RESUMO
Salicornia brachiata is one of the extreme salt tolerant plants and grows luxuriantly in coastal areas. Previously we have reported isolation and characterization of ESTs from S. brachiata with large number of unknown gene sequences. Reverse Northern analysis showed upregulation and downregulation of few unknown genes in response to salinity. Some of these unknown genes were made full length and their functional analysis is being tested. In this study, we have selected a novel unknown salt inducible gene SbSI-1 (Salicornia brachiata salt inducible-1) for the functional validation. The SbSI-1 (Gen-Bank accession number JF 965339) was made full length and characterized in detail for its functional validation under desiccation and salinity. The SbSI-1 gene is 917 bp long, and contained 437 bp 3' UTR, and 480 bp ORF region encoding 159 amino acids protein with estimated molecular mass of 18.39 kDa and pI 8.58. The real time PCR analysis revealed high transcript expression in salt, desiccation, cold and heat stresses. However, the maximum expression was obtained by desiccation. The ORF region of SbSI-1 was cloned in pET28a vector and transformed in BL21 (DE3) E. coli cells. The SbSI-1 recombinant E. coli cells showed tolerance to desiccation and salinity stress compared to only vector in the presence of stress.
Assuntos
Adaptação Fisiológica/genética , Chenopodiaceae/genética , Desidratação , Genes de Plantas/genética , Salinidade , Estresse Fisiológico/genética , Northern Blotting , Clonagem Molecular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Vetores Genéticos , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase em Tempo Real , Transformação BacterianaRESUMO
Pandanus odorifer (Forssk) Kuntze grows naturally along the coastal regions and withstands salt-sprays as well as strong winds. A combination of omics approaches and enzyme activity studies was employed to comprehend the mechanistic basis of high salinity tolerance in P. odorifer. The young seedlings of P. odorifer were exposed to 1 M salt stress for up to three weeks and analyzed using RNAsequencing (RNAseq) and LC-MS. Integrative omics analysis revealed high expression of the Asparagine synthetase (AS) (EC 6.3.5.4) (8.95 fold) and remarkable levels of Asparagine (Asn) (28.5 fold). This indicated that salt stress promoted Asn accumulation in P. odorifer. To understand this further, the Asn biosynthesis pathway was traced out in P. odorifer. It was noticed that seven genes involved in Asn bisynthetic pathway namely glutamine synthetase (GS) (EC 6.3.1.2) glutamate synthase (GOGAT) (EC 1.4.1.14), aspartate kinase (EC 2.7.2.4), pyruvate kinase (EC 2.7.1.40), aspartate aminotransferase (AspAT) (EC 2.6.1.1), phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) and AS were up-regulated under salt stress. AS transcripts were most abundant thereby showed its highest activity and thus were generating maximal Asn under salt stress. Also, an up-regulated Na+/H+ antiporter (NHX1) facilitated compartmentalization of Na+ into vacuoles, suggesting P. odorifer as salt accumulator species.