Verification And Prokaryotic Expression Of Kenaf Full-Length And Partial-Length HcPDIL5-2a Gene Interaction Protein

Kenaf(Hibiscus cannabinus L.)is an annual herbaceous cash crop.It is widely used in the textile industry,furniture manufacturing and construction industries.With the continuous innovation of technology,its use value is constantly improving.At this stage,research on kenaf male sterility has made great progress,but the molecular mechanism leading to kenaf male sterility has not yet been elucidated.We still need to continue to study in depth.In the previous study of this group,the partial-length Hc PDIL5-2a gene was transformed into kenaf by the pollen tube channel method to obtain a low temperature sensitive male sterile mutant "THMS".DNA J/Hc Hsp40 and Ca M 7 were screened for proteins that interact with the products encoded by full-length kenaf Hc PDIL5-2a and partial-length Hc PDIL5-2a.We want to verify the interaction of related proteins and further explore the functions related to the full-length and partial-length Hc PDIL5-2a gene.In order to provide more scientific basis for the molecular mechanism of kenaf male infertility.In this study,20 vectors were constructed for subcellular localization,yeast two-hybridization,two-molecule fluorescence complementation,and prokaryotic expression through enzyme cleavage ligation and Infusion homologous recombination.The following research results were obtained:(1)Bioinformatics analysis of kenaf full-length Hc PDIL5-2a and partial-length Hc PDIL5-2a was carried out through online software.The results show that the two encoded proteins are both hydrophilic and stable.The secondary and tertiary structures of the two proteins are mainly composed of random coils and α-helix.The molecular weight of the protein encoded by full-length Hc PDIL5-2a is about 48.8 k Da.The molecular weight of the protein encoded by partial-length Hc PDIL5-2a is about 36.1k Da.(2)The kenaf 722 B anther c DNA was used as a template to clone the full-length Hc PDIL5-2a and partial-length Hc PDIL5-2a genes.The subcellular localization recombinant vector was constructed and transformed into kenaf root tip protoplasts.Laser confocal microscopy observation showed that the full-length Hc PDIL5-2a and the partial-length Hc PDIL5-2a genes were located in the nucleus.(3)The full-length Hc PDIL5-2a,the partial-length Hc PDIL5-2a,Dna J/Hc Hsp40 and Ca M 7 were used as recombinant carriers for bait and prey proteins.Through yeast two-hybrid reverse complementarity verification,it was found that there is an interaction between the full-length Hc PDIL5-2a encoded product and Dna J/Hc Hsp40 and Ca M 7 proteins.There is an interaction between partial-length Hc PDIL5-2a encoded product and Dna J/Hc Hsp40 and Ca M 7 proteins.(4)The full-length Hc PDIL5-2a,the partial-length Hc PDIL5-2a,Dna J/Hc Hsp40 and Ca M 7 were C,N-terminal bimolecular fluorescent complementary recombinant vectors were successfully constructed.Through bimolecular fluorescence complementary technology,it was verified that there is an interaction between the full-length Hc PDIL5-2a encoded product and Dna J/Hc Hsp40 and Ca M 7 proteins and there is an interaction between the partial-length Hc PDIL5-2a encoded product and Dna J/Hc Hsp40 and Ca M 7 proteins.It is speculated that the Hc PDIL5-2a encoded product interacts with Dna J/Hsp40 and Ca M 7.In turn,it causes male sterility in plants transformed with partial-length Hc PDIL5-2a such as kenaf and rice.(5)Prokaryotic expression vectors of full-length Hc PDIL5-2a and partial-length Hc PDIL5-2a were constructed.Through the prokaryotic expression system,a protein of the same molecular weight as the protein encoded by the expected partial-length Hc PDIL5-2a was induced.It has laid a material foundation for the subsequent research of related proteins.