The Physiological Responses And Molecular Expression To Drought Resistance In Common Woody Plants In North China

Drought stress is one of the main factors that threatens crop production.The researches on the drought-resistant morphology,physiological mechanism and the quantitative evaluation of drought-resistant of forest trees have provided a large number of stress-resistant materials for landscaping and forestry production in China.Molecular marker-assisted breeding mechanism and technology have made some extent progresses.Based on the investigation of physiological reactions and the identification of key gene function in woody plant drought resistance,this dissertation studied common woody plants in North China on quantitative evaluation system of physiological responses with 13 physiological indicators and gene expression of DREB2A,WRKY19 and XET in response to drought stress,explored the relationship between physiological response and gene expression and revealed the key gene regulation of physiological reactions on drought stress.Cloned the DREB2A,WRKY19 and XET gene promoters and identified their cis-acting elements in response to drought,salt,low tenperature and ABA,respectively.Some enlightments achieved will be sueful to insight gene regulation of drought resistance,evaluating adaption to drought stress in molecular levels,selecting accurately harden trees for reclaiming up arid land and breeding.The main innovations are as follows.1.Integrating physiological response and gene expression revealed the molecular regulation mechanisms of forest tree genes on physiological reaction in response to drought stress.The common woody plants,selected in the north of China and trialed in the sites with serious drought and cold mountain conditions in Yanqing Beijing,several in the buried hill-plain marginal sites in Zhaoquanying town,Shunyi Beijing,respectively,39 survived species or cultivars were used for the study.13 physiological and biochelical indices related to drought resistance were tested,included leaf water content(LWC),membrane permeability(MP),stoinatal conductance(SC),chlorophyll a(Chl a),chlorophyll b(Chl b),chlorophyll a+b(Chl a + b),soluble sugar(SS).soluble protein(SP),superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),and malondialdehyde(MDA).Membership function(MF)was used to establish the relationship between physiological index variables and physiological response values,quantified and classified the physiological reactions in response to drought stress in the forest trees.According to Avarages of Mernbership function(AMF),the forest tree hardens were divided into five class:I Class,AMF? 0.6.3 species included,Rohinia pseudoacacia'Unifolia',Securinega suffruticosa,and Acer elegantulum;? class.AMF 0.5?0.6,11 species included,Fraxinus pennsylvanica,Koelreuteria paniculala.Populus tremulaxPopulus alba '717-1B4',Ulmus pumila,Xanthoceras sorbifolia.Rohinia pseudoacacia 'Idaho',Cotinus coggygria 'Cineres',Paeolia lactiflora,Lycium ruthenicum 'Ningqi No.5',Lycium ruthenicum and Lycium rulhenicum.? Class,AMF 0.4?0.5,13 trees involved,Prunus armeniaca 'Ansu',Fraxinus chinensis,Populus tremula×Populus alba,Robinia pseudoacacia 'Hungary',Paeonia ostii 'Lishizhenii',Robinia pseudoacacia 'Tetraploid',Prunus davidiana,Caragana korshinskii,Paeonia suffruticosa 'Wulong Pengsheng',Populus deltoids×Populus nigra,Ziziphus jujuba 'Apple',Crataegus cuneale and Crataegus pinnatifida.? class.AMF 0.3?0.4,11 trees involved,Ziziphus jujuba 'Jinchang-1',Ulmus laevis,Elaeagnus angustifolia,Rosa woodsii,Caragana korshinskii,Salix fragilis,Sabina vulgaris,Paeonia rokii,Nilraria tangutorum,Gleditsia japonica,and Pinus tabulaeformis.And V class,AMF<0.3,1 cultivar,Lycium ruthenicum 'Ningqi No.7'.Based on the gene function identification,the sequence characteristics of DREB2A,WRKY19 and XET conserved regions in 39 forest species were cloned and analyzed.The gene expression determined by RT-qPCR and linked to 13 physiology reaction indices.It was found that DREB2A,VVRKY19 and XET gene expression were related to the regulation of physiological responses of SOD