| Urban turf plays an important role in constructing beautiful and clean modern city, or energy cycle and transformation of matter in city ecosystem.Tall fescue(Festuca arundinaceum var.genuina Schreb.)is an excellent cool-season turfgrass,and able to applied in turf of sport ground,landscape,biological revetment,and so on.Recently,tall fescue used is increasing quickly in China,and performs function of environmental greening and ecological protection.However,water requirement of tall fescue is very high in Summer.Drought and high temperature in Summer have been regarded as main factors that restrict growth of tall fescue.So,improving environmental adaptation is one of the main objects in breeding of tall fescue currently.Plant resistance to the environmental stress is very complex,usually shows linkage of quantitative trait loci. Genetic background of tall fescue is complex,genome sequence is very long,so good cultivars with resistance are difficult to obtain by the traditional selection methods. Genetic engineering is a convenient pathway to select good cultivars with resistance. The research covers both cloning of gene related to drought resistance of tall fescue and Agrobacterium-mediated gene transformation for further research on the tolerance of tall fescue to abiotic stresses and breeding resistant cultivars by genetic engineering. The main contents are as follows.1.The relationship of epidermis wax content to drought resistance in different tall fescue cultivars was clarified.Under drought and heat stresses,comprehensive analysis of drought resistance in 14 tall fescue cultivars was carried.The results showed that epidermis wax content and leaf water use efficiency in 14 tall fescue cultivars had significant positive correlation. Epidermis wax content of leaf in tall fescue had significant effect on stomatal conductance and intercellular CO2 concentration.Under drought and heat stresses, epidermis wax content of tall fescue can decrease stomatal transpiration,increase water use efficiency by mechanism of adjusting stomatal conductance,and wax accumulation had significant effect on stomatal transpiration as decrease epidermis evaporation. Epidermis wax content of tall fescue had intimate correlation with drought resistance,so, this suggests epidermis wax content should be as a new index in selecting cultivars with strong drought resistance.'Plantation' and 'CrossfireⅡ' were tall fescue cultivars with stronger drought resistance and more epidermis wax content.2.FapDREB2-cDNA from tall fescue with strong drought resistance had been cloned successfully.A dehydration-responsive element-binding-2(DREB2)homologue gene from the 'Planation',with high drought resistance,designated FapDREB2,was isolated by TouchdownTMPCR,and sequenced.FapDREB2-cDNA was 799 bp,predicted to encode a protein of 262 amino acid residues based on ORF.Its deduced protein sequence,with a conserved AP2 DNA-binding domain,shared characteristics with the DREB2 gene family based on sequence homology,structure and phylogenetic analysis.Expression of the FapDREB2 gene in different organs indicated that its transcripts were abundant in leaves and leaf sheaths,and scarce in roots.In addition,FapDREB2 gene expression under drought or high temperature conditions was stronger than control treatment.FapDREB2 mRNA accumulated in response to various abiotic stresses, confirming that its expression was more prominently induced by drought or salt treatment than by cold stress,and did not respond to abscisicacid(ABA)treatment.The FapDREB2 gene was involved in plant responses to drought or salt stresses through an ABA-independent pathway,leading to a better understanding of the regulatory mechanism of the DREB2 transcript factor in tall fescue.FapDREB2 expression in a study plot varied irregularly under drought or high temperature conditions in contrast to the seedlings grown with drought treatment in the laboratory.This suggests that expression of the FapDREB2 transcript factor would be complex during plant responses to different stresses.3.The sterilization method for endophytic fungi of tall fescue seeds was established.The seeds of tall fescue were sterilized by marination in different temperature (40℃,50℃,60℃)water for different time(10 min,20 min,30 min)and sodium hypochlorite solution,then seed germination rate test and callus culture were carried. Statistical analysis showed that the most optimal method of sterilization was marinated in 50℃water for 10 min,and dipped in 70%alcohol for 5 min,then in sodium hypochlorite solution for 15 min.The sterilization method can decrease the contamination rate of seeds,and increase the callus rate under the high germination rate of seeds.4.The genotypic difference of the callus formation rate and the differentiation rate in 7 tall fescue cultivars was studied.The research of inducement of calli and differentiation of tall fescue embryogenic calli in 7 tall fescue cultivars showed that no significant Spearman rank correlation exhibited between the callus formation rate and differentiation rate.The genotypic difference of tall fescue had a considerable effect on the callus formation rate and the differentiation rate.The cultivars with the highest callus formation rate and highest differentiation rate in 7 tall fescue cultivars was 'AridⅢ'. 5.Agrobacterium-Mediated transformation system of tall fescue with WAX2 gene had been preliminarily established.'Plantation','CrossfireⅡ' and 'AridⅢ' were applied as the gene receptor in plant transformation system.7 transgenic tall fescue plants were obtained by Agrobacterium strains EHA105 and LBA4404 mediated transformation.The study of transformation showed that the transformation frequency of Agrobacterium strain EHA105(contain plastid pCAMBIA-1300-multi)was higher,and more transgenic plants with Hygromycin B resistance and fewer negative transgenic plants were obtained,in contrast to Agrobacterium strain LBA4404(contain plastid pCAMBIA -1300-multi).The optimized selecting concentration of Hygromycin B in Agrobacterium strain EHA105 mediated transformation system was about 150 mg·L-1.The relation of epidermis wax content and drought resistance of tall fescue in this study was analyzed firstly,the result showed that epidermis wax content of leaf can be used as a new index to evaluate drought resistance of tall fescue,which will provide theoretical basis for improving its epidermis wax content and drought resistance by biological technique.On the basis of analysis of drought resistance in 14 tall fescue cultivars,on the one hand,FapDREB2-cDNA from 'Plantation' with strong drought resistance under drought or high temperature conditions was cloned successfully,which offers excellent gene resource to improve stress resistance of tall fescue and other resource plants.On the other hand,considering drought resistance and callus formation rate and highest differentiation rate comprehensively,'Plantation','CrossfireⅡ' and 'AridⅢ' were applied as the gene receptor in plant transformation system.WAX2 gene was transformed into 3 tall fescue cultivars,and genotype,the sterilization method for endophytic fungi of tall fescue seeds,Agrobacterium strains,selecting concentration of Hygromycin B etc.,were studied in transgenic test respectively,which is the basis for genetic engineering breeding and genetic improvement of tall fescue,and helpful for breeding excellent tall fescue cultivar with strong stress resistance(drought resistance, heat resistant).The study has important significance in promoting development of lawn industry of China,protecting ecological environment,beautifying space of human existence,and so on.
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