Isolation And Identification Of A Salt-stress Responsive Gene FBPA In The Root Of Wheat

Salinity is one of the major environmental factors limiting plant growth and productivity. The expression of some genes will be changed by salt stress in the molecular level, which will promote or repress the synthesis of some relevant proteins to improve the ability to salt tolerance of the plant. It opens vast vistas for the plant slat-tolerance breeding research to isolate some relevant salt-tolerance genes from the plants and to develop slat-tolerance plant breeding by utilizing the technology of gene manipulation. However, the salt stress tolerance is controlled by quantitative trait loci (QTLs), and the mechanism of salt stress tolerance of plant is very sophisticated. Therefore, it is necessary to isolate and make an intensive study of some salt stress responsive genes, which could be transformed into plants for further studying. Finally, it is hoped that the salt-tolerant transgenic plants could be cultivated and be widely used for agricultural operation.Common wheat (Triticum asetivum L.) is one of the most important and widely planted crops in the world. A new somatic hybrid introgression line Shanrong No.3 (hereafter SR3) has been generated in our lab from hybridization of common wheat cv. Jinan 177 with wheatgrass (Thinopyrum ponticum), a salt and drought tolerant grass relative to wheat. Cytological and molecular analysis showed that some nuclear and non-nuclear DNAs and even functional genes of donor Th. ponticum were introgressed into this line. SR3 had a significantly higher yield than its parent JN177. The salt-tolerance of SR3 is controlled by the major and minor genes together. As one of the materials for salt-stress tolerance research, SR3 is different from the others with single genetic background. In our lab, the suppressive subtractive hybridization (SSH) technology was used to construct a salt-stress responsive SSH cDNA library of SR3 and JN177 wheat. According to the relevant information of the SSH cDNA library, a salt-stress responsive gene, fructose 1, 6-bisphosphate aldolase gene in wheat root (TaFBPA), was isolated and identified in this study.The fructose 1, 6-bisphosphate aldolase (FBPA) is one of the important enzymes in the carbon metabolism and sugar metabolism pathway of the organisms, which catalyses an aldol cleavage of fructose 1, 6-bisphophate to dihydroxyacetone-phosphate and glyceroldehyde 3-phosphate and a reversible aldol condensation. The full-length cDNA sequence of fructose 1, 6-bisphosphate aldolase gene (TaFBPA) was acquired in this study by Nested-PCR technology. According to the bio-informatic sequence analysis, TaFBPA belongs to the family FBPA. TaFBPA encodes a protein of 358 amino acids that shares high identity with the orthologs from Zea mays (88.55%), Arabidopsis thaliana (82.12%), Oryza sativa (77.99%), respectively. The obviously elevated levels of TaFBPA expression have been detected by RT-PCR and Real-Time PCR analysis when the wheat seedling roots exposed to high salinity stress. Arabidopsis transformation of construct expressing the TaFBPA fused with green fluorescent protein (GFP) reveals that the fusion protein is targeted to cytoplasm. The recombined plasmid with insertion of TaFBPA to vector pET32a was transformed into E.coli DE3 to analyze its expression. A large amount of fusion protein was expressed by the induction of IPTG and detected by SDS-PAGE, which makes a foundation for the further protein purification and aldolase assay. The plant over-expression vector of TaFBPA was constructed and then transformed Columbia (Co10) Arabidopsis lines with Agrobacterium-mediated floral-dip method. The experiment of seed germination shows that the seeds of transgenic lines have a lower germination rate and a delayed germination time comparing with the seeds of control lines, which demonstrates that the seeds of transgenic lines with the over-expression of TaFBPA are more sensitive to the salt stress condition. Further studies are needed to deeply illustrate the mechanism of the TaFBPA gene involving in the salt- stress responses in the plant.