Cloning Of Three Resistance-relevant Genes From Ammopiptanthus Nanus

Ammopiptanthus nanus, as well as Ammopiptanthus mongollcus, belongs to Leguminosae, Ammopiptanthus. It is one of the super drought born and evergreen broadleaved shrubs, which is a relict species at the edge of the Tarim Basin desert after the disappearance of Tethys Ocean at the Tertiary period. It distributes at the combination area between Western Tianshan and Kunlun Mountain discontinuously and takes Wuqia county Kizilsu Kirgiz autonomous prefecture Sinkiang as the center, a few of which extends to the border of Kyrgyzstan. The population of Ammopiptanthus nanus has declined seriously as the consequence of natural reasons and the influence of human economic activities, especially of overgrazing and overcut. Therefore, it has been classified as national secondary endangered plants.Ammopiptanthus tolerates extremely to abiotic stresses, such as drought、cold、 barren soil、saline-alkali soil and high temperature. At the aspect of morphological structure, it has pilose branches and leaves, thick cuticles, advanced palisade tissues, fewer and sinked stomas, big vacuoles in epidermic cells of leaves and large amount of storage of water. It can take up the water at deep layer of soil because of its flourishing taproots, dense lateral roots and well-developed conducting tissues. As the result of a great deal of antifreeze proteins of high activity as well as increasing contents of osmoregulation substance, like proline and soluble saccharides, against adversity stress, it has viscous protoplasm, high content bound water, low water potential, weak transpiration and strong ability of water absorption and retaining. So, the molecular analysis of its stress resistance and cloning of relative genes can provide reference and exogenous genes for resistance research and transgenic use.There are a number of genes related to stress tolerance have been cloned and sequenced from Ammopiptanthus mongollcus, such as the late embryos enrichment protein, the binding factor of elements responsive to ethylene, dehydrin, cell cycle related proteins, RNA-binding protein, inositol lactose chitin synthase, spermidine synthase, receptor-like kinase linked to cell wall, Na+/H+antiporter, calcineurin B-like protein1and antifreeze protein, some of which have been verificated for their ability of stress resistance by transgenosis. But, there are comparatively larger hereditary difference and lower homology of gene sequence between Ammopiptanthus nanus and Ammopiptanthus mongollcus, which leads to the difficult of homology cloning.So, this experiment used homology cloning combining with RACE to get the complete cDNA sequences of three genes from the fresh leaves of Ammopiptanthus nanus treated by500mmol/L NaCl at4℃for8hours. The three genes are molybdenum cofactor sulfurase(AnMCSU)、phospholipase Dal(AnPLDα1) and H+-PPase(AnVP1) respectively. Then, I have cloned the open reading frames (ORF) by analysis of bioinformatics software, constructed a cladogram according to homologous alignment between the amino acid sequence derived by the ORF and the homologous amino acid sequence of other specises and analysed the conserved domains in the derived amino acid sequence. Main results as follows:1. The complete cDNA sequence of AnMCSU (Ammopiptanthus nanus molybdenum cofactor sulfurase) is2544bp long, which contains a ORF of2289bp. This ORF encodes762amino acids. Homology analysis of amino acid sequence indicates that AnMCSU is most homologous with ABA3/LOS5(Arabidopsis Thaliana), that is58.01%.2. The complete cDNA sequence of AnPLDα1(Ammopiptanthus nanus phospholipase Dal) is2832bp long, which contains a ORF of2427bp. This ORF encodes808amino acids. Homology analysis of amino acid sequence indicates that AnPLDal is most homologous with MtPLDa (Medicago truncatula)、RcPLDα1(Ricinus communis) and NtPLDα1(Nicotiana tabacum), they are85.64%、84.41%and73.03%respectively.3. The complete cDNA sequence of AnVP1(Ammopiptanthus nanus H+-PPase)is2684bp long, which contains a ORF of2298bp. This ORF encodes765amino acids. Homology analysis of amino acid sequence indicates that AnVP1is most homologous with MtVP1(Medicago truncatula), that is94.51%.The sequences of three new cloned genes above have been applied for Chinese Patent, the patent application NO. are201210148464.8,201210148625.3and201210148680.2respectively. For the next step, the expression vectors will be transferred into the mutants of Arabidopsis thaliana to verificate the biotic functions, which is the basis for the transgenetic research of resistance to abiotic stress.