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Effect of Sodium Chloride on the Expression of Genes Involved in the Salt Tolerance of Bacillus Sp. Strain

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Effect of Sodium Chloride on the Expression of Genes Involved in the Salt Tolerance of Bacillus Sp. Strain Open Chemistry 2021; 19: 9–22 Research Article Jian Zhang*, Qingqing Xiao, Tingting Guo, Pengcheng Wang Effect of sodium chloride on the expression of genes involved in the salt tolerance of Bacillus sp. strain “SX4” isolated from salinized greenhouse soil https://doi.org/10.1515/chem-2020-0181 Keywords: bacteria, chemicals, gene expression, facility received June 3, 2020; accepted September 15, 2020 soil, salt resistance Abstract: Salt stress is one of the important adverse con- ditions affecting bacterium growth. How bacteria isolated from greenhouse soil cope with salt stress and regulate the genes responsible for salt tolerance are still unclear. 1 Introduction We conducted RNA transcriptome profiling of genes con- tributing to the salt tolerance of a Bacillus sp. strain Land degradation by salts is one of the major threats to [ ] (“SX4”) obtained from salinized soil. Results showed sustainable crop production in the world 1 , and soil that NaCl effectively regulated the growth of “SX4” in salinity is one of the most harmful conditions not only fi terms of cell length and colony-forming unit number in eld agriculture production but also in greenhouse [ ] - decrease. A total of 121 upregulated and 346 downregu- cultivation 2,3 . Despite that biotic and abiotic environ ff lated genes were detected under salt stress with reference mental factors greatly a ect plant growth and yield, salt to the control. The largest numbers of differential expres- stress is still considered one of the major stress factors sion genes were 17 in carbon metabolism, 13 in the bio- that seriously inhibit plant growth and microbe survival [ ] synthesis of amino acids, 10 in a two-component system, 4 . Salinized soil is widely distributed in the world and [ ] - and 10 in ABC transporter pathways for adapting to salt hinders the crop growth and productivity 5 . Approxi - ff - stress. Our data revealed that cation, electron and trans- mately one billion hectares of salt a ected soils are scat [ ] - membrane transport, and catalytic activity play impor- tered all over the world 6 , and approximately 100 mil 2 - [ ] tant roles in the resistance of bacterial cells to salt ions. lion hm saline alkali soils are distributed in China 7 .In Single-nucleotide polymorphism and the mutation of recent years, owing to rapid developments in protected - base pair T:A to C:G play potential roles in the adaptation vegetable cultivation in China, a large number of che of “SX4” to high NaCl concentrations. The findings from mical fertilizers and pesticides have been used. Despite fi ff this study provide new insights into the molecular the high yields and bene ts, diseases a ecting vegetable mechanisms of strain “SX4” and will be helpful in pro- crops have increased and seriously degraded soil quality [ ] moting the application of salt-tolerant bacteria. 8 . Currently, the proportions of secondary salinization of soil in protected vegetable fields are extensively high, and the problem of soil salinization has become one of [ ] * Corresponding author: Jian Zhang, Institute of Horticulture, Anhui the main obstacles to vegetable production 9 . Academy of Agricultural Sciences, Nongke South Road 40#, Hefei, The total salt content of greenhouse vegetable soil is 230031, Anhui, China; Key Laboratory of Genetic Improvement and much higher than that in open field conditions because of Ecophysiology of Horticultural Crops, Hefei, 230031, Anhui, China, the excessive application of chemical fertilizers, which - e mail: [email protected], [email protected], are the main causes of secondary soil salinization in tel: +86-551-6516-0817, fax: +86-551-6514-8169 greenhouse vegetable fields [10,11]. Soil salinization Qingqing Xiao: School of Biology, Food and Environment, Hefei fi University, Hefei, 230601, Anhui, China signi cantly reduces the diversity and abundance of Tingting Guo: Institute of Horticulture, Anhui Academy of microbial communities. However, soil enzyme activity Agricultural Sciences, Nongke South Road 40#, Hefei, 230031, is strongly related to soil microbial biomass and com- Anhui, China; School of Life Sciences, Anhui Agricultural University, munity abundance [12]. Therefore, soil microorganisms, Hefei, 230036, Anhui, China including phosphate solubilizing bacteria (PSB), can be Pengcheng Wang: Institute of Horticulture, Anhui Academy of Agricultural Sciences, Nongke South Road 40#, Hefei, 230031, used as a biological indicator for salinized soil quality Anhui, China; Key Laboratory of Genetic Improvement and restoration and evaluation. Salt and nutrient accu- Ecophysiology of Horticultural Crops, Hefei, 230031, Anhui, China mulation and secondary salinization are common in Open Access. © 2021 Jian Zhang et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 10 Jian Zhang et