RNA-Seq Analysis Of Industrial Hemp Under Salt Stress

Soil salinization has become the major problem in agricultural development of China. It is emergent to develop saline land for crop cultivation. The studies of mechanisms in salt resistance of crop are necessary. Hemp as one of the important crops have cultivated in China for thouands of years. It is mainly used for medicine, fiber and seed oil. The two industrial hemp cultivars ’Yunma NO.5’and’Bamahuoma’ which demonstrate growth vigor and environmental adaptability, have been primarily cultivated in Yunnan and Guangxi, China, respectively, for fiber and seeds. RNA-Seq analysis were first performed on the leaves of both cultivars sampled at four time intervals after 500mM NaCl treatment. The results are as follows:(1) Validation of qRT-PCR qRT-PCR validation of selected 18 DEGs was used to evaluate the results of RNA-Seq. Although there were some differences in the amplitude of expression of the 18 DEGs, the expression trend of qRT-PCR and RNA-Seq were consistent to each other indicated the reliability of the results of RNA-Seq.(2) Effects of salt stress on physiological parametersThe results of four physiological parameters measurement (SOD activity, soluble protein content, proline content and relative conductivity) indicated that two cultivars had differentially responsed to salt stress. The two cultivars had strongest physiological response at 2d under stress.(3) Screening and analysis of differentially expressed genes (DEGs)The results of screening of DEGs obtained from RNA-Seq showed that the number of DEGs under salt stress peaked at 2d which was consist to the results of physiological parameters measurement.220 co-regulated DEGs at 2d had been identified. The 220 DEGs were significantly enriched in p phenylalanine metabolism pathway and plant hormone signal transduction pathway, such as ethylene signal transduction pathway and abscisic acid (ABA) signal transduction pathway.22 transcription factors were identified including MYB, NAC, GATA, HSF.(4) Gene expression pattern anaylsis (clustered trend set)The expression patterns of all the differential expression genes obtained were discovered using STEM softwear. The significant profile with first increased and then decreased trends were annotated by terms of the Gene Ontology (GO) and KEGG pathway database. The results showed that two cultivars had species-specific pathway. The DEGs of’Yunma NO.5’was significantly enriched in spliceosome pathway and amino acid metabolic pathways. Thw DEGs of ’Bamahuoma’ were enriched in fatty acid metabolic pathways and amino acid metabolic pathway. Altough both the two cultivars had shared DEGs, their salt tolerance mechanisms was regulated by different metabolic pathways.The DEGs that were co-expressed in both varieties under stress may provide useful insights into the tolerance of industrial hemp and other bast fiber crops to salty stressl. These transcriptomes also represent reference sequences for industrial hemp.