Preliminary Study On The Functions Of Gastrodia Elata GeDTC,GeELM1,and GeDRP1E

Gastrodia elata is a traditional and precious Chinese medicinal herb,which has the effects of calming the wind and arresting convulsions,calming the liver yang,dispelling wind and clearing collaterals,and resisting aging.It has various pharmacological effects such as sedation and hypnosis,anti-epilepsy,improving memory and anti-dementia,regulating the cardiovascular system,and enhancing immunity.It is widely used in clinical and medical care,but the formation mechanism of the "superior form" of genuine G.elata has not yet been fully clarified.The research team conducted quantitative taxonomic research and transcriptome sequencing on the collected G.elata samples in the early stage,and selected three genes related to mitochondria: GeDTC,GeELM1,and GeDRP1 E.However,the gene function has not been studied and verified yet.In this paper,the cloning and bioinformatics analysis of the three target genes have been carried out to construct Arabidopsis complementary mutants,and preliminary verification of gene function has been carried out,Further research and exploration on the function of the target gene were carried out using the agrobacterium-mediated potato genetic transformation system,laying a foundation for further in-depth analysis of the mechanism of action of the target gene on the tuber development of G.elata and the formation mechanism of superior shape and quality of G.elata.The main research contents are as follows:1.Gene cloning of GeDTC,GeELM1,and GeDRP1 E According to the CDS sequence of the gene in G.elata transcriptome,primers were designed and PCR amplification was performed.The results showed that the ORF of GeDTC had a total length of 981 bp and encoded326 amino acids;The ORF of GeELM1 has a total length of 1275 bp and encodes 425 amino acids;The ORF of GeDRP1 E has a total length of 1899 bp and encodes 633 amino acids.The sequencing result of the positive clone was compared with the original transcriptome sequence,with a similarity of 100%,indicating that the cloning was successful.2.Bioinformatics analysis of GeDTC,GeELM1,and GeDRP1 E Software such as Ex PASY was used to predict relative molecular masses,theoretical isoelectric points,and other coefficients of the genes of interest.It was found that GEDTC belongs to a stable hydrophilic protein,exists one transmembrane structure,has no signal peptide,and is localized in the inner mitochondrial membrane,showing the highest homology of 85.89% with Dc DTC;GeELM1 belongs to a stable hydrophilic protein,does not present a transmembrane structure,has no signal peptide,and localizes to the cytoskeleton,showing the highest homology with Dc ELM1 at 82.61%;GeDRP1 E belongs to a labile hydrophilic protein with no transmembrane structure,no signal peptide,and localizes to the cytoplasm,showing the highest homology of 90.05% with Dc DRP1 E.3.Phenotype statistics and physiological index determination of the GeELM1/elm1 and GeDRP1E/drp1 e Arabidopsis complementation lines The GeELM1/elm1 and GeDRP1E/drp1 e Arabidopsis complementation mutants were constructed,and the high-grade phenotypes of the wild-type,mutant,and complementation mutant Arabidopsis plants grown during the same period,as well as the related physiological indexes under cold and heat stress,were calculated.The results showed that GeELM1 restored the short stature,bolting and early flowering time in Arabidopsis plants caused by the ELM1 mutation and played an important role in heat stress;GeDRP1 E can restore the reduced seed setting rate of Arabidopsis caused by DRP1 E mutation,which plays an important role in growth and development.4.Agronomic traits of GeDTC,GeELM1,and GeDRP1 E potato transgenic tubers Using Agrobacterium mediated potato genetic transformation system,we harvested the targeted genetically modified micro potato and found that the GeDTC transgenic line micro potato was smaller in size,lighter in weight and had significantly lower organic acid contents than the wildtype line,initially speculated that GeDTC most likely accelerated organic acid efflux from potato tubers;The GeELM1 and GeDRP1 E transgenic lines exhibited larger volumes,heavier weights and significantly higher starch contents than the wild-type lines,and it was initially speculated that geelm1 and gedrp1 e might be involved in the process of mitochondrial fission,thereby accelerating potato growth and development.