Cloning And Analysis Of Potassium Absorption And Potassium Transporter POT-X1 And Potassium Channel Protein POT-?1 In Gastrodia Elata

Gastrodia elata(G.elata)is a kind of precious traditional Chinese medicine.It belongs to the orchid plant with degenerated roots and leaves,which can not carry out photosynthesis and absorb nutrients from soil.The nutrients needed for seed germination and vegetative growth are mainly from Mycena and Armillaria mellea(A.mellea).A.mellea invaded the vegetative stem of G.elata by Rhizomorph was digested and absorbed by G.elata to provide itself nutrition.In addition,G.elata can also obtain nutrients from the surrounding soil through epidermal infiltration.Studies have shown that G.elata can absorb inorganic and organic nutrients from the environment in both vegetative growth and reproductive growth periods without armillaria companion culture.There are more than 40 kinds of mineral elements in G.elata.The content of potassium in mineral elements is the highest,indicating that G.elata is a potassium-loving crop.Potassium promotes the growth of G.elata and is conducive to the formation and accumulation of G.elata and G.elata polysaccharides.The absorption of potassium by G.elata is affected by ecological environment and has certain regional characteristics.However,there are few reports on potassium absorption mechanism of G.elata.In this study,the potassium content,plasma membrane(PM)H+-APase activity and pump activity of G.elata were analyzed under different temperatures and different development periods,and the potassium absorption mechanism of G.elata was discussed.At the same time,based on the transcriptome analysis of A.mellea and G.elata,the potassium channel protein gene and transporter gene were screened.The gene sequences were cloned and analyzed,and the functions of these genes were identified,which laid the foundation for further research on the absorption,transportation and regulation of potassium nutrition of G.elata and the influence on the yield and quality of G.elata.The main results are as follows:1.The content of potassium,PM H+-ATPase activity and hydrogen pump activity were analyzed.The results showed that the potassium content of both indoor and outdoor G.elata was higher than that of commercial G.elata.H+ ATPase activity and H+ pump activity in G.elata cultivated under Zhaotong natural environment were higher than that of G.elata cultivated indoor 23 ° C and 13 ° C,H+ ATPase activity and H+ pump activity of G.elata cultivated indoor 23 ° C was 13 ℃ higher than that of Gastrodia elata cultivated indoor 13 ℃.The H+-ATPase activity of G.elata was 1.3 times that of commercial G.elata.H+-Atpase activity and H+ pump activity promoted the potassium absorption of G.elata.2.Based on the transcriptome analysis data of G.elata and A.mellea,we screened the potassium transporters with significant differentially expressed genes POT-X1,and find out its CDS sequence.Using RACE-PCR amplification,POT-X1 gene was cloned,which contained 2361 bp and encoded 786 amino acids.The protein molecular weight is 88 Kilodalton.The theory of PI value of 9.22,which contain mostly hydrophobic domain structure and more alpha helix as well as beta-fold,indicating that it has good stability and resistance to protease.This gene has high homology with dendrobium.The cloning and sequence analysis of this gene laid a foundation for further study on its function and transcriptional regulation mechanism.3.Based on G.elata and A.mellea transcriptome database,The significantly differentially expressed potassium channels protein gene was screened out and its CDS sequence was found.Using RACE-PCR amplification technology,987 bp of this gene was cloned,which coded 328 amino acids has the molecular weight of 36.400 KiloDalton.The theory of PI value of this protein is 5.73.N and C terminal of the peotian has obvious hydrophilic structural domain.The plant overexpression vector pH2wG7-POT-β1 of this gene was constructed.Homozygotes were screened out from the arabidopsis mutants purchased from the United States by the three-primer method,which laid a foundation for the functional identification and transport regulation mechanism analysis of POT-β1 gene in the next step.