BpZK-2 Transiently Generated Betula Platyphylla Suk.optimization And Phase Separation Of Disordered Regions

Betula platyphylla Suk.is the main afforestation tree species in north-east of China.It has the characteristics of fast growth,good ecological benefits,and is widely used in carbon sink afforestation and pulp and paper afforestation.At present,the completion of Betula platyphylla Suk.genome sequencing,the development of gene editing and transgenic breeding technology have provided new ideas for the breeding of Betula platyphylla Suk.Through the transient transformation technology,we can take the lead in conducting molecular biology research on Betula platyphylla Suk.,and express the target gene in the receptor in a short time,laying a solid foundation for obtaining excellent plants through transgenic methods in the future.The establishment of an efficient receptor system can promote the smooth development of transgenic research.As the main undertaker of life activities,protein is the executor of gene function.The relationship between protein structure and function is generally considered to be"The amino acid sequence determines the protein structure,and the protein structure determines the biological function".However,in recent years,a class of proteins which lack a three-dimensional stable structure under physiological conditions but have normal functions and are involved in various biological processes such as transcriptional regulation,signal transduction,and stress response have been continuously discovered.Such proteins are called"intrinsically disordered proteins（IDPs）"and play important roles in protein interactions and protein-nucleic acid molecule interactions.BpZK-2 is an intrinsically disordered protein,but the function and mechanism of BpZK-2 are still unclear.In this study,by researching the ratio of plant hormones in the process of callus induction,subculture,induction of adventitious buds,and rooting of adventitious buds,I optimize the regeneration system of Betula platyphylla Suk.tissue culture.On this basis,I investigate the effects of bacterial concentration,infection time and acetosyringone addition on the expression rate of BpZK-2 transiently transformed Betula platyphylla Suk.tissue culture plants;the effects of cellulase concentration,enzymatic hydrolysis time and mannitol concentration on the the effects of Betula platyphylla Suk.protoplast extraction;the effects of PEG species,PEG concentration and Betula platyphylla Suk.protoplast source on the expression rate of Betula platyphylla Suk.protoplasts transiently transformed by BpZK-2.In this study,BpZK-2 protein purified in vitro with His-tag was obtained by prokaryotic expression and protein purification of BpZK-2.On this basis,I investigate the effects of PEG concentration,temperature and BpZK-2 protein concentration on the"liquid-liquid phase separation"phenomenon of BpZK-2 protein.The specific research results are as follows:（1）Optimized Betula platyphylla Suk.tissue regeneration culture system:The isolated leaves of Betula platyphylla Suk.were cultured on MS solid medium supplemented with 1mg/L 6-BA+0.1 mg/L NAA,and the callus induction rate was the highest,reaching 81.67%;Tissue cultured on MS solid medium supplemented with 0.5 mg/L 6-BA+0.1 mg/L NAA had the highest growth rate,reaching 68.33%;callus was cultured on MS solid medium supplemented with 0.5 mg/L 6-BA On the other hand,the budding rate was the highest,reaching 90%;when the Betula platyphylla Suk.shoots were cultured on MS solid medium supplemented with 0.5 mg/L 6-BA+0.25 mg/L NAA,the rooting rate of adventitious shoots was the highest,reaching 88.89%.（2）BpZK-2 cloning and bioinformatics analysis:Sequence analysis of the BpZK-2 showed that the coding region of the BpZK-2 is 1842 bp,encoding a total of 614 amino acids;the BpZK-2 protein produced by translation contains six Zn F＿C2HC domains,indicating that BpZK-2 protein is a zinc finger protein.Since proteins with zinc finger structure can bind to DNA or RNA,BpZK-2 protein is involved in the regulation of gene expression.（3）The transient transformation system of Betula platyphylla Suk.tissue culture plants was optimized and the transient transformation of BpZK-2 was successfully carried out:the agrobacterium tumefaciens EHA105 strain carrying the p BI121-BpZK-2-EGFP plant expression vector was cultured to OD₆₀₀=0.4,and the cells were grown for 30 minutes.Day-old Betula platyphylla Suk.tissue culture plants were immersed in Agrobacterium solution,and after 30 minutes of infection,they were cultured in the dark for 48 hours in Betula platyphylla Suk.transient transformation medium.After optimization,the instantaneous conversion rate can reach 43.33%.（4）The transient transformation system of Betula platyphylla Suk.protoplasts was established and the transient transformation of BpZK-2 was successfully carried out:using the enzymatic hydrolysate with cellulase concentration of 0.9%and mannitol concentration of 0.4mol/L to enzymatically hydrolyze Betula platyphylla Suk.tissue culture grown for 30 days In the leaves of the strain,the protoplast concentration could reach 2.87×10⁵ ml^-1 after 4 hours,and the activity could reach 85.04%.Under the mediation of 40%PEG4000,the p BI121-BpZK-2-EGFP plasmid was transformed into protoplasts,and then transferred to Betula platyphylla Suk.transient transformation medium for 48 hours of dark culture,and the transient transformation rate reached 35.54%.（5）pGEX-6×His-BpZK-2 recombinant expression plasmid and protein purification:The recombinant expression plasmid pGEX-6×His-BpZK-2 was constructed and expressed in prokaryotic E.coli BL21（DE3）;nickel column affinity layer was used the BpZK-2recombinant protein containing the 6×His tag was purified by analytical technology,and the concentration of the obtained recombinant protein was about 3.5～3.7 mg/m L.（6）pGEX-6×His-BpZK-2 protein forms"liquid-liquid phase separation"in vitro:pGEX-6×His-BpZK-2 protein contains inherent disordered regions,and the total number of disordered amino acids is 479,accounting for 78.01%.The purified pGEX-6×His-BpZK-2 protein can form"liquid-liquid phase separation"in vitro,and its degree becomes more and more obvious with the increase of PEG8000 concentration,temperature and protein concentration.In this study,I optimize the tissue culture regeneration system of Betula platyphylla Suk.and the transient transformation system of Betula platyphylla Suk.tissue culture plants,and establishe the transient transformation system of Betula platyphylla Suk.protoplasts,and obtaine BpZK-2 transiently transformed plants and protoplasts successfully;find the pGEX-6×His-BpZK-2 protein was purified in vitro and observe the phenomenon of"liquid-liquid phase separation"happened.These may lead the foundation for the future functional analysis of BpZK-2.