Transcriptome analysis of differential genes associated with the ectoine synthesis pathway of Halomonas campaniensis under salt stress and expression verification by qRT-PCR

Abstract:

[Objective] The study explored the differentially expressed genes of Halomonas campaniensis under salt stress and identified the differential genes associated with anabolism of ectoine.[Methods] Three experimental groups were set up:no-salt group (NS, 0 mol/L NaCl), medium-salt group (MS, 1.5 mol/L NaCl), and high-salt group (HS, 2.5 mol/L NaCl), and then H. campaniensis XH26 was respectively cultured. Illumina HiSeq was employed for transcriptome analysis, and differential genes associated with ectione metabolism were screened and then verified by qRT-PCR.[Results] The accumulation of ectione in XH26 was closely related to salinity and the maximum (419.2 mg/L) was achieved in MS (1.5 mol/L NaCl). According to the transcriptome sequencing (n=3) result, total mapped reads accounted for 87.24%-95.87% of the clean reads. A total of 748 operons (2 182 related genes) and 941 transcription start/stop sites were annotated and 456 new transcripts were predicted in addition to 385 up-regulated genes and 326 down-regulated genes (involving 245 KEGG pathways). For the differential genes between NS and MS, synthetic ectABC and lysC were up-regulated to promote the ectoine production, and associated genes gltB, gltD, davT, hisD, alh-9, betA, acnB, pckA, and gadA were up-regulated to modulate the upstream of ectoine synthesis pathway. As for the differential genes between MS and HS, ectA, acnB, pckA, gadA, and gdhA were down-regulated, thus decreasing ectoine production. qRT-PCR result supported the transcriptome sequencing result.[Conclusion] The biosynthesis of ectoine was closely related to the aspartic acid (or aspartic acid hemiacetal), the upstream amino acid metabolism networks (e.g., asparagine, glutamate, glutamine, and histidine), and tricarboxylic acid cycle (succinic acid, fumaric acid, and oxaloacetic acid). These results can serve as a reference for the optimization or integration design of ectoine synthesis pathway in the future.