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Plant vacuoles are the largest organelles in cells, which play a role in regulating homeostasis in the cytoplasm under the action of adversity stress, and play a regulatory role in the regionalization of toxic substances and the cell signal transduction system.
In desert plants, many plants are under environmental stress, resulting in reduced crop yield and reduced quality, but species containing stress resistance genes can greatly reduce the damage to plants by adjusting the expression of relevant genes in cells through external stress signals.
In order to adapt to the extreme environment, C. huahuahua has a strong tolerance to abiotic stress and other stresses, and is a beneficial plant for saline-alkali soil improvement.
Therefore, as a desert plant, firewood plays an important role in the development and utilization of its stress-resistant resources.
Materials and methods of the experiment
The leaves of young flowers and firewood were collected, and the leaves were brought back to the laboratory after liquid nitrogen quick-frozen, and stored in an ultra-low temperature freezer at -80 °C for later use.
The extraction of total RNA from young leaves of C. huahuahua is a kit for the extraction of total RNA from polysaccharide polyphenol plants, a kit for the synthesis of the first strand of cDNA, and the obtained cDNA is cryopreserved at a temperature of -20 °C.
The sequence of KcVP1 gene cloned by RACE method was designed by Prime5.0 software, and the first strand of cDNA reverse transcribed by Huahuachai RNA was used as a template for PCR amplification, reaction system and reaction program.
The PCR amplification product was subjected to agarose gel electrophoresis, and the target DNA fragment was recovered with agarose DNA recovery kit, which was ligated to the pMD-19T cloning vector, transformed into E. coli competency, and incubated at 37°C for 16 h.
The obtained cDNA sequence was used as a template and PCR amplified by primers pET28a-KcVP1-F/pET28a-KcVP1-R.
The target fragment and the vector fragment were recovered by double digesting the empty vectors of KcVP1 and pET28a with restriction enzymes SalI and XhoI, respectively, for 3 h.
The product was ligated overnight at 16 °C, reconverted into E. coliDH5α competency, coated onto LB solid medium containing 50 μg/mL kanamycin antibiotic, and incubated at 37 °C for 16 h.
The white single colony was picked for PCR verification and plasmid double digestion verification, and the successfully identified positive recombinant plasmid was transferred into DE3 host cells.
Analysis of experimental results
The KcVP1 gene was amplified by PCR, and the fragment size of the KcVP1 gene was about 2286 bp by 1% agarose gel electrophoresis.
After ligation with the cloning vector pMD-19T and transfer to E. coliDH5α competency, the colony PCR verification and sequencing analysis were performed, and the results showed that the cloning vector was successfully constructed.
The recombinant plasmid pMD19-T-KcVP1 was used as a template for PCR amplification, and the target fragment was recovered by gelatin, and the restriction enzymes SalI and XhoI were used to double digest the pET-28a empty vector and the target fragment, respectively.
After digestion, the target fragment was recombinantly ligated with the pET-28a empty vector, and the recombinant plasmid pET-28a-KcVP1 was transformed into E. coliDH5α.
Colony PCR validation shows a bright band, approximately 2286 bp, with a pET28a no-load band at about 5000 points on agarose gel electrophoresis, and a band of interest for the KcVP1 gene at 2000-3000 points.
The recombinant plasmid pGBKT7-KcVP1 was then transformed into E. coliDH5αBL21, and the results showed that there was a target band and sequencing analysis of KcVP1 gene at about 2000-3000 in agarose gel electrophoresis, indicating that the prokaryotic expression vector was successfully constructed.
The recombinant plasmid pMD19-T-KcVP1 was used as a template, PCR amplification, and the target fragment was recovered by gel, and the pGBKT7 empty vector and the target fragment were double-digested with endonuclease SalI and NotI.
After the recombination ligation was transformed into E. coliDH5α competency, and then the positive recombinant plasmid pGBKT7-KcVP1 was transformed into Y2HGold competency, a bright band of approximately 2286 bp could be seen as shown by colony PCR.
The double digestion identified that there was a no-band for pGBKT7 at about 8000 and a target band for KcVP1 gene at about 8000 and 2000-3000 for the KcVP1 gene.
The growth of pGBKT7 and recombinant yeast pGBKT7-KcVP1 in the control group was basically the same in the control group without any stress, indicating that the initial concentrations of the two groups were the same.
Under different concentrations of PEG under drought stress, tolerance stress determination showed that the colonies of yeast pGBKT7 and recombinant yeast pGBKT7-KcVP1 in the control group had died at 25% PEG concentration.
However, under the drought stress treatment of 20% PEG, the number of colonies of recombinant yeast was significantly higher than that of the control group, and the survival rate of the control group gradually decreased with the increase of dilution factor.
When the dilution factor reached 1000-fold, the control yeast colony did not grow at all, while the recombinant yeast had a good growth trend.
Under the stress of different concentrations of NaCl, the tolerance pressure was measured, and it was found that the colonies of yeast pGBKT7 and recombinant yeast pGBKT7-KcVP1 in the control group had died at the concentration of NaCl of 0.5mol/L.
However, under the salt stress of 0.4mol/LNaCl, the resistance of recombinant yeast pGBKT7-KcVP1 to high salt was significantly stronger than that of the control yeast pGBKT7 with the increase of dilution factor.
Under the stress of different concentrations of NaCl, tolerance stress determination was carried out, and it was found that the colonies of the control yeast pGBKT7 and the recombinant yeast pGBKT7-KcVP1 had died at a high temperature of 52°C.
Under the treatment of 48°C, the dilution factor became larger and larger, and the number of colonies in the experimental group was significantly higher than that in the control group.
