Identification Of Novel Highly Tolerant NaHCO₃ Microalgae And The Role For Acyl-coA-binding Protein （ACBP） Under Stress Tolerance

In northeast China, large-scale tree clear-cuts and subsequent intensive grazing have induced soil erosion and increased evapotranspiration, resulting in salt accumulation from groundwater on the soil surface. However, the soil in the Songnen Plain contains mainly salts like sodium bicarbonate and sodium carbonate, also known as "Soda-Saline-Alkali Soils". Plants are "carbonate stressed" by NaHCO3 and Na2CO3. The extreme saline-alkali region has many "barren" lands with a pH of about 10.5, and only few plant species are sporadically distributed. Through identifying and characterizing microalgae from extreme saline-alkali soils, which are rich in carbonate (NaHCO3 and Na2CO3), we aimed to explore the microalgae carbonate stress mechanism and acquire possible extreme saline-alkali microalgae as genetic resources for crop improvement and cultivation.1. Isolation and identification of microalgae from extreme saline-alkali soil environment(1) 20 microalgae were isolated from extreme saline-alkali soil environment. We identified the microalgae based on optical microscopy (OM) and scanning electron microscopy (SEM). The results showed the twenty isolated microalgae had enormous morphological diversity, including unicellular, coenobic, and filamentous forms.(2) In order to test and supply the morphological classification, we identified 20 novel strains of microalgae as six groups by class level based on 18S rRNA sequence analysis, classified them into 13 genera.(3) In order to obtain the extreme tolerant microalgae resource, we determined strains’ salt-tolerance abilities with a range of NaCl and NaHCO3 concentrations. The intracellular ultrastructure was observed with transmission electron microscopy (TEM) at NaCl and NaHCO3. We observed strain growth response by detecting the growth, dry weight, and chlorophyll-a (chl-a) content at different NaHCO3 concentrations. Most isolates possessed saline tolerance, especially two stains in the class Trebouxiophyceae, which could grow on solid Bold’s basal medium (BBM) with high NaHCO3 concentrations. The TEM results showed cell walls of the two Trebouxiphyte strains (JB6 and 17) were more stability than reference Chlorella. Further analysis showed the growth rate, dry weight, and chl-a of two Trebouxiphyte strains were much better than the reference Chlorella in liquid BBM with different NaHCO3. The two Trebouxiphyte strains both survived in 1,000 mM NaHCO3. The results revealed that the two Trebouxiphyte strains showed extreme tolerance to high concentrations of NaHCO3.2. The cDNA library construction and the cloning of tolerant NaHCO3 gene(1) In order to construct tolerant NaHC03 Chlorella cDNA library, the cDNA Library Construction Kit was used to synthesize full-length double stranded cDNA. Specific primers were designed to obtain the pYES2 linear vector by means of PCR. The cDNA double chains were connected with the linear vector by using the In-Fusion technology, and transformed into the yeast strain to obtain full-length cDNA yeast library.(2) In order to obtain tolerant saline-alkali Chlorella genes, the transgenic yeaast were screened from NaHCO3 treatment. The tolerant NaHC03 related genes were isolated. The results showed isolated 20 gene were distributed between 500 to 2000 bp. Gene Ontology (GO) annotation results suggested that these genes were mostly relevant with single organism process (2), response to stimulus (7), metabolic process (2), biological regulation (2), cellular processes (7) and the location (2). Seven genes may be the stress (such as high salt, oxidation, heavy metals and toxic) response related genes.3. The role for acyl-coA-binding protein (ACBP) under stress tolerance(1) ACBP gene was isolated from tolerant saline-alkali Chlorella full-length cDNA library. In order to understand the gene characteristics, the bioinformation method was used to analyze the ACBP gene. The full-cDNA was 525 bp in length with an open reading frame of 264 nucleotides, encoding a deduced protein of 87 amino acids. The ACBP sequence of Chlorella (ChACBP) shared 50-82% identities with the previous published ACBP sequences of other species.(2) In order to analyze the gene expression under abiotic stesses, northern blot analysis showed that the expression of ChACBP gene in Chlorella was strongly induced to increase by multiple abiotic stresses from high salinity, oxidant, heavy metals and low temperature.(3) In order to analyze the gene stress tolerance, over-expression of ChACBP in yeast and Arabidopsis plants increased the resistance to high salt, oxidative, heavy metals and low temperature stresses. Taken together, these data raised the possibility of using ChACBP to genetically improve environmental stresses tolerance in plants. In vitro filter-binding assays indicating that histidine-tagged ChACBP bound phosphatidylcholine, implied that ChACBP may participate in phospholipid metabolism in Chlorella, including the possibility of ChACBP in the cytosolic transportation of phosphatidylcholine, and perhaps played an important in protection and stability of cell membrane.These studies provide the theoretical basis for screening special characteristics of algae germplasm resources, to explore the microalgae carbonate stress mechanism and acquire possible extreme saline-alkali microalgae as genetic resources for crop improvement and cultivation.