al. greenhouse vegetable soil [13]. Salt ion composition plate with yeast extract, 5.0 g L−1; peptone, 10.0 g L−1,agar, related to secondary salinization soil in vegetable soil 15.0 g L−1 for solid, and 10% NaCl at pH 6.9–7.1. The sur- 3− − 2− 3− 2+ includes eight major ions (HCO ,Cl,SO4 ,NO ,Ca , vival of Bacillus sp. “SX4” at various salt concentrations (up K+,Mg2+,andNa+)[10]. The high content of soluble salts to 20%) was examined using a subculture on a nutrient in greenhouse soil not only has become the main limiting liquid medium at 25 ± 2°C for 3 days. Cells in a nutrient factor for vegetable production [10] but also affects the solid agar plate were counted. The individual morphology distribution of bacterial communities [14]. of bacteria was visualized with a light microscope. Fresh Dissolved organic matter (DOM) is an important com- cells were stored with 40% glycerol at −80°C for subse- ponent of soil organic matter [15,16]. More importantly, quent analyses. ions such as Na+,Ca2+,K+, and Mg2+, the important inor- ganic components of DOM [17], facilitate the accumula- tion of ions in soil. The effects of salt stress on plant or microbe growth are ion toxicity by sodium or chloride 2.2 Induction of salt tolerance and and imbalanced nutrition [18,19]. Increase in Na+ and collection of bacteria Mg2+ ions cause structural damage to plant or microbes’ cells [20]. Despite that the ability of plants to tolerate salt Two kinds of culture methods were used in activating stress can be regulated by multiple biochemical path- bacterial cultures in solid media. The media containing ways, a large amount of soil salt can cause toxic inter- salt (10%) and that without salt (SX4.CK) were used. mediate products and result in the weakening of crop or Initially, the concentrations of the bacteria were adjusted bacteria cell metabolism [21,22]. Both plants and micro- to 108 colony-forming units (CFU) mL−1, and the bacteria organisms have to adapt to salt stress and form their own were grown on an agar plate for 3 days at 25°C. Then, strain unique way to survive in saline soils. growth was observed. The growing bacteria were col- The expression of salt-tolerant genes in bacteria effec- lected and transferred to a 1.5 mL tube, frozen with liquid tively improves the chance of bacteria to survive. For nitrogen, and stored at −80°C until further analyses. example, an Azospirillum strain obtained from saline soil exhibits high salt tolerance [23].Therefore,anin-depth study of the regulation of salt on bacterial genes is useful in improving the application of beneficial bacteria because 2.3 RNA extraction salt-adapted strains may have higher potential to alleviate saline stress [23,24].Beneficial bacteria in the soil can Fresh “SX4” cells from two treatments were used in pre- survive under high salt conditions and potentially pro- paring RNA. Total RNA was extracted from the cells mote plant growth under salt stress. In our previous with TRIzol® Reagent (Invitrogen, Shanghai, China) study, a large number of salt-tolerant bacterial strains according to the manufacturer’s instructions. Genomic were isolated from salinized vegetable soils [25],and DNA was carefully removed using DNase I (TaKara, Dalian, some of them showed an ability to dissolve phosphorus China). RNA purity was determined using ND 2000 and fix nitrogen. The purpose of this study was to ana- (NanoDrop Technologies, Wilmington, USA). The obtained lyze the expression and regulation of the salt tolerance high-quality RNA samples were selected according to the gene of strain “SX4” by NaCl. Our findings will be con- following criteria: OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, ducive to the application of beneficial bacteria and soil RIN ≥ 6.5, and 28S:18S ≥ 1.0. Finally, the purified RNA was improvement. used in constructing a sequencing library. 2 Materials and methods 2.4 Library preparation and Illumina HiSeq sequencing 2.1 Bacterial strain and cultivation RNA-seq strand-specific libraries were prepared accord- Bacillus sp. strain “SX4” with accession number MF431750 ing to the procedures in TruSeq RNA sample preparation was previously isolated from salinized greenhouse soil [25]. kit (Illumina Inc., San Diego, USA). RNA (5 μg) was used. The salt tolerance of “SX4” was determined. A nutrient agar rRNA was removed using a RiboZero rRNA removal kit Genes in Bacillus sp. “SX4” respond to NaCl 11 (Illumina Inc., San Diego, USA) and fragmented using a was carried out using Fisher’s accurate test (P < 0.05). fragmentation buffer. cDNA synthesis, end repair, and False positive rate was reduced by using four multiple the A-base addition and ligation of Illumina-indexed test methods (Bonferroni, Holm, Sidak, and FDR) to adaptors were conducted according to Illumina’s proto- correct the P value. When the Bonferroni-corrected cols. Libraries were selected and constructed on the basis P value was less than 0.05, DEGs were considered sig- of the sizes of cDNA target fragments (200–300 bp) on 2% nificantly enriched in GO terms and metabolic pathways.
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