The Function Study For Potassium Channel FaTPK1 Gene In Strawberry (Fragaria×Ananassa) Fruit Ripening And Quality Formation

Potassium(K+),an abundant cation in plant cells,is important in fruit development and plant resistance.However,how cellular K+ is directed by K+ channels in strawberry(Fragaria x ananassa)fruit ripening and quality formation is not yet fully clear.Strawberry 'Sweet Charlie,was used as material to explore the function of potassium channel FaTPK1 gene in strawberry fruit ripening and quality formation.The main results and conclusions are as follows:1.A two-pore K+(TPK)channel gene in strawberry,FaTPK1,was cloned using reverse transcription-PCR.A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane.A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit,which was coupled with the fruit's red colour development,suggesting that FaTPK1 is related to fruit quality formation.2.The down-and upregulation of the FaTPK1 mRNA expression levels using RNA interference(RNAi)and overexpression(OE),respectively,inhibited and promoted fruit ripening,respectively,as demonstrated by consistent changes in firmness and the contents of soluble sugars,anthocyanin and abscisic acid,as well as the transcript levels of several ripening-related genes,including fruit solfting related genes,such as GAL6(beta-galactosidase),XYL2(D-xylulose reductase)and PG1(polygalacturonase),the pigment synthesis genes,CHS(chalcone synthase)and CHI(chalcone flavanone isomerase),sugar transport gene SUT1(sucrose transporter).Results demonstrated that FaTPK1 promoted the strawberry ripening.3.Additionally,the regulatory changes of FaTPK1 influenced fruit resistance to Botrytis cierea.The spore suspension of Botrytis cinerea(106 CFU·ml-1)was inoculated into the strawberry fruit.After 5 days,the lesions appeared,while the FaTPK1-RNAi fruit showed only slight infection.In contrast,the control fruit had a moderate level of infection and the FaTPKI-OE fruit was severely infected 5 days after inoculation.Compared with the control,the FaTPK1-RNAi fruit had obvious resistance to Botrytis cinerea.On the contrary,the FaTPK1-OE fruit was more sensitive to pathogens,indicating that FaTPK1 may be involved in strawberry fruit resistance to pathogens.4.An isothermal calorimetry(iTC)analysis showed that the Escherichia coli-expressed FaTPK1 recombinant protein could bind K+ with a binding constant of 2.1×10-3 M-1 and a dissociation constant of 476?M.K+ uptake deficient Escherichia coli mutant LB2003 was used to analyze the functional properties of FaTPK1.The FaTPKl was cloned and expressed in LB2003 E.Coli.The expression of channel in bacteria was analyzed by RT-PCR.Our results show that FaTPK1 is restoring the LB2003 growth on low K+ media.The analysis of potassium uptake exhibited elevated level of K+ uptake in FaTPK1 transformant.Thus,the strawberry TPK1 is a ubiquitously expressed,tonoplast-localised K+ channel that plays important roles in the regulation of fruit ripening and quality formation.