Cloning, Characterization And Expression Analysis Of STT3A And STT3B Of Spartina Alterniflora

Background:Salinity is a major abiotic factor in inhibiting plant growth and crop yields and the fundamental way to solve the problem is to breed salt-tolerant cultivars. Since salt tolerance is a quantitative trait which is controlled by multiple genes and metabolic pathways, improvement of plant salt tolerance through operation on a specific gene is often far from expectation. Asparagine(N)-linked glycosylation of proteins is one of the most crucial post-translational modification in cells. In Arabidopsis thaliana, loss of function of STT3a, catalytic subunit of oligosaccharyltransferase, leads to the salt-hypersensitive phenotype of plants. On the other hand, Spartina alterniflora hasbeen paid more attention because of its superior salt tolerance and genetically close to important economic crops, though little is known about its exact molecular mechanism against salinity stress.ln this thesis, STT3A and STT3B gene were cloned from S.alterniflora and their functions were verified by genetic complementation. These work supply the essential basisfor elucidating the potential role of N-glycosylation involved in plant salt tolerance.Methods and Results: Firstly, SaSTT3A and SaSTT3B were cloned from S.alterniflora using regular PCR, rapid amplification of cDNA ends (RACE), and high-efficiency thermal asymmetric interlaced PCR for amplification (hi-TAIL PCR). The full length of the CDS sequences of SaSTT3A and SaSTT3B are 2361bp and 2163bp, corresponding to 786 and 720 amino acids, respectively. Genomic SaSTT3A spanned over 5829bp in length, containing 23 exons and 22 introns. However, SaSTT3B spanned over 4230bp in length, containing 6 exons and 5 introns. The evolutionary and phylogenetic relationship showed the gene structure of SaSTT3A and SaSTT3B are conserved among analyzed plant species. SaSTT3A and SaSTT3B belong to the same group with their each counterpart in the phylogenetic tree. Subcellular localization and transmembrane structure analysis revealed that SaSTT3A and SaSTT3B should locate on the ER membrane and the possible transmembrane helices of them are 13 and 11, respectively. Second, SaSTT3A and SaSTT3B cDNA sequences were cloned into the binary vector to rescue Arabidopsis stt3a-2 mutant. The result showed 35S-SaSTT3A transgenic plants could complement stt3a-2 to wild-type and relieve UPR response as well.Conclusions: There exists a conserved catalytic subunit of oligosaccharyltransferase in S.alterniflora and its ability to rescue A.thaliana salt-sensitive stt3a-2 phenotype suggests N-glycosylation might be involved in salt tolerance ofS.alterniflora.