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Halophyte is an annual herbaceous plant of the phylum Angiosperm, Chenopodaceae, halophyllum genus. It is mainly distributed in the foothills and Gobi Desert in northwest China, and the altitude of the growing area is 700~1000m.
Due to the extreme growth environment of halophyte grass and the infestation of abiotic stress, its aboveground part of the plant has fleshy leaves, multi-branched stems and dense and strong root system in the underground part, so that it has a strong salt tolerance and drought resistance.
In the salt-tolerant mechanism, the root, as a salt-expelling organ, must exclude most of the dissolved Na+ and Cl- from the soil solution.
The root system is the first tissue organ to sense soil salinity, and a large amount of salt in the soil accumulates in the fleshy leaves, and then separates it in the vacuolar tissue in the leaves.
Shaker voltage-dependent K+ channel proteins are tetrameric membrane proteins composed of β subunits in response to changes in membrane potential by controlling the flow of K+ in and out of the cell membrane, with the core domains of Kvβ1 and Kvβ2 being the aldehyde ketone reductase AKR.
The aldehyde and ketone reductase protein superfamily is a group of enzymes that metabolize aldehydes and ketones to the corresponding alcohols with NADPH as a cofactor.
Materials and methods of the experiment
The seeds of halophyllum used in the experiment were taken from saline-alkali soil, evenly sown in flower pots filled with distilled water soaked culture medium, covered with plastic wrap and punctured for ventilation, and placed in an artificial climate chamber for cultivation.
Water an appropriate amount of Hoagland's nutrient solution every day to ensure the moist state of the culture medium, and after the seedlings grow to 2 months, use 200mmol· L-1NaCl was treated with salt stress for 7 days.
The strong roots of Halophyllum salinus were selected, cleaned and disinfected, and the RNA RNA of the roots of Halophyllum was extracted using the Root Extraction Kit, and the Root Reverse Transcription Kit was used to reverse transcribe into cDNA for cloning.
NCBIPrimer-BLAST was used to sequence the protein-coding region of the HgAKR6C gene in the root system of halophyllum grass, and the multiple cloning sites on the pBI121-eGFP and pYES2 vector plasmids.
Specific primers containing enzyme cleavage sites, HgAKR6C-F1, HgAKR6C-R1, HgAKR6C-F2 and HgAKR6C-R2, were designed to construct subcellular localization expression vectors and yeast heterologous expression vectors, respectively.
The qRT-PCR primers HgAKR6C-q1F, HgAKR6C-q1R, and HgActin were designed by NCBIPrimer-BLAST, and HgActin was used as the reference genes.
The reaction program was performed according to the two-step method of SuperReal Fluorescence Quantitative Premix Reagent: pre-denaturation at 95°C for 15 min, denaturation at 95°C for 10 s, annealing at 60 °C for 32 s, 40 cycles.
Reaction system: 2× SuperRealPreMixPlus 10 μL, forward and reverse primers 0.6 μL each, cDNA template 1 μL, RNase-freeddH2O 7.8 μL. Three technical replicates were set up, and the expression level of HgAKR6C gene under different salt stress times was calculated by 2-ΔΔCT method.
Analysis of experimental results
In this study, 200 mmol· The cDNA of the roots of L-1NaCl treated with one week was used as template, and HgAKR6C-F1, HgAKR6C-R1, HgAKR6C-F2 and HgAKR6C-R2 were used as primers for PCR amplification.
The amplified product was electrophoresis by agarose gel, and after the fragment size was verified, the gel was recovered and purified and detected by electrophoresis again, and the band was 951 bp consistent with the size of the target gene.
The target band gel recovery product was connected to pMD19-T, transformed into E. coli and blue and white plates for screening, and the target fragment was successfully amplified with a size of 951 bp by PCR amplification using the bacterial solution as a template.
The positive bacterial solution was sent to the company for sequencing, and the sequencing results were compared with the DNAMAN9.0 sequence, showing 100% homology, indicating that the target gene HgAKR6C was completely amplified.
The plasmid of the target gene was successfully cloned, as well as the activated pYES2 vector, pBI121-GFP plasmid, and HgAKR6C gene.
After the verification of the gel with two restriction enzymes, the pBI121-HgAKR6CGFP vector was constructed by T4 ligase and XbaI. and SmaI.
