Enzymatic Properties And Gene Expression Of Key Sucrose Metabolizing Enzymes In Leaves Of ‘Hongyang’ Kiwifruit

’Hongyang’kiwifruit is a dioecious vine plant of Actinidia in Actinidiaceae.’Hongyang’kiwifruit is rich in high dietary fiber and high vitamin C.Compared with other kiwifruits,it has the advantages of bright flesh color,high sugar and low acid,and is widely loved by people.According to the relationship between sink and source,the quality and yield of fruit are closely related to the sucrose input by sink tissue,the sucrose input by sink tissue is affected by the sucrose metabolism of source tissue leaves.Leaf is the main place for photosynthesis of higher plants.Carbon assimilates synthesized by photosynthesis are mainly transported from source leaves to fruits in the form of sucrose and are hydrolyzed into hexose or derivatives in fruits to participate in the growth and development of fruits.The regulation and change of sucrose synthesis,transport and utilization in source leaves affect fruit growth and sucrose accumulation.It is generally believed that Sucrose Synthase,Sucrose Phosphate Synthase,Invertase are the key enzyme of sucrose metabolism in leaves.In this experiment,the key enzyme activity and enzymatic characteristics of sucrose metabolism,the expression of key enzyme genes of sucrose metabolism and the dynamic changes of soluble sugar and starch content of’Hongyang’kiwifruit at different development stages were studied.The main research contents and results are as follows:1.The optimum p H,optimum temperature,substrate K_m,V_maxand enzyme stability of SPS,NIN,VIN,SUSs and SUSc were explored.The optimum p H of SPS was 6.5,the optimum temperature was 30°C,the V_maxwas 37.87 umol/g（FW）h when the substrate K_mwas 4.98 m M.The optimum p H of SUSc was 8,the optimum temperature was 50°C,the V_maxwas 2.58 mmol/g（FW）h when the substrate K_mwas4.87 m M.The optimum p H of SUSs was 5.5,the optimum temperature was 55°C,the V_maxwas 22.75 umol/g（FW）h when the substrate K_mwas 1.73 m M.The optimum p H of NIN was 7.5,the optimum temperature was 45°C,the V_maxwas 2.43 mmol/g（FW）h when K_mwas 75.23 m M.The optimum p H of VIN was 4.0-4.5,the optimum temperature was 60°C,the V_maxwas 11.98 mmol/g（FW）h when K_mwas 7.89 m M.The key enzyme activities of sucrose metabolism in leaves at different developmental stages were studied.It was found that SPS,NIN,VIN,SUSs and SUSc all showed an upward trend with the development of leaves.In the early stage of leaves development,SUSc played a major role in the decomposition of sucrose.In the late stage of leaves development,the activities of NIN,VIN and SUSs were higher.NIN and VIN decomposed sucrose and SUSs synthesized sucrose,which act synergistically to complete the metabolism of sucrose in the leaf,enabling the transport of sucrose from the leaf to the fruit.2.Soluble sugars in leaves mainly include fructose,glucose and sucrose.The content of fructose increased from bud stage to stable stage,and reached the maximum in stable period.The content of glucose reached the maximum in the stable period,the least in the bud period,and the content of the brown period and the pale green period was almost the same.The sucrose content was almost the same in the bud stage and the brown period,and the content increased from the pale-green period to the stable period,and reached the maximum in the stable period,indicating that the leaves began to accumulate sucrose in the pale-green period.Starch content was higher than sucrose content in all developmental stages of leaves,and it was found that starch was the main storage substance in kiwifruit leaves.3.High-throughput sequencing technology was used to sequence transcriptome in four different development stages of’Hongyang’kiwifruit leaves.A total of108.79 Gb data was obtained from 12 leaf samples,with GC content more than 45%and Q30 value more than 93%.A total of 109832 Unigenes were obtained by Trinity sequence assembly,with a minimum of 301 bp,a maximum of 16929 bp,a medium length of 654 bp,and an average length of 1135 bp.The number of Unigenes in300-500 bp was the largest,with a total of 39866,and the number of Unigenes greater than 2000 bp was the least,with a total of 17559.Perform functional annotations on the obtained Unigene and obtain a total of 58379 annotation information.14982differently expressed genes were screened and subjected to GO analysis and KEGG metabolic pathway enrichment analysis.These differentially expressed genes can be divided into 131 metabolic pathways,and the starch and sucrose metabolic pathways contain 355 differentially expressed genes.Among them,11 differentially expressed genes related to sucrose metabolism were screened for Quantitative Real-time PCR.4.Using q RT-PCR technology to analyze the relative expression levels of various Ac SPS,Ac SUS,Ac NIN,Ac VIN genes in the leaves of’Hongyang’kiwifruit at different development stages.The expression of Unigene51090 and Unigene51091genes in different developmental stages of leaves was basically the same as the change trend of SPS enzyme activity,which increased significantly in the pale-green period.It was speculated that Unigene51090 and Unigene51091 genes were likely to be the key genes expressed in leaves sucrose metabolism.The relative expression levels of Unigene38045,Unigene26958 and Unigene51089 genes decreased significantly from the bud stage to the brown stage,reached the lowest value in the brown stage,and increased in the pale green stage.The expression of Unigene38045,Unigene26958 and Unigene51089 genes is basically the same as the change trend of enzyme activity of SUS synthesis.It is speculated that Unigene38045,Unigene26958and Unigene51089 genes are likely to be the key genes expressed in leaves sucrose metabolism.The relative expression levels of Unigene19589,Unigene36426,Unigene47299 and Unigene47292 genes were the least in the bud stage.From the bud stage to the pale green stage,the relative expression level showed an upward trend and reached the maximum in the pale green stage.It is speculated that Unigene19589,Unigene36426,Unigene47299 and Unigene47292 genes are likely to be the key to expression in leaves sucrose metabolism.