Expression Analysis Of AtLTPG16 Gene In Arabidopsis Thaliana And Identification Of MtLTPG Gene Family In Medicago Truncatul

Glycosylphosphatidylinositol-anchored lipid transfer protein(LTPG)is a small molecular basic protein widely existing in plants.It plays an important role in many biological activities such as plant lipid transport,sexual reproduction,seed development and germination,resistance to pathogen invasion and abiotic stress,and signal transduction.The Arabidopsis At2g48140 gene encodes EDA4 protein,also known as AtLTPG16 protein,which belongs to the LTPG family.In this study,members of the LTPG gene family were chosen for the present study.First,the expression pattern of the Arabidopsis AtLTPG16 gene and activity of the AtLTPG16 promoter were analyzed,and then the distribution,evolution and expression of LTPG family members in other species such as Medicago truncatula were further analyzed.In short,the studies above provide important information and clues for the functional analysis of the LTPG gene family,promoter activity analysis and utilization,and provide diverse gene,promoter and regulatory element resources for crop genetic improvement,which have important theoretical significance and potential application prospects.The main experimental results of this study are as follows:1.The analysis of gene expression pattern showed that the AtLTPG16 gene had root-specific expression activity and was induced by salt stress.Total RNA was extracted from different tissues of wild Arabidopsis thaliana,such as roots,leaves,flowers and seeds,and expression level of the AtLTPG16 gene in different tissues was analyzed by q RT-PCR.The results showed that the AtLTPG16 gene had the highest expression level in roots and had root-specific expression activity.Wild type Arabidopsis were treated with 200 mM NaCl for 0 h,4 h,8 h and 12 h,respectively,then different tissues were collected to analyze the expression activity of the AtLTPG16 gene under salt stress.The results showed that the expression of the AtLTPG16 gene was induced by salt stress,and the highest expression was induced at 4 h,indicating that the AtLTPG16 gene might participate in the process of plant salt stress response.2.Homozygous plants of transgenic Arabidopsis and tobacco with AtLTPG16 promoter were obtained.pAtLTPG16-GUS plant expression vector was transformed into Agrobacterium tumefaciens,and then wild type Arabidopsis was transformed by floral dip procedure;In addition,tobacco was infected by leaf-disk transformation.Finally,the homozygous lines of transgenic Arabidopsis and transgenic tobacco with AtLTPG16 promoter were obtained by screening for antibiotic resistance.3.The AtLTPG16 promoter was a root-specific promoter.The roots,leaves,flowers,seeds and other organs or tissues of the transgenic Arabidopsis thaliana with AtLTPG16::GUS were selected for histochemical staining and fluorometric assays.The results showed that the AtLTPG16 promoter could drive the expression of the reporter gene in the roots,while the expression level in other tissues was relatively low,indicating that the AtLTPG16 promoter was a root-specific promoter.4.The transient expression assays showed that the AtLTPG16 gene promoter could promote the expression of the gene in the heterologous tobacco.Furthermore,different organs or tissues of AtLTPG16::GUS transgenic tobacco were selected for GUS staining and activity analysis.The results further confirmed that the AtLTPG16 promoter is a root-specific promoter,which can drive the tissue-specific expression of foreign reporter genes in the root.5.Identification and analysis of LTPG gene family members in Medicago truncatula.In this study,HMM model comparison,two-way blast search analysis,conservative domain screening and GPI prediction were used as the basis and method for identification of Arabidopsis homologues of the Medicago truncatula LTPG gene family The study found that there are 40 members of LTPG protein family in Medicago truncatula,the molecular weight range is 9 KDa ～ 24 KDa,the isoelectric point range is 4 ～ 11,and the average number of amino acids is 146 aa.In the collinearity analysis,there are 6 paralogous gene pairs in Medicago truncatula,and 14 orthologous gene pairs between Arabidopsis and Medicago truncatula.The cis-acting element analysis found that there are a large number of regulatory elements related to abiotic stress response.6.Analysis of gene expression patterns of MtLTPG gene family members of Medicago truncatula.In this study,10 members of the MtLTPG gene family of Medicago truncatula,MtLTPG4,MtLTPG6,MtLTPG16,MtLTPG19,MtLTPG21,MtLTPG24,MtLTPG26,MtLTPG27,MtLTPG30 and MtLTPG31 were selected for gene expression detection.Real-time quantitative PCR was performed and the results showed that most of the MtLTPG gene family members were highly expressed in flowers.Wild type Medicago truncatula was treated with 200 mM NaCl for 0 h,4 h,8h and 12 h respectively,then various tissues were selected for qPCR analysis.The results showed that the expression of 9 genes was increased under salt stress treatment,but the expression of MtLTPG24 was decreased,indicating that the members of MtLTPG gene family may play important roles in plant stress response pathway.