Studies On Molecular Cloning Of Functional Genes From Special Organisms In Xinjiang And Their Application

Studies on Molecular Cloning of Functional Genes from Special Organisms in Xinjing and Their ApplicationWith the accomplishment of the human genome project and others mode organisms such as Arabidopsis thaliana and rice, it has become a shortcut way to clone new genes in a gene family via homology sequences. Salt stress is the primary effect that limits and decreases the output of crops in many parts of the world, particularly irrigated land. Overly high soil salinity can cause salina and limit the utility of soil. Today, ~20% of the world's cultivated land and nearly half of all irrigated land are affected by salinity. Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the salt tolerance of crops, keeping steady-state growth and increasing the output in the saline environment. NHX1 , a tonoplast Na+/H+ 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. This research contains: cloning of NHX1 gene from Atriplex dimorphostegia in Xinjiang by RT-PCR and RACE; cloning of orf25 gene, nhaA gene, nhaB gene and AtNHX2 promoter; expression analysis and molecular testing.At first, the conservative region of the variable NHX1 gene sequences published in Genbank were analyzed, and seven primers (4 upstream, 3 downstream) were designed for amplifying core fragment of NHX1 according to these conservative region. Total RNA was isolated from the plant tissue and cDNAs were synthesized by reverse transcription. The different combinations of these primers were used to amplify the core fragment of NHX1. The core fragments were cloned into pMD18-T cloning vector, positive clones were checked with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the core fragments from Atriplex dimorphostegia were similar with the AtNHX1 (short for NHX1 from Arabidopsis thaliana) sequence (GI: 30690553). With a set of primers designed according to the sequence of the cloned core fragments, the 3' and 5' ends of the cDNAs were obtained by 5' and 3' rapid amplification of cDNA ends (RACE), respectively. Combining the sequences of the 3' ends, 5' ends and cloned core cDNA, the full-length sequence of the NHX1 cDNA was assembled. To obtain the open reading frame (ORF) of the NHX1, a pair of primers was designed according to the assembled cDNA sequence. PCR product was cloned into pMD18-T cloning vector and sequenced. The result shows that ORF of the AdNHX1 was obtained. And then it was registered in Genbank (GI: AY211397). The deductive amino acid sequence of AdNHX1 (short for NHX1 from Atriplex dimorphostegia) is significantly different from AtNHX1. The result 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.Specific primers for orf25 gene, nhaA gene and nhaB gene were designed according to published sequences and RT-PCR or PCR were conducted to amplify orf25 gene, nhaA gene and nhaB gene. Sequence analyses suggest that cloned genes contain entire coding frame of orf25 gene, nhaA gene and nhaB gene, respectively. In order to detect whether AtNHX2 promoter can be induced by salt stress, a pair of primers were designed to amplify a 2.8kb DNA fragment upstream of ATG start codon of AtNHX2 gene according to the sequence of Arabidopsis thaliana genomic DNA. Sequence analysis shows that the amplified fragment conform to the published sequence. AtNHX2 promoter was cloned into the plant expression vector pCAMBIA1301-1. The promoter activity was detected by transient e...