoplast would be done as shown by (Ouaked et al., 2003).
GST Fusion Protein Expression and Purification
E. coli strain BL-21 codon and (Stratagene) would be altered using the expression construct cloned into the Pgex4-T1 vector (that can be obtained from Amersham Pharmacia Biotech or related manufacturers) and conveyed as GST fusion proteins. The bacterial development and isolation of recombinant GTS fusion protein would be done based on the method provided by Matsuoka et al. (2002).
Arabidopsis Protoplasts and Seedlings for Extraction of Protein
Protoplasts would be used to prepare protein extracts using the previously described by Cardinale et al. (2002). Alternatively, the extracts may be obtained from 200 mg of frozen seedlings in 200 μL of Lacus buffer and sea sand (Dóczi et al., 2007). Samples would be obtained from 50 mM Tris, pH seven point eight, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, and proteinase inhibitor mix (Roche) for coimmunoprecipitation (Dóczi et al., 2007).
Coimmunoprecipitation from Plant Extracts
Protein 50 μL would be used to preclear protein extracts (500 μg). This will have A–Sepharose beads and the process will take 2 hours at 4°C. The product will then undergo immunoprecipitation throughout the night in the presence of 50 μL of beads containing antibodies. The sample will undergo a thorough cleaning, at least three times using buffer (50 mM Tris, pH 7.8, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, and zero point 1% Tween 20), and will be subjected to immunoblotting. The anti-MPK7 antibody raised from rabbits will be obtained for the study.
Immunocomplex Kinase Assays
The method provided by Cardinale et al. (2002) (as cited by Dóczi et al., 2007) would be used for immunocomplex kinase assays. In addition, the same amount of protein extracts would be immunoprecipitated with 30 μL of protein A–Sepharose globules while the globules would be cleaned twice using buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EGTA, 5 mM EDTA, and 0.1% Tween 20). Beading washing will also be done using kinase buffer (20 mM HEPES, pH seven point four, 10 mM MgCl2, 5 mM EGTA, and 1 mM DTT).
The reaction involving kinase immunoprecipitated proteins will be conducted in 15 μL of kinase buffer containing 5 μg of MBP, 0.1 mM ATP, and 3 μCi of [γ-32P] ATP. Autoradiography will be used to observe phosphorylation of MBP following isolation by 15% SDS-PAGE (Dóczi et al., 2007).
The same amount of protein extracts would be isolated using SDS-PAGE, taken to polyvinylidene difluoride membranes (Millipore), and then assessed with HA or myc monoclonal antibodies for protein gel blotting. A secondary antibody – Alkaline phosphatase–conjugated anti-mouse IgG (Sigma-Aldrich) will be applied and the process would be analyzed using fluorography using CDP-Star (Amersham Pharmacia Biotech) as a substrate (Dóczi et al., 2007).
MKK9 Experiment for Protein-Protein Interaction
Plant Materials, Growth, and Treatment Requirements
Arabidopsis thaliana Col-0 genetic background would be used in this experiment. All seeds shall be sown in Petri dishes with 0.53 Murashige and Skoog salts and 0.8% (w/v) phytoagar enriched with the recommended antibiotics (Guo, Gan, Zhou, & Cai, 2009). Two-week-old plants would be taken to soil mixture containing 3:2:1 peat moss:vermiculite:perlite, and later subjected to 220C with 60 percent relative humidity under sustained light (100–120 mmol m22 s21 light from a mixture of fluorescent and incandescent bulbs) (Guo et al., 2009). The T-DNA insertion lines and transgenic lines would also be subjected to similar conditions just like the wild type.
Glucocorticoid processes would be performed based on the method previously described by Aoyama and Chua (1997), as cited by Guo et al., (2009). About 30 mM DEX would be used to spray plants after three weeks, and after 96 hours, the leaves would be used for molecular and physiological analyses.
Yeast Two-hybrid Assays
The presumed interaction between MKKs and MAPKs will be analyzed using Clontech matchmaker Gold Yeast Two-Hybrid system based on the manufacturer’s instructions (Zhang et al., 2016). The gene specific primers from G. hirsutum acc. TM-1 and cloned into pGBKT7 and pGADT7 vectors will be used. Further, eight MKK proteins will be altered into yeast two-hybrid Gold yeast strains and the auto-activation process will evaluated.
Aureobasidin A (ABA) will be applied as auto-activation inhibitor while each MKK-MAPK pair would be moved into yeast cells, and the formed colonies would be analyzed on selective medium specific medium (SD/-Trp/-Leu and SD/-Trp/-Leu/ABA). Further analysis would be conducted using quadruple dropout medium (SD/-Ade/-His/-Leu/-Trp) and another quadruple dropout medium augmented with X-α-Gal and Aureobasidin A for 7 days at 30°C (Zhang et al., 2016). It is expected that only yeast colonies with interactions between MKKs and MAPKs would develop in the used medium (Zhang et al., 2016).
