| 其他摘要 | Halophilic archaea is an intermediate group between prokaryotes and eukaryotes. They can produce substances of development value, such as strong antioxidants bacterioruberin, bacteriorhodopsin (BR), polyhydroxy fatty acid (PHA), and osmoprotectant trehalose and glycine betaine, etc. In-depth study of halophilic archaea can broaden people's understanding of the evolution of biological systems and provide important evidence for the origin and evolution of life. Through transgenic technology, using its osmoadaptative gene, it is expected to provide new ways to improve the stress resistance of crops.
In this study, the halophilic archaea Halorubrum kocurii 2020YC7 was used as the material, combined with whole-genome sequencing and expression level analysis, to determine the effect of NaCl concentration changes on endogenous and exogenous osmoprotectants (potassium, trehalose, glycine betaine, etc.) The special halophilic mechanism of halophilic archaea was explored, and the main results obtained were as follows:
1.Whole-genome sequencing results showed that: the full-length gene of H. kocurii 2020YC7 was 3,727,755 bp, and its GC content was high, accounting for 66.24%, which was more conducive to its DNA stability and survival in a hypertonic environment; The proportion of acidic amino acids was also very high, accounting for 17.14% of the total amino acids. Acidic amino acids were negatively charged, which were conducive to binding cations for osmotic regulation. In addition, 3513 protein function annotation genes were obtained, which were related to osmotic regulation. There were 33 copies of osmoprotectant transport-related in total, involving potassium transport, sodium transport, trehalose synthesis, glycine betaine, and trehalose transport, and no glycine betaine synthesis genes were found.
2.The expression of osmoadaptative genes was analyzed by RT-qPCR, and it was found that the expression of potassium uptake genes trkH, trkA, kch and potassium export gene kefB were positively correlated with NaCl concentration. Among them, the transmembrane protein encoding gene trkH of the Trk potassium uptake system changed the most, and the relative expression level of the 250 g/L group was nearly 500 times than 50 g/L group. The relative expression levels of genes trkA, kch and kefB in the 250 g/L group were 449 % ± 101 %, 339 % ± 22 % and 266 % ± 28 % of the 50 g/L group, respectively. The relative expression of trehalose synthesis gene treS was negatively correlated with NaCl concentration. In the absence of exogenous compatible solutes, the glycine betaine transporter bcct and the trehalose transporter sugA did not change with the NaCl concentration.
3.The detection results of osmoprotectants showed that the cells of the strain contained a large amount of potassium, and the potassium concentration increased with the increase of NaCl concentration. The maximum appeared in the 200 g/L group, and the concentration was 28.67 ± 3.91 μmol/mg protein, about 7.5 times that of the 50 g/L low-salt group. Trehalose was detected in the cells of the strain, but the changing trend with NaCl was opposite to that of potassium. The higher the NaCl concentration, the lower the trehalose concentration. The presence of endogenous glycine betaine was not detected, indicating that the strain did not have the ability to synthesize glycine betaine de novo.
After exogenous addition of glycine betaine, a large amount of glycine betaine was detected in H. kocurii 2020YC7 cells, and it was positively correlated with the concentration of NaCl. The concentration of glycine betaine in the 250 g/L NaCl group was as high as 15.27 ± 1.20 mg/mg protein. At the same time, the potassium concentration in the 150, 200, and 250 g/L NaCl groups was significantly decreased (P<0.05). The potassium concentration decreased from 23.79 ± 0.29, 28.67 ± 3.91, 25.70 ± 3.59 without addition to 11.70 ± 0.23, 15.36 ± 0.80, 14.93 ± 0.77 μmol/mg protein, respectively, At the same time, there was only little change in trehalose concentration, and when trehalose was added exogenously, the decrease in potassium concentration was not significant (P>0.05), indicating that trehalose did not participate in high-salt osmotic balance.
The osmoprotectant glycerol has a significant inhibitory effect on the growth and bacteriocin accumulation of H. kocurii 2020YC7. Studies had confirmed that the inhibitory effect of the above glycerol was not directly related to its concentration, but is related to a significant change in pH. After the addition of glycerol, the pH of H. kocurii 2020YC7 medium decreased significantly and became acidic. Through genome exploration of glycerol metabolic pathways, combined with the results of oxygen electrodes, it was speculated that glycerol metabolizes intracellularly to produce lactic acid or acetic acid, which reduces the TCA cycle substrate acetyl-CoA, inhibited aerobic respiration rate, and hinders energy generation, which in turn led to the inhibition of growth and bacterioruberin synthesis.
4.In addition, studies showed that the content of bacterioruberin in H. kocurii 2020YC7 was closely related to the concentration of NaCl. When the salinity was 150 g/L, the accumulation of bacteriocin per unit strain was the highest (A494/A600 was up to 0.93 ± 0.09). Acetic acid significantly promoted the accumulation of bacteriocin in halophilic archaea (P<0.05).
In summary, the halophilic mechanism of H. kocurii 2020YC7 was summarized as follows: (1) When the concentration of NaCl concentration was 150 - 250 g/L, K+ played an role as the main osmoprotectant. The high proportion of acidic amino acids in the cells of the strain was the basis for binding with these K+. Potassium transport channels were activated in the NaCl concentration range of 100 - 150 g/L. (2) When the NaCl concentration was 200 - 250g/L, if glycine betaine existed in the external environment, the strain could transport them into cells through the BCCT family transporter, and cooperate with K+ for osmotic balance. (3) When the NaCl concentration dropped to 100 even 50 g/L, the polysaccharide of the strain was disintegrated and then catalyzed by the amylase/trehalose synthase TreS to produce trehalose, which explained why it cannot survive in a low-salt environment. Trehalose did not act as the main osmoprotectant of H. kocurii 2020YC7 in high salinity environment (above 100 g/L). (4) In addition, the high GC content of DNA and the accumulation of bacteriorhodopsin provided protection for the survival of H. kocurii 2020YC7 in a hypertonic environment. |
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