Identification Of Phloem Sap Proteins Of Mulberry And Functional Study Of Mul-BRD1

Mulberry is the sole feed species of silkworm,and has high ecological,economic and medicinal values.Salt and drought are important environmental factors that affect growth and development of mulberry.Improving the resistance of mulberry to these stresses is conducive to the development of sericulture industry and the realization of ecological value,economic value as well as medicinal values of mulberry.The phloem sap of mulberry is rich with proteins,which play important roles in signal transmission,material transport and plant defense.Therefore,it will be help for revealing the resistance mechanism of mulberry and providing candidate genes for mulberry resistance breeding to identify the phloem sap proteins and study their functions.In this study,high-throughput transcriptomic proteomic analysis was conducted on the mulberry phloem sap,and the gene(Mul-BRD1)of bromodomain-containing protein 1 identified was cloned,and its expression characteristics and biological functions were studied.Moreover,the interaction proteins of Mul-BRD1 protein were identified.The information provided will help us to understand the function,molecular mechanism and signal transduction pathway of phloem in response to environment stress,and provide a candidate gene for mulberry resistance breeding.It also shed light on the study of the function mechanism of MulBRD1 gene.Results presented in this thesis can be summarized as follows:(1)Proteomic analysis of mulberry phloem sapLiquid chromatography-tandem mass spectrometry was conducted to analysis the proteome of mulberry phloem sap,and 712 proteins were identified which can be divided into 20 classes based on their functions.Among the classes,the most abundant category is associated with protein post-translational modification and transport.The other proteins belong to categories such as h stress-related and defense mechanisms,signal transduction,secondary substances metabolism,etc.These indicated that the phloem was associated with diverse physiological functions.The results provide a phloem sap protein database with reference value and provide a basis for better understanding of the physiological functions of the phloem.(2)Expression characteristics and biological functions of Mul-BRD1 geneIn this study,the gene of mulberry phloem sap protein Mul-BRD1 identified was cloned,and a full length cDNA of 2355 bp was obtained,which encoded a protein 784 aa residues in length with a predicted size of 85.30 kDa.Mul-BRD1 protein is predicted to contain multiple phosphorylation sites and N-glycosylation sites.It does not contain signal peptides and transmembrane regions,but it is hydrophilic and located in the nucleus.Mul-BRD1 gene was expressed in all the mulberry tissues and has no tissue-specific expression.Moreover,the promoter of Mul-BRD1 gene was cloned and named as pMulBRD1,which contains some core components of promoter and multiple cis-acting elements.Transient expression system of tobacco leaf and GUS histochemical staining were used to analyze the activity of pMulBRD1,and the results showed that it has light and ABA induced expression activity.The function of Mul-BRD1 gene was studied by using transgenic technology and it was indicated that the expression of Mul-BRD1 gene in Arabidopsis thaliana enhances transgenic plant seeds resistance to ABA,mannitol and NaCl,and enhance the transgenic plants resistance to NaCl and drought stresses.(3)Screening and identification of Mul-BRD1 protein interactionsThe bait vector of Mul-BRD1 was successfully constructed,and the toxicity and selfactivating activity of the recombinant vector were detected,and the results showed that the MulBRD1 protein had no toxicity and self-activating activity.Six proteins interacting with MulBRD1 were identified using yeast two-hybrid system.These proteins included the glyceraldehyde-3-phosphate dehydrogenase B,COP9 signalosome complex subunit 5A isoform X1(CSN5A),aldehyde oxidase GLOX,GDSL esterase/lipase APG,histone acetyltransferase of the MYST family 1 and an uncharacterized protein.These proteins are involved in diverse biological processes such as carbohydrate metabolism,constitutive photomorphogenesis,biological oxidation,lipid metabolism and mediated acetylation.In addition,the interaction between Mul-BRD1 and Mul-CSN5 A,as well as Mul-BRD1 and MulMYST were proved by yeast two-hybrid backfilling experiments.