The OD600 values of yeast pGBKT7 and recombinant yeast pGBKT7-KcVP1 in the control group were basically the same under no stress treatment, indicating that the initial concentrations of the two were the same.
The OD600 value of recombinant yeast pGBKT7-KcVP1 was significantly higher than that of control yeast pGBKT7 under different concentrations of drought stress, and the OD600 of recombinant yeast decreased from 1.8 to 0.8 at 20% PEG concentration.
The OD600 of the control group decreased from 1.5 to 0.01, and the survival rate of the control group tended to be close to 0, indicating that the recombinant yeast had a certain effect on resisting drought stress.
The OD600 value of pGBKT7-KcVP1 in the heavy wedge group was significantly higher than that of the control yeast pGBKT7 at 0.4mol/L, and the OD600 value of the recombinant yeast pGBKT7-KcVP1 was 1.6 times that of the control yeast pGBKT7 at 0.4 mol/L.
The results indicated that the recombinant yeast pGBKT7-KcVP1 had a strong salt resistance, and the OD600 value of the recombinant yeast pGBKT7-KcVP1 was significantly higher than that of the control yeast pGBKT7 under high temperature stress.
At 44°C, the OD600 value of recombinant yeast pGBKT7-KcVP1 was 2.1 times that of the control yeast pGBKT7, which inferred that 44°C was the most sensitive temperature of yeast, indicating that the gene expression of recombinant yeast pGBKT7-KcVP1 had a certain effect on high temperature resistance.
Further research was carried out on the OD600 values of the recombinant yeast pGBKT7-KcVP1 and the control yeast pGBKT7.
The results showed that under the drought stress of 20% PEG, the gap between the two groups increased first and then decreased with the increase of time, and at 8 h of stress, the experimental group was 1.5 times that of the control group.
Under the salt stress treatment of 0.4mol/LNaCl, the OD600 value difference between the two groups was the largest at 4 h, and the experimental group was 1.8 times higher than that of the control group.
Under the high temperature stress of 52°C, with the increase of time, the experimental group was 1.6 times that of the control group at 4 h, and the OD600 of the two groups approached 0 at 24 h, indicating that the survival rate of the experimental group and the control group was the lowest.
Discussion of experimental results
At present, the global saline-alkali land area has reached 954.38 million hm2, and China's saline-alkali land accounts for about 10% of the world's total, of which the Tarim Basin in Xinjiang is the main saline-alkali zone, with arid soil, low precipitation and high temperature, which causes serious damage to plants.
The study found. Saline-alkali tolerant plants such as Asteraceae, Chenopodaceae, Leguminaceae and Poaceae in the desert can effectively adapt to the adversity environment through their own stress resistance mechanisms under the environmental pressure of strong drought, high temperature and high salinity.
Therefore, it is of great significance to develop and utilize stress-tolerant plants and their genetic resources, study the stress resistance mechanism of plants, and select and breed stress resistance breeding through in-depth excavation of stress-tolerant plants.
Vacuolar membrane pyrophosphatase, which is one of the proton pumps, uses pyrophosphate as a substrate to hydrolyze to produce two molecules of Pi, which converts H+ from the cytoplasm into vacuoles.
Electrochemical gradients and pH gradients are formed inside and outside, and can actively transport, cations, anions, amino acids and sugars, solute molecules and exclude accumulated ions.
By regulating the osmotic pressure and regulating the intracellular ion balance, the excessive Na+ and Cl- in the cytoplasm are separated in the vacuole, so as to reduce the damage caused by the adverse environment to plant cells and maintain intracellular homeostasis and physiological growth.
Ion segregation is one of the most common ways to regulate plant stress, in which vacuolar membrane H+-PPase plays a key regulatory function.
Studies have shown that overexpression of vacuolar membrane H+-PPase gene AVP1 can enhance the functions of transgenic Arabidopsis thaliana in salt tolerance, drought resistance and high temperature resistance.
At present, in addition to model plants Arabidopsis thaliana and tobacco, apples, rice, cotton, tomatoes and other crops are also widely used, and it has been found that H+-PPase can improve the drought resistance, salt tolerance and high temperature tolerance of the recipient material.
At the same time, it was found that in Arabidopsis, tomato, tobacco and other plants, excessive expression of AVP1 could enhance the growth ability of their roots, expand the leaf area, increase the yield of kernels, and improve the yield of cotton fiber.
These studies showed that H+-PPase can improve plant stress resistance and crop yield, and the sequence of KcVP1 gene, which is 2286 bp long and encodes 761 amino acids, was cloned using Huahuachai as a material.
The prokaryotic expression vector and yeast expression vector of KcVP1 were constructed, and the recombinant strains were transferred to Escherichia coli BL21 and yeast strain Y2HGold, respectively.
The results showed that the recombinant yeast expression vector pGBKT7-KcVP1 had stronger resistance to high temperature, salt tolerance and drought tolerance, indicating that KcVP1 was highly responsive to drought, high temperature and high salt stress, and played an important role in the ion separation process of C. huahua.
Therefore, vacuolar membrane H+-PPase plays a key role in the growth and development of plants, and its function and mode of action lay the foundation for the development and utilization of plant stress-resistant resources.
At the same time, it has a wide range of applications in real agricultural production, such as regulating the growth and development of plants, improving crop yields, increasing stress resistance, etc., and can also reduce energy consumption and environmental impact.
Therefore, the exploration of vacuolar membrane H+-PPase has broad research prospects and has potential value in genetic engineering applications for the improvement of plant stress tolerance traits.