| There is a large scale of arid region in China, which is limited in water resource. As two major environmental hazards which play a restrictive role in crop production and species distrbition, desertification and secondary salinization are rapidly increasing due to population growth, economic development, forest destruction and soil erosion etc in China. It is an effective way to improve the environment that selecting plant species with strong resistance to stress from nature and use them in vegetation restoration. Sophora davidii (Franch.) Skeels, a perennial deciduous shrub, is distributed widely in most of the semi-arid and arid regions of central and western China. This species is proved to be ideal material in project of soil and water conservation because of its great resistance to drought and its deep roots. However, little studies have been conducted on resistant mechanism of S. davidii. In the present study, the physiological and morphological mechanism of seed germination and seedling growth under drought and salt stress were researched. We also cloned three genes related with drought resistance from S. davidii. All of the achievement will lighten the work of understanding comprehensively on physiological and molecular mechanism in S. davidii to stress. The main contents and results are as follows:1. Two seed lots of S. davidii were used as materials:seeds stored in room temperature and4℃respectively for two years. The responses of seed germination to different concentrations of PEG and NaCl solution were evaluated. The results indicated that seed germination of S. davidii has strong reisitance to drought and salt stress. Opposite to the inhibition of20%PEG on seed germination, lower concentration of PEG solution improved germination percentage, germination rate and seedling fresh weight of S. davidii. The NaCl treatments inhibited seed germination slightly except the200mM NaCl. A higher ability to germination under drought ans salt stresses was found in seeds stored in4℃for two years. The result suggested that the improved accumulation of proline and increased activities of antioxidant enzymes were the important reasons for seed germination of S. davidii under drought and salt stresses.2. Seeds stored for one month and two years in room temperature were used as materials. Effect of pre-sowing treatment of PEG on seedling emergence and growth was studied. The results showed two years storage decreased significantly the seed emergence percentage, seedling survival rate and seedling growth. The ability of seeds to emerge quickly and to grow into vigorous seedlings could be restored by15%PEG pretreatment and the effect was more obvious on seeds with lower vigour than on that with higher vigour. The increasement imposed on seeds by15%PEG was thought to be related with PEG pretreatment improved proline accumulation and raised activities of SOD, POD and CAT. This study will promote the application of S. davidii in the project of soil conservation and vegetation reestablishment.3. The changes in growth, morphology and physiology of S. davdii were investigated under dehydration and rehydration. After growing in well-watered conditions for four month, drought was imposed on seedlings by withholding water for21days, followed by rewatering for7days. The results indicated that S. davidii obtained its strong tolerance to drought stress through several ways. Although21days of withholding water decreased biomass accumulation of shoot in S. davdii, the root maintained nomal growth under this condition. Both shoot and root started to growth after rewatering for7days. As the drought stress continued, the epicuticular wax layers on both sides of leaves were becoming thicken and densed as well as more stoma closed. After rewatering, the epicuticular wax layers still kept thicken. Photosynthesis was inhibited and chlotoplast structure was destroyed to a ceitain extent by prolonged dryness, and rehydration could ameliorate the situation. Both the activities of SOD, APX, POD and GR and MDA content in leaves and roots of S. davidii were increased by long time water withholding. An obvious higher accumulation of proline was also found in dehydrated seedlings compared to non-dehydrated ones. The content of MDA and proline, and the activities of the four antioxidant enzymes decreased to normal level as the seedlings without stressed.4. Degenerate primers were designed according to homologous sequences from other plants. Three stress-related genes were then isolated from S. davidii through RT-PCR and RACE-PCR technologies.(1) A2671bp full-length cDNA fragment named SdP5CS was isolated from S. davidii seedlings, with the GenBank accession number JX307643. The ORF consisted of2262nucleotides and encoded a protein of753amino acids. The predicted protein had a calculated molecular mass of81.9kDa and a theoretical isoelectric point of6.29. The composition analysis showed that this enzyme mainly composed of hydrophobic amino acids like leucine and alanine etc. Sequence analysis revealed that SdP5CS protein included six conserved domain, which are an ATP-binding site, two leucine zipper, a conserved Glu-5-kinase domain, a NADPH-binding domain and a conserved GSA-DH domain. Semiquantitative RT-PCR was used to detect the expression level of SdP5CS gene in seedlings of S. davidii under conditions of drought and rewatering. The results showed expression of SdP5CS was increased in response to drought stress in both leaves and roots, and turned back to normal level after rewatering.(2) A novel transcription factor DREB (dehydration responsive element binding) was identified from S. davidii seedlings, which was named SdDREB2(GenBank accession number JX307644). The930bp full-length cDNA fragment was composed of an ORF of600nucleotides which encoded a protein of199amino acids. The predicted protein had a calculated molecular mass of21.4kDa and a theoretical isoelectric point of8.69. Sequence analysis suggested that SdDREB2protein had a highly conserved AP2/ERF DNA binding domain consisted of58amino acids. The14th and19th amino acids in this domain were valine and glutamate which were both important for the binding of TF to target DNA. Phylogenetic analysis showed SdDREB2was a member of A-2group of DREB sumfamily in AP2/EREBP superfamily. Semiquantitative RT-PCR was used to detect the expression level of SdDREB2gene in seedlings of S. davidii under conditions of drought and rewatering. The results showed expression of SdDREB2was increased slightly in response to drought stress in both leaves and roots, and turned back to normal level after rewatering.(3) A novel dehydrin gene was identified from S. davidii seedlings, which was named SdDHN (GenBank accession number JX307645). The1009bp full-length cDNA fragment was composed of an ORF of615nucleotides which encoded a protein of204amino acids. The predicted protein had a calculated molecular mass of19.6kDa and a theoretical isoelectric point of5.87. The composition analysis showed that this protein mainly composed of basic hydrophonic residues like glycine and threonine etc. Sequence analysis suggested that SdDHN protein composed of a K-segment which was necessary for dehydrin, and two conserved Y-segment without S-segment. SdDHN had low indentity in sequence with its homologous sequences from other plants except Y-and K-segment. SdDHN was proved to be a type of Y2K dehydrin from YK subgroup in DHN protein family though phylogenetic analysis. Semiquantitative RT-PCR was used to detect the expression level of SdDHN gene in seedlings of S. davidii under conditions of drought and rewatering. The results showed expression of SdDHN was increased strongly in response to drought stress in both leaves and roots, and turned back to normal level after rewatering.In one word, S. davidii has strong tolerance to drought stress during stages of seed germination and seedling growth. The whole results in this paper indicated that S. davidii obtained its drought resistance through three main ways. Strong growth of root and quickly accumulation of proline improved the ability of S. davidii to uptake water under drought conditions. Dense epicuticular wax layers and closed stoma avoided water loss as much as possible. The destruction of peroxidation on chloroplast and other structures in cell was also ameliorated by effective ROS-scavenging system. In addition, the resistance of S. davidii to drought was related to induced expression of SdDREB2、SdP5CS and SdDHN gene in leaf and root under stress conditions. |
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