| Vascular bundles are distributed in the roots,stems,leaves and other parts of plants,used to transport water and nutrients,and provide mechanical support for plants.Studying the development patterns and regulatory mechanisms of vascular bundles has important theoretical significance.The START(The Steroidogenic Acid Regulatory Protein related Lipid Transfer)conservative domain is composed of about 220 amino acids.In Arabidopsis,there are 35 proteins with START domain,and only 9 proteins with START domain.Members of the protein family that have reported HD-ZIP-START multi domain play an important role in regulating the establishment of vascular bundles in Arabidopsis.However,it is unknown whether the 9 proteins with only the START domain have the function of regulating vascular bundle formation.The paper focuses on proteins with only the START domain subfamily as the research object,and uses bioinformatics analysis to analyze the gene expression characteristics and physicochemical properties of proteins with only the START domain subfamily,as well as the characteristics of each START domain.On this basis,the development of vascular bundles in mutants with only 9 members of the START domain in Arabidopsis was observed and compared,and it was determined that only the START domain protein had the function of regulating vascular bundle formation.Using qRT-PCR technology to analyze the influence of family members on the expression level of known vascular bundle building genes,and yeast two hybrid technology to screen the interaction proteins of the START domain protein subfamily members,revealing the mechanism of action that only has the START domain protein.For the convenience of description,the members in Arabidopsis that only have a START domain are named PCST(Protein Containing START domain),with 9 members named from 1 to 9,namely PCST1 to PCST9.The results are as follows:1.Bioinformatics analysis with only members of the START domain protein familyBioinformatics analysis shows that proteins with only the START domain subfamily are mostly unstable proteins,with PCST1/2/3/6 having one transmembrane segment,PCST4/5 having two transmembrane segments,and PCST7/8/9 having no transmembrane segments.The start position and size of the START domain vary among various proteins,with different ligand binding sites.Among them,PCST1/2/3/6 protein has a phosphatidylcholine binding site,PCST4/5 protein has a hydrophobic ligand binding site,PCST9 protein has a lipid binding site,PCST7 protein has one lipid binding site,and no ligand binding sites have been found for the PCST8 protein.Chromosome localization analysis shows that PCST1/3 is located on chromosome 3,PCST2/6 is located on chromosome 1,PCST4/8 is located on chromosome 4,and PCST5/7/9 is located on chromosome 5.PCST1-PCST9 has different spatiotemporal expression characteristics,and each gene is expressed in different tissues at different stages,but exhibits significant tissue specificity.2.Subcellular localization analysis of only members of the START domain subfamilySubcellular localization shows that the vast majority of proteins with only the START domain are located in the cell plasma membrane,while PCST1 and PCST8 proteins are both located in the cell membrane and nucleus.3.functional analysis of vascular bundle formation regulated by protein with START domain only(1)Analysis of Vascular Bundle Phenotypes in Mutants with Only START Domain subfamily Gene Function Acquisition and Function DeletionThe observation of cotyledon vein showed that the shape of the cotyledon vein of the overexpression and mutant had changed.The cotyledon vein of the overexpression plant had only three secondary veins,forming three complete interveinal regions with the main vein,while some cotyledons of the mutant plant had only two secondary veins,pcst1,pcst2,pcst3 and pcst6,forming two interveinal regions with the main vein,which affected the development of the secondary vein.Observing the veins of lotus leaf,it was found that compared with wild-type Arabidopsis,the number of secondary veins in overexpressed plant leaves,except for PCST3-OE,did not show significant changes,while the number of tertiary and fourth veins increased.The number of secondary veins in mutant leaves,except for pcst8,did not show significant changes,and the number of tertiary and fourth veins decreased.The number of tertiary and fourth veins in psct7 did not show significant changes.Compared with wild type Arabidopsis thaliana,the number of stem vascular bundles in overexpression plants increased significantly,the volume of fiber histiocyte between bundles increased,and the degree of lignification increased;The stem of the mutant plant became thinner,and there was no significant change in the number of vascular bundles.(2)Analysis of Vascular Bundle Phenotypes in Mutants with Multiple Domain Protein PH-START1 Gene Function Acquisition and DeficiencyObserving the veins of cotyledons,it was found that both PH-START1-OE and ph-start1 cotyledons have three lateral veins,forming three closed interveinal regions with the main vein.The morphology of cotyledons has undergone changes.Observing the true leaf veins,the number of second,third,and fourth level leaf veins in the PH-START1-OE plant significantly decreased,while the number of second,third,and fourth level leaf veins in the ph-start1 mutant plant significantly increased.The number of stem vascular bundles in PH-START1-OE plants did not change significantly,while the number of stem vascular bundles in ph-start1 mutant plants increased significantly,and the size and number of xylem and fiber histiocyte cells between bundles changed.4.Molecular Mechanism Analysis of Regulating Vascular Bundle Formation by Proteins with START DomainThe qRT-PCR technique was used to analyze the expression levels of known vascular bundle related genes CVP2,SFC,AXR1,ATHB-8,PIN1,LAX2,WRKY13,DET3,and HDT1 in PCST1 and PH-START1 overexpressions and mutants.The results indicate that changes in the expression levels of PCST1 and PH-START1 can cause changes in the expression levels of these genes,which in turn regulate the development of vascular bundles by regulating their expression levels.The yeast two hybrid results showed that there was no interaction between the PHSTART1 and KNAT1 proteins.PCST1 screened 5 candidate interacting proteins,among which EXLB1 and CALS1 are related to vascular bundle development;PH-START1 screened 9 interacting proteins,among which POM1 is associated with vascular bundle development.5.Response of START Domain Proteins to flg22Different concentrations of flg22 were used to treat wild-type Arabidopsis thaliana that had grown for 4 days,as well as mutants containing only the overexpression of START domain subfamily members and T-DNA insertion.The main root inhibition was observed,and the results showed that flg22 had different levels of main root inhibition for each mutant,indicating that only members of the START domain subfamily may participate in the immune response of Arabidopsis thaliana. |
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