MKK9 and MPK6 T-DNA Insertion Lines
An insertion line for MKK9 (SALK_017378) for T-DNA and other two T-DNA insertion lines for MPK6 (SALK_073907 and SALK_127507) would be acquired from the Salk T-DNA Collection.
For genotyping involving the putative null lines, gene-specific primers G2165, G2420 (for mkk9-1), G2709, and G2710 (for mpk6-2 and mpk6-3) and T-DNA left border primer G2325 will be used for this study. Insertion lines involving MPK3 would perhaps require a different approach (Pitzschke, Datta, & Persak, 2014).
Plasmid Production and Molecular Cloning
MKK9 opening reading frames and different MPKs would be enhanced using PCR obtained from Arabidopsis cDNA and cloned using the pGEM-T vector and later verified using sequencing. Alteration of assumed phosphorylation sites would be used to produce essential active form of MKK9. The mutation process will produce an essential active kinase (MKK9-EE). On the contrary, inactive types of MKK9 and MAPKs will be produced by altering the unspoiled Lys remnants in the ATP-linking domain of each kinase to Arg.
The post-alterations are most likely to produce kinase-inactive types. These mutants would be developed through a PCR-initiated site-controlled mutagenesis method.
Coding areas of MKK9 or every MPK would be cloned in E. coli expression vector for pMAL-c2 to create a maltose-binding protein (MBP) fusion protein for in vitro assessment. In vivo assessment would require coding areas of MKK9 and MPKs cloned into pRTL2-S65TGFP to create GFP fusion proteins (Guo et al., 2009). Cloning involving pGL800, a binary vector obtained from pPZP211 would be used for constitutive overexpression of MKK9, MKK9EE, or MKK9KR (Guo et al., 2009). DEX-inducible overexpression would require the binary vector pGL1152 for cloning.
Transgenic Plants Generation
Based on the previous description given by He and Gan (2000) as cited by Guo et al. (2009), the above-mentioned binary vectors would be factored in Agrobacterium tumefaciens strain ABI. Agrobacterium cells with various constructs would then be applied to change Arabidopsis ecotype Col-0 wild-type or mutant plants through vacuum infiltration (Guo et al., 2009). Transgenic plants would then be chosen on medium of Skoog and Murashige.
RNA Gel-Blot and RT-PCR Analyses
All RNA extractions obtained from the leaves and RNA gel-blot would assessed as described by He and Gan (2002) (Guo et al., 2009). Hybridization will be conducted at 650C with cDNA. RT-PCR process will be conducted using various primers, including for MKK9, G2419 and G2420 (Guo et al., 2009).
In Vitro Kinase Assay
The expression constructs cloned using pMAL-c2 vector and presented as MBP fusion protein will be used to change E. coli. Further, the fusion proteins will be expressed and washed using amylose-affinity Chromatography and progressively measured by Bio-Rad protein assay reagent. Every MBP-MPK (1 mg) or MBP-MPKKR protein would preserved using kinase 20 mL of a reaction buffer in the presence of or without MBP-MKK9 or MBP-MKK9EE for 30 minutes at 300C. The reaction of kinase would be stopped by introducing 4 mL of 63 SDS sample buffer and heating for 5min at 950C. Ten percent (w/v)
SDS-PAGE, autoradiographed, and stained with Coomassie Brilliant Blue R250 would be used to stop the reaction.
Protein Extracts and Immunoprecipitation Construction
The method previously used by Ichimura et al. (2000) would be used for protein preparation (Guo et al., 2009). Immunoprecipitation will be obtained from crude extracts from plants.
In-Gel Kinase Assay
Different protein previously mentioned would be separated on 10 percent SDS-polyacrylamide gels surrounded with 0.5 mg mL21 myelin basic protein on a splitting gel as a kinase factor. Once electrophoresis has been conducted, the gel will be purified thrice with cleaning buffer (25mM Tris-HCl, pH 7.5, 0.5mM DTT, 0.1mM Na3VO4, 5mM NaF, 0.5 mg mL21 bovine serum albumin, and 0.1% Triton X-100) to eliminate SDS. The protein would later be denatured in a denaturing buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF) immediately at 40C with three changes of the buffer. Further, the gel would require incubation using reaction buffer (25 mM Tris-
HCl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4) at room temperature for 30 minutes, then phosphorylated in 20 mL of the same reaction buffer containing 0.5 mM ATP and 50 mCi [g-32P]ATP at room temperature for one hour. The reaction would ended by moving the gel into stop solution (5% trichloroacetic acid and 1% sodium pyrophosphate). Unguents would then be cleaned using stop solution for five hours at room temperature using four deviations of clean solution, dried on 3MM Whatman paper, and then autoradiographed.
Protoplast Separation and Transient Expression Analyzes
Separated protoplasts obtained from Arabidopsis mesophyll cells will be used for transient expression analyzes. The separation, alteration, and nurturing of protoplasts will be done based on to protocol described by Sheen. MKK9-1 null mutant protoplasts will be rapidly changed using polyethylene glycol with 10 mg of plasmid. After 12 hours following change, the protoplasts would be gathered via centrifugation and suspended in protein removal buffer for supplementary examination.
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