Cloning And Expression Analysis Of SERK Gene In The Somatic Embryogenesis Of Manchrian Ash

Manchrian ash(Fraxinus mandshurica Rupr.)is a deciduous tree which belongs to oleaceae,fraxinus.It is a precious hardwood species in Northeast china.Manchrian ash not only has high economic value,but also has important ecological value.In recent years,the research group has carried out a detailed study on the tissue culture and somatic embryogenesis of Manchrian ash,but a lot of malformed embryos have been found in the process of somatic embryogenesis.In the process of inducing somatic embryogenesis,protein kinase gene is a very important kind of protein in biological life activity,in which SERK gene can be used as a gene that can mark embryogenic cells in the process of somatic embryogenesis,marking the process from the formation of embryogenic cells to embryogenesis.At the same time,SERK gene may participate in other biological processes except plant growth.In order to insight the mechanism of disintegrated cell embryogenesis,this paper uses the mature embryo and immature embryo of Manchrian ash as explants to induce somatic embryos and callus.The homologous cloning method is used to isolate and clone the SERK gene related to somatic embryogenesis,and the full length of cDNA is obtained.Fluorescence quantitative PCR is used to detect the expression of SERK gene in different organs,tissues and somatic embryos at different stages and at different times of induction of somatic embryos,hoping to lay a foundation for the research of the mechanism of somatic embryogenesis inManchrian ash.The results of the study are as follows:1.The immature embryo and mature embryo of Manchrian ashare used as explants respectively.NAA(3 mg/L,5.0mg/L),6-BA(1.5 mg/L,2 mg/L),casein(CH)400mg/L,sucrose 70g/L,and agar 6.5g/L are obtained respectively in the MS1/2 medium,and the somatic embryos are obtained,which provides the experimental materials for the following-up study.2.The target gene SERK fragment is isolated from the genomic DNA ofManchrian ash by the homologous cloning method.The sequence analysis fragment is 920 bp and contains 2 introns.By Blast analysis of NCBI database,the SERK gene fragment gets higher homology with homologous SERK gene of other species.3.RACE technique is used to successfully amplify the full-length cDNA sequence of the SERK gene from the genome of Manchrian ash.The results show that the full length of the cDNA is 2580 bp,the length of the sequence(CDS)is 1884 bp and the 627 a is encoded,the 5 'end and the 3' end have the non translation region of 122 bp and 599 bp respectively,and the CDS length of the SERK homologous gene of Manchrian ashis obtained.Its homology with Arabidopsis(Arabidopsis thaliana(L.)Heynh.)and tobacco(Nicotiana tabacum L)SERK homologous gene sequences is above 80%.It indicated that the full-length SERK gene has been cloned from the genome of Manchrian ash,and the gene is named FmSERK(GeneBank login number: KT071544).4.The structure of FmSERK gene protein is analyzed by DNAMAN software and DNAClub and other bioinformatics software.The results show that the homology of the amino acid sequence of the FmSERK gene encoded by the amino acid sequence of the other species is above 89%,indicating that the related FmSERK gene of the somatic embryogenesis is in the evolution of the SERK homologous gene,SERK/LRR-RLK gene family;the gene encodes 627 amino acids;its protein molecular weight is 69.3916 KDa,the theoretical half life is 30 h,the unstable parameter is 39.06,indicating that the protein is an unstable protein;the theoretical isoelectric point 5.57,the aliphatic amino acid index: 96.56.Analysis of the composition and physicochemical properties of FmSERK amino acids shows that the content of leucine Leu(L)is13.9%,which is the most,Gly(G)and Pro(P)is 7.2% and 7% respectively,and no pyrrolysine Pyl(O)and cysteine Sec(U)are detected.The total number of negatively charged residues(Asp+Glu)of the protein is 74,among which the total number of positively charged residues(Arg+Lys)is 61.The average coefficient of total hydrophilicity is-0.148,and it predicts that the protein is a hydrophilic protein.5.Real-time fluorescent quantitative PCR technique is used to detect SERK gene expression in different organs and tissues of Manchrian ash,the results showthat the gene expresses in different organs and tissues,but the highest gene expressionquantity is in the embryonic callus,the minimum gene expressionquantity is in the non-embryonic callus,even no expression;Analysis of SERK gene expression quantity in different periods of Manchrian ash somatic embryos concludes that minimum SERK gene expression is in globular embryo period,along with the development of embryo,expression quantity in heart embryo and torpedo embryo stage increase,in cotyledon embryo period SERK gene expression quantity reaches the peak.In mature embryo period SERK geneexpression quantity gradually decreases;Quantitative analysis of different time of induction of Manchrian ash somatic embryos shows that the early training 0 d~15 d,with the stimulation of exogenous hormones,the cells begin to dedifferentiate,the expression of FmSERK gene is in a stable expression stage.With the increase of induction time,in the train 20 d~30 d,SERK gene expression quantity increases gradually,in the induction training 35 d,FmSERK gene expression quantity reaches the peak,It is speculated that the FmSERK gene regulates the expression of cells in different culture stages of of Manchrian ash somatic embryos,and is the marker gene associated with somatic embryogenesis.