Identification And Functional Analysis Of Salt Stress-Related Genes,GarCIPK And SbHKT1in Plant

Plants are often faced with the damage from salt, drought, cold and other abiotic stresses during growth and development. Saline as the nature one of the major abiotic stresses is one of the most serious factors affecting crop yields. Saline land area throughout the world is about1billion hectares, accounting for10%of the total cultivated area in the world, and getting bigger. Cotton as a most important fiber crop and oil crop, is the pioneer crop in saline land area. However, when salt concentration exceeds0.3%in soil, growth and development of cotton will be severely affected. The overall salt tolerance of upland cotton is not high. Modern upland cotton has narrow genetic basis on salt resistance, which makes it difficult in improvement of salt tolerance in upland cotton. Therefore, the mining of some stress resistance genes will provide potential candidate genes for transgenic resistance breeding in cotton.Some salt stress-related genes such as protein kinase and high-affinity potassium transporter play an important role in plant salt resistance pathway. In this study, we cloned a protein kinase gene from a Gossypium wild species, G. aridum, and transfer the gene into tobacco to carry out an initial functional analysis. Meanwhile, a putative high-affinity potassium transporter gene SbHKTl was isolated from halophyte Salicornia bigelovii by RACE cloning in an earlier research. In this study, we conducted a preliminary functional analysis by transferring the gene into tobacco and cotton with agrobacterium-mediated transgenic technology. The main results were as follows:1. Cloning and sequence analysis of GarCIPK geneA protein kinase-like sequence was obtained from a salt induced transcriptome of Gossypium aridum. In silico cloning technology was used to extend the sequence and obtained a full-length cDNA gene (GarCIPK). The open reading Frame (ORF) of GarCIPK is1350bp, encoding a51.12kD protein with449amino acids and theoretical pI of8.13. Alignment analysis showed GarCIPK protein hada25.11%～42.09%identities to Arabidop-sis CIPK protein and31.66%～73.95%identity to rice CIPK protein. Real-time fluorescence quantitative PCR analysis showed the relative expression level of GarCIPK gene in leaves reached the peak after48h salt treatment, while it reached the peak after24h salt treatment in the root. Its promoter region contains stress response elements (ARE and LTR), development of relevant (CAT-box), hormones (GARE-motif), gibberellin response element (GARE-motif) regulatory domain. It was speculated GarCIPK gene may be involved in response to salt stress during cotton seedlings2. Function analysis of GarCIPK geneThe GarCIPK gene which was driven by CaMV35S promoter and stress-specific promoter RD29A were transferred into tobacco separately by agrobacterium-mediated transgenic technology. In MS medium without NaCl, the root length of all transgenic lines didn’t show any significant difference with that of the wild-type. However, in the medium with150mM or200mM NaCl, The root length of transgenic lines was significantly longer than that of wild-type. In the PEG stress experiment, when nutrient solution contained0%PEG or10%PEG, the growth of transgenic plants showed no obvious difference with that of wild-type tobacco. However, when nutrient solution containe20%PEG, the growth of transgenic plants were greatly restrained.The results showed that transgenic plants were more tolerant to salt and drought stress than wild-type plants.3. SbHKT1transformation in tobacco and cottonBy the use of Agrobacterium tumefaciens high-affinity K+SbHKTl gene was transferred into tobacco, and resistance plants were screened. By PCR detection of To generation regenerated plants,20transgenic tobacco seedlings were obtained. Positive plants by RT-PCR, four transgenic tobacco plants were successfully expressed at the mRNA level. Identification salt tolerance of transgenic tobacco indicated the transgenic plants may be more resistant to the salt tolerant.Then, SbHKT1gene was transportered into cotton and29transgenic tobacco seedlings were obtained. Southern blot analysis of positive plants shows the gene SbHKTl has been integrated into the cotton genome.