Biological Function Of Stress Response A/B-barrel Domain Protein HS1 Gene From Mulberry

Mulberry is not only an important food plant of silkworms,but also the important material basis of silk industry.In addition to beening picken leaves for silkworms,mulberry has high edible,medicinal,feeding and ecological value.In the process of growth and development,mulberry plants are often infected by various pathogens,which will seriously affect the development of sericulture and the realization of mulberry ecological and economic values.Explore the disease resistant genes and cultivate new mulberry varieties using genetic engineering technology will contribute to realize the economic and ecological values of mulberry trees.As a kind of defense response gene,the stress response A/B-barrel domain protein HS1 gene is closely related to systemic acquired resistance of plants,but its specific biological activity and resistance mechanism remain largely unknown.Based on the obtained transcriptome information,Mul-HS1 gene was cloned and its biological function was studied.Meanwhile,the promoter of Mul-HS1 gene was amplified and its induction activity was analyzed,and the expression profile and regulation of Mul-HS1 gene was discussed.The results provided a candidate gene for mulberry disease resistance molecular breeding and also laid a foundation for the study of the function and regulatory mechanism of stress response A/ B-barrel domain protein HS1 gene.The main results of this study are as follows:(1)Functions of stress response A/B-barrel domain protein HS1 gene Mul-HS1In this study,the stress response A/B-barrel domain protein HS1 gene Mul-HS1 was successfully cloned using PCR.The Mul-HS1 gene encodes a 110 amino acid protein which is a hydrophobic protein with more hydrophobic amino acids and charged amino acids.Its theoretical molecular weight is 12.5 kDa and its isoelectric point is 5.45.It was found that the secondary structure of Mul-HS1 protein was rich in α-helix and irregular curl,followed by a small number of extension fragments and β-folding,which together constituted the α/β barrel domain.The protein has no signal peptide and transmembrane structure,but it has multiple phosphorylation sites,four N-glycosylation sites and one O-glycosylation site.Mul-HS1 protein has higher amino acid sequence consistency with HS1 proteins of other plants.The predicted results of the interaction proteins of Mul-HS1 protein showed that Mul-HS1 protein interacts with multiple stress response related proteins.The Mul-HS1 gene was inserted into the expression vector pBI121,and the plant expression vector pBI121-Mul-HS1 was successfully constructed andused to transforme Arabidopsis,and the transgenic plants were successfully obtained.It was found that transgenic Mul-HS1 Arabidopsis had enchanced resistance to the pathogens Pst.DC3000,Botrytis cinerea and Phytophthora capsici,which indicated that the Mul-HS1 gene may play important roles in improving the resistance of plants to these pathogens.(2)Analysis of the tissue expression characteristics of Mul-HS1 gene and subcellular localization of Mul-HS1 proteinThe expression profile of Mul-HS1 in mulberry various tissues were analyzed by fluorescence quantitative PCR with the cDNA extracted from the stems,leaves,male flowers,female flowers,unripe and ripe mulberry fruits as templates,respectively.The results showed that the expression of Mul-HS1 was the highest in male flower,followed by that in stem,ripe mulberry fruits and female flowers,and the expression level of Mul-HS1 in the leaves and unripe mulberry fruits was relatively low.The Mul-HS1 was inserted into an expression vector pROKⅡcontaining GFP,and the expression vector containing the Mul-HS1 gene fused with GFP was constructed for transformation into tobacco epidermal cells by using agrobacterium-mediated method.Then the transgenic tobacco epidermal cells were observed through fluorescence microscopy and the results showed the Mul-HS1 localizated in cell cytoplasm and nucleus.(3)Cloning and functional analysis of the promoter pMul-HS1The promoter sequence of Mul-HS1 was cloned successfully and designed as pMul-HS1.The sequence analysis of pMul-HS1 with online promoter analysis tool showed that the sequence contains some core elements of promoter and a variety of environmental factor response elements,indicating that Mul-HS1 may be involeved in the response process of plants to various stresss through different signal pathways.The expression activity of promoter pMul-HS1 was analyzed by transient expression system of tobacco leaves and GUS histochemical staining,and the results showed that pMul-HS1 had significant SA,JA,ABA,light,low temperature and pathogen induced activities.