EcoRI. and XbaI. performed double enzyme digestion to construct pYES2-HgAKR6C, transformed Escherichia coli, picked monoclonal and PCR amplification of bacterial solution, and the band size was consistent with the target gene.
After sequencing, the sequence was successfully aligned with the help of DNAMAN software, and the bacterial liquid plasmid was extracted for double digestion verification, and the band size was consistent with the target gene.
The analysis results showed that the HgAKR6C gene was 1657 bp in length, the longest open reading frame was 987 bp, and the CDS region was 951 bp, encoding 317 amino acids.
The theoretical isoelectric point value of the protein encoded by HgAKR6C was 6.25, which was an acidic protein, the molecular weight was 35151.21Da, and the molecular formula was C1575H2473N419O468S12.
The negative charge residual base was 36, the positive charge residual base was 34, the fat coefficient was 90.41, and the instability coefficient was 32.06, which was a stable protein.
The secondary structure analysis of the protein encoded by HgAKR6C gene showed that the structure with the largest proportion was α-helix, accounting for 52.68%.
This was followed by random coiling, accounting for 25.87%, extended chains accounting for 14.2%, and the remaining 7.26% being β-turns, which was consistent with the results of the tertiary structure of the protein predicted by Swiss-Model.
TMHMM software analyzed the transmembrane region of the protein encoded by the gene and showed that the HgAKR6C gene had no transmembrane region, that is, the protein encoded by the gene was a non-transmembrane protein.
The analysis results showed that the probability of the presence of signal peptide in HgAKR6C gene was 0.0038, and it was speculated that the protein encoded by HgAKR6C gene did not have a signal peptide and could not guide the newly synthesized protein into cells or subcellular organs, so it was a non-secreted protein.
Protein hydrophobicity was analyzed using ProtScale, and the ratio of each point was judged to be hydrophobic above 0 and below 0, so the protein encoded by HgAKR6C was a hydrophilic protein.
The results of phosphorylation site prediction showed that the protein encoded by HgAKR6C gene had serine, threonine and tyrosine phosphorylation sites.
pBI121-GFP and pBI121-HgAKR6C-GFP were transiently transformed into tobacco, and the subcellular localization of genes in tobacco cells was observed by laser confocal microscopy.
The pBI121-GFP empty vector was expressed in all positions in tobacco cells, while pBI121-HgAKR6C-GFP was mainly observed in the cytoplasm with green fluorescence and a small amount distributed in the nucleus, confirming the expression of HgAKR6C gene in the cytoplasm and nucleus.
By real-time fluorescence quantitative analysis, 200mmol· The expression of HgAKR6C gene in roots of L-1NaCl treatment at 0, 2, 6, 24 and 72 h.
It was found that HgAKR6C gene was significantly expressed under salt stress at 2, 6, 24 and 72 h, and the expression level increased first and then decreased with the extension of treatment time, reaching a peak at 24 h and then decreasing.
The concentration of K+ in AP medium was 1 mmol· L-1 and Na+ concentrations≤ 30mmol· In the case of L-1, it was evident that the yeast strains transformed with the HgAKR6C gene grew better when the bacterial solution of the transformed empty vector and the recombinant vector was diluted 1000-fold.
The concentration of K+ in AP medium was 1 mmol· The concentration of L-1 and Na+ was 50mmol· In the case of L-1, it was evident that the yeast strain transformed with the HgAKR6C gene grew better than the strain transformed into the empty vector when the solution was diluted 10 times.
The results indicated that the transfer of HgAKR6C gene reduced the toxicity of Na+ to yeast cells, indicating that HgAKR6C gene was involved in regulating Na+ efflux.
When the concentration of K+ in AP medium was 10 and 100 mmol· L-1, both yeast strains transformed with empty vector pYES2 and yeast strains transformed with HgAKR6C gene could grow, and K+ was absorbed in AP medium.
The Na+ concentration in the growth environment of the strain had a significant effect on the contents of Na+ and K+ in PYES2 and AXT3-HgAKR6C, and the K+ and Na+ contents in the AXT3-HgAKR6C strain decreased when the Na+ concentration in the growth environment of the yeast strain increased.
