| Soluble sugar,an important signaling molecule and osmotic regulator in plants,is involved in the response to various stresses and its content is highly regulated by sugar metabolism.Fructokinase is the key enzyme that regulates the sugar metabolism and the Md FRK2 gene,which is induced by drought stress and a key gene to determine the activity of fructokinase in apple.In order to explore the relationship between Md FRK2 gene and drought resistance of plants,and the mechanism of regulating drought resistance,we analyzed the drought resistance and drought resistance mechanism of Md FRK2 genetically modified apple seedlings with the help of the phenotype and transcriptome data of apple seedlings,and it was proved that the Md FRK2 gene changed the drought resistance of apple seedlings through the root system using genetically modified ’Gala’ apple seedlings as material and adopted two drought treatment methods of pot culture and PEG6000 simulated hydroponic.In order to ascertain the relationship between the Md FRK2 gene and drought resistanc ein apple seedlings,we analyzed the activity of the promoter of Md FRK2 gene and transformed poplar to thoroughly illuminate the function in response to drought.The main results were as follows:Drought treatment of potted pot culture and hydroponic seedlings showed that the over-expression of Md FRK2 gene enhanced drought resistance of ’Gala’ apple seedlings.Under normal conditions,there were no obvious differences in morphological characteristics of the leaf and root between transgenic apple seedlings and wild-type apple seedlings.However,under drought stress,the net photosynthetic rate and water content of transgenic apple seedlings of leaves were significantly higher than wild-type apple seedlings which showed a significant increase in drought resistance of transgenic apple seedlings,but the stomatal conductance and transpiration of transgenic apple seedlings were significantly higher than wild-type apple seedlings.Further studies on the roots of apple seedlings showed that the contents of fructose,glucose,sucrose and sorbitol in the roots of transgenic apple seedlings were significantly lower than wild-type apple seedlings under drought conditions and there was no significant accumulation of carbohydrates.In the terms of root structures,the total length,root surface area and number of root tips of transgenic apple seedlings were significantly higher than wild-type apple seedlings.The research results above indicated that the increase of drought resistance of Md FRK2 transgenic apple seedlings and the ability to maintain root growth under drought conditions were related to the increase of water absorption capacity of apple seedlings.The hormone content indicated that there was a high content of auxin in root system of transgenic apple seedling under drought condition.In order to further explore the mechanism of increased drought resistance of Md FRK2 transgenic apple seedlings,transcriptome analysis was carried out on the roots of apple seedlings.The results showed that the genes involved in glucose metabolism,hormone signaling pathway and root growth and development of transgenic apple seedlings were changed under drought stress compared with wild-type apple seedlings.The analysis of differentially expressed genes showed that differentially expressed genes were mainly enriched in the KEGG pathways which including plant hormone signal transduction,starch and sucrose metabolism,amino sugar and RNA metabolism,amino acid biosynthesis,carbon metabolism,benzene propane biosynthes and sugar glycolysis or sugar dysplasia.Analysis of genes involved in sugar transport showed that the expression of related genes of FRK,SDH,SUT and SOT were increased,but the expression of genes related to Invertase were decreased.In general,the expression of HK,SUSY and SPS genes in transgenic plants were up-regulated even more,suggesting that the over-expression of FRKs genes enhanced the utilization of sugar metabolism in the roots of transgenic apple seedlings under drought stress.The analysis of genes related to hormone pathway showed that differentially expressed genes were mainly involved in the auxin signaling pathway,and the genes of SAUR family showed the most significant changes.The analysis of genes involved in the regulation of root development pathway showed that the differentially expressed genes were mainly related to root hair elongation,root development and lateral root morphogenesis.In order to further investigate the relationship between the increase of drought resistance and the root system of Md FRK2 transgenic apple seedlings,the root stock of Md FRK2 transgenic apple was grafted with "Fuji champion" to study the change of drought resistance.Under normal water supply condition,the aboveground part and root morphology of the graftedseedlings showed no obvious change.After moderate drought stress of long-term,the plants with Md FRK2 transgenic apple seedlings as rootstock showed more stronger drought resistance,and the root length and number were significantly higher than those with wild-type plants as rootstock.These results demonstrated that the increased drought resistance of Md FRK2 transgenic apple seedlings was related to root growth.In order to further explore the relationship between Md FRK2 gene and drought resistance,We analyzed the activity and expression patterns of Md FRK2 promoter Md FRK2 p under drought stress,and the model plant poplar was transformed.The activity analysis of Md FRK2 p showed that the Md FRK2 p was induced by drought in tobacco leaves.In poplar,Md FRK2 p was mainly expressed in stem tips,aged leaves and roots,but not in young and functional leaves.The results of paraffin section of stem tip showed that Md FRK2 p was expressed in vascular tissue of poplar,which suggested that Md FRK2 may enhance the ability of water drainage by affecting the development of vascular tissue and its function was also related to the growth and development of overground and root systems.By constructing the promoter vector of Md FRK2 gene,the analysis results of transformed poplar showed that the expressionlevels of Md FRK2 gene were different in different parts of the poplar stem segments,and the expression levels were the most highest in cambium and primary xylem.The contents of sugar in different parts of the abovemention part of poplar were also different,and the soluble sugar content increased in cambium and primary xylem of stem tips and stem segments.On the contrary,the soluble sugar content in the old leaves showed a decreasing trend.Over-expression of Md FRK2 gene promoted the growth of poplar and enhanced its photosynthetic capacity.The over-expression of Md FRK2 gene promoted the development of vascular tissue in the stem segment of poplar to facilitate the water drainage. |
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