and POD activities,MP capacity,SS content,respectively.The correlation between each gene expression quantities of DREB2A,WRKY19,and XET and 13 indexes of physiological responses were analyzed in each of genus species,individually.In Robinia species,WRKY19 gene expression correlated with SS content,while XET gene expression correlated with SOD activity.In Paeonia species,WRKY19 gene expression correlated with SC,while XET gene expression correlated with FP and SP content.In Populus species,DREB2A gene expression correlated with MP capacity while XET gene expression correlated with CAT activity.In Lycium species,XET gene expression correlated with SC and FP contents,and SOD activity,respectively.2.Promoters of PoDREB2A,PoWRKY19 and PoXET genes in P.ostii 'Lishizhenii' were cloned and identified cis-acting elements in response to osmotic,salt and low temperature.P.ostii 'Lishizhenii' has strong drought resistance and wide adaptability in Section Moutan ofPaeonia.The gene expression of PoDREB2A was highest,PoWRKY19 regulated leaf SC,and PoXET regulated FP and SP contents,respectively.These three genes involved in the plant drought resistance formation.The promoter sequences of PoDREB2A,PoWRKY19,and PoXET were 2342 bp,1974 bp,and 2025 bp,respectively.Bioinformatics analysis indicated that all three promoters contained drought,salt,low temperature and ABA signal response cis-acting elements.Identified these cis-acting element function followed by deleted 5'-end promoter sequence,constructed into GUS gene expression vector for transformation into tobacco,subjected to PEG,NaCl,4? and ABA treatment,analyzed GUS gene expression activities qualitatively and quantitatively,respectively.As the results indicated below.The PoDREB2A gene promoter has drought response elements,undetermined element and CANTNTG,in the region of-1541 bp--1833 bp and-1137 bp--1541 bp,salinity response elements,undetermined element and GRWAAW,in the region of-1833 bp--2240 bp,-1028 bp--1137 bp and-679 bp--1028 bp,a low temperature stress response element,undetermined element,in the region of-1541 bp--1833 bp,and an ABA responsive element,ACACNNG,in the region of-1028 bp--1137 bp,respectively.The PoWRKY19 gene promoter has drought response elements CANNTG and CNGTTR in-1264 bp--1461 bp and-422 bp--718 bp,respectively.The promoter sequence ranging from-1461 bp--1901 bp,-953 bp--1264 bp,to-718 bpP-953 bp has the saline response elements GRWAAW and undetermined element.The low-temperature response element CANNTG was in the region of-1264 bp?-1461 bp and-422 bp?-718 bp.There was an ABA response undetermined element in the-422 bp?-718 bp region.The PoXET gene promoter has two drought response elements,CATGTG and CANNTG in the regions of-1173 bp--1570 bp and-425 bp--591 bp,respectively.There were saline response elements GRWAAW and undetermined element in the regions of-1173 bp--1570 bp and-425 bp--591 bp.The low temperature response element CANNTG was in the regions of-1774 bp--2025 bp and-125 bp--425 bp,undetermined element was in the regions of-425 bp--591 bp.In the regions of-1570 bp--1774 bp,-591 bp--1173 bp and-125 bp--425 bp were ABA response elements,CATGCA,ACACNNG,respectively.3.The invention of Extraction Method of Total RNA from Plant Leaves solved the serious interference problems of nucleic acid extraction of woody plant materials was authorized China State Patent ZL201410444486.8.Woody plant leaves rich in polysaccharide polyphenols were decomposed twice in succession,extracted with water-saturated phenol and chloroform mixed solution,and precipitated total RNA with isopropanol.This method was no water bath or cryogenic centrifuge and avoided the influence of LiCl residue in conventional precipitation method on subsequent experiments,met the needs of RNA reverse transcription PCR and qPCR experiments.The above results provide insights into the tree physiological mechanism of drought resistance formation and its molecular regulation mechanism.It provides theoretical and technical approaches for molecular physiological selection and breeding of drought-resistant high-quality forest trees.