The K+ concentration in the growth environment of the strain had a significant effect on the Na+ and K+ contents in PYES2 and CY162-HgAKR6C, and when the K+ concentration of yeast strains increased in the growth environment, the K+ content in CY162-HgAKR6C strain increased, while the Na+ content decreased.
Discussion of experimental results
Bioinformatics analysis of the protein encoded by HgAKR6C gene showed that the total length of HgAKR6C gene was 1657 bp, and the total length of CDS region was 951 bp, encoding 317 amino acids.
Protein phosphorylation can change the viability of proteins and play an important role in signal transduction, growth and development in organisms.
By comparing the physiological and transcriptomic data of Zygosaccharomyces lucephala, it was found that salt stress led to a significant increase in the content of four amino acids in yeast cells.
Using phosphorylation-modified proteomics, the Arabidopsis model was analyzed and a series of phosphorylation sites were discovered, influenced by high abscisic acid-induced phosphatase 1, to modulate the signaling and defense response mechanisms used by plants in response to abiotic stresses.
It is speculated that the protein encoded by HgAKR6C gene includes three phosphorylation sites, which may play a role in cross-protection or signal regulation to resist salt stress through abiotic stress response or dephosphorylation of protein phosphatase.
The expression of HgAKR6C gene in the roots of Halophyllum salviphytes was analyzed under salt treatment at different times, and it was found that HgAKR6C had the most significant expression level at 24 h of salt treatment, and the expression of the protein encoded by HgAKR6C gene was closely related to salt stress.
The domain prediction results showed that the protein encoded by HgAKR6C gene contained AKR_SF superfamily domains, and the specific sites AKR_AKR6C1_2 voltage-gated K+ channel β subunits, which were from Arabidopsis thaliana and Aquaticus, respectively, and belonged to the initial members of Aldehyde Ketone Reductase Family 6 C1 and C2, respectively.
Aldehyde ketone reductase, on the other hand, is a large superfamily of proteins, mainly NAD-dependent oxidoreductases, which are involved in carbonyl metabolism and whose main purpose is to reduce aldehydes and ketones to primary and secondary alcohols.
KCAB, also known as the K+ channel subunit, or KAB1, is a possible accessory K+ channel protein that regulates the activity of the pore-forming α subunit, with consistent related functions with ketoaldehyde reductase and K+ channel proteins.
According to the NCBI protein sequence alignment and domain prediction, the existing AKRs family protein sequences were selected to construct a phylogenetic phylogenetic tree.
Phylogenetic analysis showed that HgAKR6C was most closely related to AtAKR6C1 in Arabidopsis, which was consistent with the domain-predicted results AKR_AKR6C1_2 with specific loci, so it was speculated that HgAKR6C may be a functional protein belonging to the AKR6C1 family.
Therefore, it is inferred that the protein encoded by HgAKR6C gene is a functional gene in the voltage-dependent K+ channel β subunit that relies on AKRs as a cofactor to exert salt stress tolerance.
AXT3 is a strain of yeast that is sensitive to Na+ and does not contain ATPase, so it is extremely sensitive to Na+, and CY162 is a K+-deficient yeast strain that does not contain the K+ transporters TRK1 and TRK2, and cannot grow under low potassium conditions, which are often used for preliminary verification of genes.
According to the results of this study, it can be reasonably predicted that HgAKR6C is a stable protein acting on the cytoplasm, with AKR gene family domain and tends to AKR6C1 family, and Kvβ can maintain ion homeostasis to improve the salt tolerance of plants.
In the early stage of the experiment, the transcriptome data of salt stress in halophytes were screened, and the gene HgAKR6C was cloned, and it was found that the protein encoded by this gene was expressed in both the nucleus and the cytoplasm.
Domain analysis showed that the protein encoded by HgAKR6C gene had AKRs family domain, which was highly homologous to the AKR6C1 family protein sequence in phylogenetic analysis.
The results of qRT-PCR showed that the expression of HgAKR6C could be increased due to a certain concentration of salt stress.
The transfer of yeast expression vectors into defective strains showed that the gene had the function of Na+ efflux and K+ transport, and it was determined that HgAKR6C could be used as the research object of the regulatory mechanism of salt tolerance genes in halophyte roots, which provided a theoretical basis for the functional analysis of HgAKR6C in the later stage.