| Based on a large-area(267 hectares)introduction of Zhongke series of Leymus chinensis in Ningxia,in order to screen out high-quality and high resistant strains of L.chinensis adapted to arid conditions,a multi-site and multi-angle systematic evaluation of the drought resistance and their adaptabilities to Ningxia site of Zhongke series of L.chinensis(8 different strains)was conducted using the method of drought resistant physiological and biochemical characteristics combining genetic engineering and adopting the test approach of flowerpot+field(simulated drought+natural water control).A target material,namely,the QF10 high drought resistant strain of L.chinensis,was screened out and identified.At the same time,based on an analysis of the early 454 transcriptome sequencing data of L.chinensis and the cloning of the family gene of part of the WRKY transcription factors,an gene expression profiling analysis was made on the WRKY family gene cloned to select the LcWRKY5 gene induced obviously by drought as target gene to make further biological function analysis.The purpose is to make a systematic exploration on the response of Leymus chinensis to drought stress from different angles and different levels.8 L.chinensis strains,including Y1,Y3,B,BG-2,SZ-3,SF4-2,XQ1 and QF10 bred by Institute of Botany,Chinese Academy of Science,were selected as the research objectives.The physiological and biochemical characteristics,such as the chlorophyll SPAD value,stable isotope 8 13C value,POD value,proline,soluble sugar and membrane permeability,of various strains of L.chinensis were studied under the conditions of 5%-35%concentration PEG-6000 stress and the nonlinear regression relationship between the nutritional quality and SPAD value of L.chinensis and its adaptability to the arid area of Ningxia was analyzed.It was the first to put forward the new method that the forage nutritive quality can be identified quickly and easily by field testing of grass leaf SPAD values.The test result showed that the average values of membership function of seeds germination of various strains of L.chinensis were in following order:QF10>XQ1>SF4-2>SZ-3,and the biggest was that of QF10 strain(0.983).It indicates that QF10 strain has a poor sensitivity to drought stress.So the drought resistant gene can be extract from the L.chinensis QF10 strains The ?13C values(long-term water use efficiency)of various strains of L.chinensis were in following order:Y1 strain>Y3 strain>BG-2 strain>B strain.The 8 strains of L.chinensis introduced into Ningxia all could live through the winter safely and successfully complete the life history of jointing stage,heading stage,milk stage and mature stage without any plant diseases and insect pests.Among which the production performances of the Y1 and Y3 strains were the highest and their drought resistances were relatively higher.The Stable isotope 813C Value,SPAD value,dry matter,crude protein,total amino acid,proportion of essential amino acid and energy of Y1 and Y3 strains were all higher than those of the other strains but their content of crude fiber was relatively low.They are high quality strains worthy of being extended and grown widelyThe study also shows that the drought stress had made the contents of proline,soluble sugar,POD,membrane permeability of L.chinensis up first and then down.The indices such as the high peak value/control value of the curve,the time node appearing in the high peak,the downward threshold range of the changing curve all could reflect the osmotic adjustment ability,antioxidant enzymes activity and the strength of physiological and biochemical metabolism of L.chinensis under stress condition which are one of the important physiological mechanisms by which L.chinensis.plants adapt to drought.At the same time,with QF10 strain of L.chinensis with stronger drought resistant features as a testing material,the target gene's expression profile in different abiotic stress conditions and different parts of tissues was analyzed.The results of the expression profile analysis shows that LcWRKY5 gene was induced significantly by drought,the LcWRKY5 gene of L.chinensis was expressed dramatically in roots and leaves,but there is no obvious expression in other tissues and organs.A phenotypic analysis of the T3 homozygote strain and wild Arabidopsis thaliana have been done through transforming the plant expression vector pBI1302-W5P-LcWRKY5 into A.thaliana by agrobacterium mediated floral dip method.The results show that in the normal growth condition,there is no significant difference between the transgenic plants and the normal growing a A.thaliana plants,but in the drought stress condition,the germination rate(green cotyledon number)and ability of drought resistance(survival rate)of the A.thaliana transformed by the expression LcWRKY5 gene are higher significantly compared with that of the wild type A.thaliana.The results of an analysis of the proline synthetase gene(P5CS)expression of the LcWRKY5 gene-overexpressed A.thaliana plants show that in the drought stress condition,the P5CS1 gene expression of the transgenic A.thaliana was significantly higher than that of the wild type,but the expression of P5CS2 gene in transgenic plants is lower obviously than that of wild-type plants.Though the expression analysis of the marker gene DREB1A,DREB2A,RAB18,RD29A and DREB1A related to the stress,it has been found that the expression of DREB2A and RD29A gene in transgenic plants under the drought stress was higher than that of the wild type.While the expression of DREB1A and RAB18 in transgenic plants was lower than that of the wild type.So it was inferred that the improvement of drought resistance of A.thaliana by transgenic LcWRKY5 gene is mainly by upgrading the expression of proline synthetase P5CS1 gene of the plant under stress conditions and the expression of stress-related DREB2A and RD29A genes so as to improve the drought resistance ability of transgenic plants.So the LcWRKY5 gene is the key factors deciding the drought resistance of L.chinensis. |
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