Cloning,Sequence Analysis And Preliminary Function Assay Of The Salt-tolerant Genes From Species Of Xinjiang Halophytes

Salt stress is the major factor that limits and decreases the output of crops in many parts of the world, particularly for the irrigated land. Overly high soil salinity can cause salina and limit the utilization of soil. Advances in molecular genetics and plant transformation in recent years have made it possible to employ biotechnological strategies in crops of salt-tolerant characters, keeping normal growth and without loss of production in the saline environment. Crops are able to tolerate salt stress by many ways, such as regulation of the pathways of biological synthesis, expression salt-related genes, altering genes'reaction characters regarding to the stress, etc.. So far, it has been proved that the SOS(Salt Overly Sensitive) pathway plays key role in plant salt tolerance and the ionic homeostasis. In this process, the Na+/H+ antiporter gene (NHX1) has been noticed widely, this antiporter energized by the ?pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na+ into the vacuole, which results in avoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis. Based on previous research, we employed local halophytes in Xinjiang as materials, performed the following research work: cloned NHX1 gene from Salicornia europaea,Kalidium foliadum and Halostachys caspica by RT-PCR and RACE; cloned calidium genes from Karelinia caspica, Kalidium foliadum and Betula halophila, carried out sequence analysis and preliminary function assay. At first, the conservative regions of various NHX1 gene sequences published in Genbank were analyzed, and seven primers (4 upstream, 3 downstream) were designed for amplification of core fragment of NHX1. Total RNA was isolated from plant tissue and cDNAs were synthesized by reverse transcription. Different combinations of these primers were used to amplify the core fragment of NHX1. The core fragments were cloned into pMD18-T cloning vector, then sequence analysis was conducted with positive clones. Sequencing results showed that the core fragments from Salicornia europaea,Kalidium foliadum and Halostachys caspica were similar to the AtNHX1 (abbreviation of NHX1 for Arabidopsis thaliana) sequence (GI: 30690553). With a series of primers designed according to the sequences of the cloned core fragments, the 3'ends of the cDNAs were obtained by 3'rapid amplification of cDNA ends (RACE), respectively. For the 5'unknown regions of these genes, the up-stream primers at the start-codon region was designed, the down-stream primers are the primers used to cloning the core cDNA. Combining sequences of the 3'ends, 5'ends and core cDNA, the full-length sequences of the NHX1 cDNA were assembled. To obtain open reading frame (ORF) sequence of NHX1 gene, three pairs of primers were designed according to the assembled cDNA sequence. PCR products were cloned into pMD18-T cloning vector and sequenced. Results confirmed that ORF of the NHX1 gene were completely involved in the full-length sequence. Three genes shared high homology. Comparing with the other plants, especially with the plants in Chenopodiaceae, NHX1 gene were highly conservative. The results may have important implications for the elucidation of the different abilities of halophytes and glytophytes for salt tolerance, and may present a feasible method to utilize much more salina area in Xinjiang. In addition, plant expression vector harboring with NHX1 gene of sarlicornia was constructed, and transformation of explants of brassica was carried out to investigate the function of this gene further more. Specific primers for calidium (CaM) gene were designed according to published sequences. RT-PCR were conducted to amplify calidium genes from Karelinia caspica,Kalidium foliadum and Betula halophila. Sequence analysis revealed that the cloned fragments contained entire calmodulin ORF. Sequence similarity was performed by BLAST network service. The results showed that high similarity (about 85%) among three calmodulin genes in terms of evolution status was observed. The homology of calmodulin genes between these three plants and the other plants was up to 80%. It indicated that calmodulin gene was relatively stable in plant evolution. Our research work no doubts has promoted the utilization of salt-resistant genes isolated from local halophytes in improving the ability of plant in salt tolerance and in the near future developing salt-resistant crops, and we believe , by genetic engineering, more and more salt-resistant plants will be developed and in application.