Study On The Function And Mechanism Of FpHXK1 That Regulates Strawberry Fruit Sugar Accumulation And Plant Drought Resistance

As a bifunctional enzyme,hexokinase（HXK）not only regulates the first step of glycolysis,but also acts as a glucose sensor,which regulates the plant growth and development,secondary metabolism and stress response.The current studies on the mechanism of the sugar signaling function of HXK are mainly constrained in Arabidopsis,but studies have been scarcely carried out in strawberry HXK.In this study,the glucose signaling function of the strawberry HXK1 was explored.The explements such as bioinformatics analysis,subcellular localization,and complementation of Arabidopsis mutants gin2-1 were conducted,we gave deep insights of the importance of the FpHXK1and its kinase activity on the sugar metabolism and responses to drought stress.We also created the transgenic strawberry plantlets,which will provide materials to dig the interacting proteins of FpHXK1 with Co IP-MS.The main results were as follows:1.Through bioinformatics analysis,four HXK genes were identified in the Fragaria vesca and Fragaria pentaphylla genome respectively.The results showed that the Fv HXK1（FpHXK1）was the homolog of Arabidopsis At HXK1 base on amino acid sequence similarity and dynamic characteristics.We cloned the m ORF from FpHXK1 of F.pentaphylla.FpHXK1::e GFP recombinant plasmid was constracted,and then transiently expressed in tobacco,the red fluorescent nuclear marker m Cherry-Fa HY5 and mitochondrial marker m Cherry-At SRT2 were used as controls.The Fluorescence confocal observation showed that most of FpHXK1 proteins are localized in mitochondria,and a small part are localized in nucleus.2.We mutated the 177th Serine of FpHXK1 to Alanine（S177A）with the method of SOE-PCR,and constructed the protein expression plasmids p EAQ-FpHXK1 and p EAQ-FpHXK1^S177A.The two proteins were transiently expressed in tobacco leaves,and their kinase activities were measured.Finally,a significant decrease in the kinase activity of FpHXK1^S177A was discovered.These result showed that the 177th Serine was one of the key residues for the kinase activity of FpHXK1.3.We transiently overexpressed FpHXK1 and FpHXK1^S177A proteins in strawberry fruit.Compared with the empty vector control,the levels of glucose and sucrose did not fluctuate much in 35S::FpHXK1-OE fruits,but the sugars were significantly abundant in35S::FpHXK1^S177A-OE fruits.Also,the content of anthocyanin was significantly higher in35S::FpHXK1.However,the level of total phenolic compounds of 35S::FpHXK1^S177A-OE fruits was significantly lower than the control and 35S::FpHXK1-OE fruits.These results showed that FpHXK1 participates in the regulation of both primary metabolism and secondary metabolism in strawberry fruit,which is affected by its kinase activity.4.Under drought stresses mimic by PEG-6000,the plantlets in which the kinase activity was inhibited by NAG were severely suffered compared with the control.The peroxidase system,the synthesis of ABA and the expression of drought-induced genes were inhibited.In Arabidopsis,similar results were observed when compared with Ler wild-type plantlets and the hexokinase mutant gin2-1,the 35S::FpHXK1 and 35S::FpHXK1^S177A plants were more tolerance to drought stress.5.We constructed the recombinant plasmids 35S::FpHXK1 and 35S::FpHXK1^S177A,and complemented the Arabidopsis HXK1 function-deficient mutant gin2-1.In the MS medium supplemented with 6%glucose,the transcript expression of photosynthesis related genes CAA/CAB of 35S::FpHXK1 was inhibited,which was consistent with the wild type.However,the 35S::FpHXK1^S177A complementation seedlings were insensitive to the high level of glucose,which grew normally with green cotyledons and even superior to gin2-1 in the 6%glucose medium.The transcriptional repression of CAA/CAB by high levels of glucose was abolished in 35S::FpHXK1^S177A complementation seedlings.The number of branches and stem thickness of 35S::FpHXK1 complementation plants were similar to those of the wild type,but the bolting stage was significantly delayed.On the contrary,the growth of 35S::FpHXK1^S177A was similar to that of gin2-1,with less bolting,slender branches,and shrinking leaves.These results showed the FpHXK1 acts as a glucose sensor depending on its kinase activity.6.We also constructed transgenic strawberry plantlets:p TA7001-FpHXK1,in which FpHXK1 can be induced by dexamethasone;and FpHXK1::3×Flag tag in which FpHXK1is constantly over expressed.These plants provide important materials for Co IP-MS test to identify interacting proteins of FpHXK1.