Study On The Function Of START Domain Regulating The Growth And Development Of Arabidopsis

START(The Steroidogenic Acute Regulatory Protein-related Lipid Transfer)domain is composed of about 220 amino acids and is widely found in animals,plants and microorganisms,and its function is relatively conservative.In animals,abnormal START domains can lead to metabolism changes of lipids and glucose,lead to diseases such as congenital lipoid adrenal hyperplasia,liver cancer etc.,so its function and mechanism in the process of regulating animal development have become research hotspots.Family members of the START domain are also widely present in plants,but there are few related studies.There are 35 proteins containing START domain in Arabidopsis thaliana,A few members of the multi-domain subfamily have other domains so that their functions were studied(for example,some members of the HD-ZLZ or HD-ZIP transcription factor family contain START domain).The role of the only START domain subfamily and the START domain in regulating plant growth and development were not clear.In this study,the function of START domain in Arabidopsis thaliana was tested.The bioinformatics method were used to analysis the physicochemical property,promoter elements and the composition of START domains;On the basis of bioinformatics analysis,we analyzed the expression characteristics of subfamily members containing START domains only in different developmental stages and different tissues of Arabidopsis thaliana using qRT-PCR technology.By phenotypic analysis of transgenic plants overexpressing and lossing functional of the START domain,we found that START domain had important role in regulating the polar growth of plant leaves,flower development,seed development and other biological functions.The exprsssion levels of the subfamily members containing START domain were analyzed in the transgenic plants overexpressing the START domain and in the mutant using qRT-PCR technology.We also clarified the regulatory relationship between subfamily members containing only the START domain.The contents of protein,sugar and fatty acids in mutants with and without START subfamily functions and wild type controls were analyzed,to explain the effect of the START domain on proteins,sugars,and Fatty acid metabolism.The results of the study are as follows:1.An analysis of the bioinformatics:there are 35 members in the START domain family in Arabidopsis thaliana,while 9 members in the only START domain subfamily(They are named PCST1-PCST9).However,the phylogenetic tree analysis shows that PCST7 and PCST8 are not in the same branch with the other 6 members,and their hydrophobic binding site is different.The instability coefficient of PCST3 is less than 40 and most members are unstable proteins.Gene expression profiling analysis showed that they were expressed in roots,stems,leaves,flowers and fruits,but showed significant organ specificity.2.Through the observation of the phenotype of the gain-of-function and mutant plants with subfamily member containing START domains only,the over-expression transgenic Arabidopsis thaliana and T-DNA insertional mutants of subfamily members showed significant seed size differentiation.The size of large seed is similar to the wild type,but the size of small seed is over 40%smaller that the seed size of wild type.Large seeds similar in size to the wild type were selected for subsequent phenotypic observation.(1)All seeds of subfamily members containing START domains only with size similar to the wild type can germinate after over expression,but the two cotyledons curl downwards,and the first pair of true leaves showed obvious changes in developmental stage.The 2 true leaves of wild type are fully extended,but transgenic Arabidopsis thaliana overexpressing only the member of the START domain produced only one true leaf,and the other true leaf was delayed in development.The over expressed plants with subfamily members had different degrees of asymmetric development.The symmetry of T-DNA inserted mutants also changed,but it was not as serious as the phenotype of over expressed transgenic plants.(2)After overexpression of genes from members of the subfamily containing only the START domain,the symmetry of the floral organs changes,the characteristics of 4 long stamens and 2 short stamens are no longer obvious.There are 6 filaments in the floral organs of wild-type Arabidopsis thaliana,but after the members of subfamily containing START domains only are overexpressed,the number of filaments decreases(4-5),and the pollen vitality.The pollen vigor of T-DNA insertion mutants also decreased.(3)The study showed that after only the genes of each member of the START domain subfamily were overexpressed,seed development was significantly affected:the seeds in the pod appeared size differentiation,and the seed development was abnormal.T-DNA insertional mutants also showed abnormal seed size differentiation and partial seed development.It shows that change of expression of subfamily members containing START domains only will have a significant impact on seed development.3.Analyzed the metabolic levels of storage proteins,soluble sugars and fatty acids in the seeds shows that the content of soluble sugars in each over expressed member and in T-DNA inserted mutants increase significantly;The content of seed storage protein of over-expression plants increased slightly;the content of fatty acid of overexpression plants decreased significantly,the content of seed storage protein of mutants did not change significantly,and the content of fatty acid decreased slightly.Further analysis of the content of each component of fatty acid show that changes in the expression level of START domains members will affect the synthesis of unsaturated fatty acids or long-chain fatty acids from primary fatty acids,indicating that subfamily members containing START domains only play an important role in the metabolism of internal storage materials in seeds.4.The analysis of the expression level of the other members of over expressed family members with START domains only and the other members in the T-DNA inserted mutants using qRT-PCR technology shows that changes in the expression levels of any member will regulate the expression levels of other members.5.Regulation of hormones on expression of subfamily members containing only the START domain.Compared with CK,the expression of each member of the subfamily is significantly different after being treated with IAA,GA,BR and ABA for 0.5 h,and the expression of different members is not consistent under different treatments.