Cloning And Functional Analysis Of Rtlea Gene From Reaumuria Trigyna

Reaumuria trigyna is endemic to the Eastern Alxa-Western Ordos area in China. It is well adapted to salty deserted land and can complete life cycle in high salinity stress. LEA proteins (late embryogenesis abundant proteins) are highly aggregated in the seed late embryo stage and widely distributed in the biosphere. It can be induced by developmental stage、dehydration signal and abscisic acid(ABA). LEA protein family is a large family that is closely associated with resistance to abiotic stresses in organisms.In this study, a full-length cDNA of RtLEA was cloned from Reaumuria trigyna by rapid amplification of cDNA ends (RACE). The full-length cDNA of RtLEA contains a315bp open reading frame(ORP), encoding a polypeptide of104amino acids with a calculated molecular mass of11.282kDa and a theoretical PI of7.92. Bioinformatic analysis revealed that RtLEA contains a Pfam LEA-3domain, belonging to the Group5B of LEA family.Homologous alignment and phylogenetic analyze showed that RtLEA is most related to TaLEA which is isolated from Tamarix androssowii.Semi-quantitative PCR analysis shows that RtLEA gene can be induced by high salinity, PEG, low temperature and ABA. Promoter sequence analyze of RtLEA shows that it includes two endosperm expression required element(Skn-1motif) and cis elements response to salt PEG, low temperature and ABA.In order to explore the function of RtLEA,T-32a-RtLEA recombinant vector was constructed and transducted to Escherichia coli Rosetta. After induced by IPTG and analysised by SDS-PAGE, a fusion protein consistent with the expected size was obtained. RtLEA enhanced the tolerance of E.coli recombinant to high salinity, heat, freezing-thaw, PEG, ABA and sorbitol. The results indicated that RtLEA protein may play a protective role in E.coli under abiotic stress.A full length cDNA of sulfate transporter(RtST) was isolated. The2232bp sequence comprised a1962bp open reading frame, encoding a653amino acids protein with the molecular weight of71.18kDa Bioinformatics analyze reveals that RtST contains12membrane-spanning domain(MSD) and the STAS domain. The sulfate transporter protein domain is located in the middle region, the N-terminal and C-terminal are in the inner membrane of the sequence. Homologous alignment and phylogenetic analysis showed that RtST is related to Group3sulfate transporters in Arabidopsis thaliana.