Cloning And Functional Study Of Calcium-dependent Protein Kinase (MaCDPK1) From Morus Atropurpurea Roxb.

As the main feed source of the silkworm,mulberry is widely planted nationwide,which is of great significance to the development of the silkworm industry.Besides,some studies have shown that mulberry trees have good adaptability in saline-alkali land,dry land,and even flood and waterlogged areas,and little research has been done on the ecological value.Therefore,research on their related stress resistance genes and molecular mechanisms are particularly important.To adapt to the environment,the mulberry tree has formed a set of defense mechanisms.This process involves stress signaling,the transmission of related second messengers,and the participation of related protein kinases.Calcium-dependent protein kinases（CDPKs）,as important calcium sensors in plants,are widely involved in regulating plant growth and development,pathogen defense responses,and signal transmission processes of biological or abiotic stress.Currently,CDPKs are reported in various plants,such as wheat and rice,but it has rarely been reported on the mechanism of mulberry stress resistance.In this study,Morus atropurpurea Roxb was selected as the research object to explore the resistance function of its calcium-dependent protein kinase MaCDPK1.The research results are as follows:1.Through molecular biology experimental techniques,the full-length CDS sequence of MaCDPK1 was cloned from leaves of Morus atropurpurea Roxb,and bioinformatics analysis was performed showed that the sequence encodes 573 amino acids with an isoelectric point of 5.36.The phylogenesis analysis showed that it mainly cluster with Mn CDPK1,At CDPK1,and At CDPK2 in Group I.MaCDPK1 has a classic kinase domain and an EF-hand domain,and a hydrophobic domain appears in the central sequence.There is no signal peptide,and there are 23 serine phosphorylation site and 12 threonine phosphorylation sites.2.The recombinant prokaryotic expression vector p ET28a-MaCDPK1 of MaCDPK1protein was constructed to express the recombinant protein MaCDPK1,and the mutant expression vector p ET28a-mMaCDPK1 was constructed by PCR site-directed mutation method to ensure The 234th aspartic acid in the amino acid sequence of MaCDPK1 was mutated to alanine to express the mutant recombinant protein mMaCDPK1.The E.coli BL21（DE3）strain was used for prokaryotic expression of them.The MaCDPK1 and mMaCDPK1 were obtained after purifing by affinity chromatography.The kinase activity test showed that MaCDPK1 has complete enzymatic activity and exhibits Mg²⁺and Ca²⁺dependence,Km is 30μM,and Vmax is 100,000[RLU]/min/μg.However,mMaCDPK1does not show kinase activity,which means that aspartic acid at position 234 is essential for enzymatic activity of Ma DPK1.3.The 1,500 bp upstream of the start codon of the MaCDPK1 gene was cloned and used as a promoter sequence for cis-acting element analysis.The results showed that the sequence contains core promoter elements TAAT-box and CAAT-box,and contains components related to stress signals such as drought and abscisic acid（ABA）.Analysis of the transcription level of the MaCDPK1 gene in different tissues（drought,salinity and ABA）of mulberry under different stress conditions by q RT-PCR showed a complex expression pattern,which was significantly different under different stresses,implying that MaCDPK1 gene showed different functions to different stress signals.It shows the basic characteristics of MaCDPK1 in Morus atropurpurea Roxb,and provides a theoretical basis for its anti-stress function.This report laid the foundation for the improvement of mulberry genetic engineering.