Cloning And Functional Analysis Of MaTET Gene Of Banana

Tetraspanins (TETs) are transmembrane proteins characterized with four transmembrane structures and present in all multicellular organisms. In animals, TETs play an important role in diverse biological processes and get a comprehensive research. Despite their established importance in diverse biological processes and organisms, their function in plants is still poorly understood. In this study a TET from banana was cloned and its function was analysed which will attribute to the comprehensive acquaintance for plant TETs. The content of this study is abstracted below.1. A full length cDNA was isolated based on a EST sequence obtained from the suppression subtractive hybridization (SSH) library in the early stage of the banana’s ripening by the approach of RACE. Blast aligment shows this gene belongs to TET family and designated as MaTET which was1234bp in length with an open reading frame of852bp encoding283amino acids.2. Bioinformatics analysis showed that MaTET is unstable protein containing four transmembrane regions and two extracytoplasmic regions, a small extracellular loop (SEL) and a large extracellular loop (LEC). Two protein kinase C phosphorylation site, four casein kinase Ⅱ phosphorylation site four N-myristoylation site, one prokaryotic membrane lipoprotein lipid attachment site and a prenyl group binding site exist in MaTET demonstrate that MaTET is a membrane protein and associated with signal transduction and transport. Amino acid sequence alignment of MaTET showed high identity with Oryza sativa, Setaria italica. Zea mays, Fragaria vesca, Cicer arietinuml which belong to monocotyledons or herbaceous plant.3. A IFA method was conducted to elucidate the subcellular localizition of MaTET in banana, and the result demonstrated that MaTET was located on plasma embrane of leaf epidermic cells, which is consistent with the bioinformatic analysis.4. The expression characteristic of MaTET in different organs was analyzed by RT-PCR approach. The results showed that MaTET possessed a constitutive expression character with higher level in leaf than in other organs, such as root, flower, fruit and corm.5. MaTET expression level did not increased after wounding, invasion of FOC4-GFPand in leaves infected by black sigatoka, shows that MaTET is not involved in the molecular response to wounding and pathogens.6. MaTET expression level get enhanced in ethylene treated fruits as well in ABA treated fruits, but was inhibited in1-MCP treated fruit until the later ripening stage, which means that MaTET is induced by ethylene and associate with the postharvest ripening process.7. The expression level of MaTET increased in senescened leaves treated by thephon, induced by darkness and irradiated by UV-B, illustrate that MaTET is a senescence associated gene. MaTET is induced at the late stage treated by UV-B and ethephon indicate that MaTET is involved neither the senescence directly nor the damage of plasma membrane8.The expression level of MaTET increased in root responsed to stresses of salt, salt and drought, indicate that MaTET is induced by abiotic stress but not biotic stress illustrate that MaTET involved in the resiatant molecular response to abiotic stress.9. H2O2and paraquat treatment enhanced the expression level of MaTET when plasmam membrane was damaged elucidate MaTET’s function is associated with the membrane damage’s maintaining and renovation.10. To understand MaTET’s function, we inserted the coding region of MaTET to the pCAMBIA1304, a plant expression vector, and constructed the plant expression vector of pCAMBIA1304-MaTET driven by35S for heterologous expression analysis in Arabidopsis thanian. The expression vector was transformed into Arabidopsis thanian by the method of agrobacterium tumefaciens mediated dipping. The IFA analysis showed that MaTET localized on plasma embrane of leaf epidermic cells and root tip.11.The transgenic Arabidopsis thanian plants displayed a delaying senescence phenotype and a higher salt tolerance than wild type with a enhanced antioxidant system, in which the activity of SOD、POD、CAT was enhanced but REC was lower in transgenic plant than in wild type. Nevertheless, transgenic plants did not displayed remarkable drought resistance than the wild type but exhibited a higher seed germination rate.12. In conclusion, MaTET is involved in the physiological process associated with senescence of leaves, salt and cold resistance and seed storability by participating in signal transduction of SOS type which needs further analysis to clarify.