Preliminary Study On The Function Of SikCDPK1 Gene In Response To Abiotic Stress

Calcium dependent protein kinase(CDPK)acts in concert with Ca²⁺to regulate plant growth and development and respond to various stresses.The EF-hand motif in its C-terminal regulatory region makes the activation of CDPK dependent on Ca²⁺key structure.Sasussured involucrata(Sasussured involucrata Kar.et Kir.),as a precious plant with strong stress resistance,is of great significance to the study of its stress resistance gene function and molecular mechanisms.So far,researches on the function of Saussurea tianshanensis SikCDPK is very limited,and there are few studies on the EF-hand motif of SikCDPK1 gene and its response to abiotic stress.Therefore,in this study,site-directed mutations(Ca²⁺binding sites)were performed on the N-terminal and C-terminal EF-hand motifs of SikCDPK1 by PCR site-directed mutagenesis technology.The E.coli expression system was used to induce and express in vitro proteins with different EF-hand motifs in SikCDPK1 to explore the solubility.The four recombinant proteins were purified and concentrated by using a nickel column affinity chromatography system combined with an ultrafiltration centrifuge tube,and then the Ca²⁺binding capacity and enzyme activity were tested.On the other hand,two different habitat plants SikCDPK1 gene and GhCDPK1 gene were genetically transformed at the same time to compare the growth and development phenotype differences of the T1 generation transgenic tobacco at the same growth period,and the stress treatment was performed at the same time,through the phenotypic difference and stress resistance comparative observation of related physiological indicators,functional analysis of SikCDPK1gene.The main results are as follows:1.Using PCR site-directed mutagenesis technology,the amino acids at positions 435,471,507,and 540 of the SikCDPK1 gene were respectively changed from E(glutamic acid)to Q(glutamine)by site-directed mutations,and successfully constructed the prokaryotic expression vector p ET32a-SikCDPK1-N-EF,p ET32a-SikCDPK1-C-EF,p ET32a-SikCDPK1-EF_N+C,p ET32a-SikCDPK1 for site-directed mutation of EF-hand motif at different positions of SikCDPK1 gene.Bioinformatics analysis found that mutations at these sites will make The spatial conformation of the protein changes.The expression strain was transformed,and the four recombinant strains were able to successfully express the target recombinant protein through IPTG induction,and the soluble expression of the recombinant protein was better induced and cultured under 18?1 mmol/L IPTG induction for 16 h.After purification by nickel column affinity chromatography and concentration by ultrafiltration centrifuge tube,four recombinant proteins with good purity were obtained,and there were differences in electrophoretic mobility,indicating that the obtained recombinant protein had changed in spatial conformation.2.The Ca²⁺binding capacity and enzyme activity of the obtained recombinant protein were tested.The results showed that the electrophoretic mobility of SikCDPK1 and SikCDPK1-N-EF became faster after Ca²⁺treatment,but the change of SikCDPK1 was more obvious.In contrast,SikCDPK1-C-EF and SikCDPK1-EF_N+C showed no significant changes in electrophoretic mobility before and after treatment.It shows that after the mutation of SikCDPK1,its Ca²⁺binding ability has changed.The detection of kinase activity showed that there was no significant change in kinase activity in the presence of EGTA.In the presence of Ca²⁺,the activity of SikCDPK1 protein kinase was released.As time passed,its activity gradually disappeared,and the mutant protein SikCDPK1.The enzyme activities of N-EF and SikCDPK1-C-EF both changed to different degrees,and the mutant protein SikCDPK1-EF_N+Conly changed significantly after 5 minutes of treatment.The above results indicate that the EF-hand motifs at different positions have different effects on the spatial conformation and enzyme activity of SikCDPK1.The C-terminal EF-hand can bind to Ca²⁺to affect the spatial conformation,while the N-terminal EF-hand has a greater influence on the kinase activity.3.The SikCDPK1 gene and the GhCDPK1 gene were genetically transformed at the same time.After phenotypic comparison,it was found that there were obvious phenotypic differences between the two transgenic tobacco plants and the wild-type tobacco plants.Transgenic tobacco plants with SikCDPK1 gene are taller,with more leaves and wide oval leaves,shorter leaf lengths and thinner stems.The plant heights of GhCDPK1 transgenic tobacco plants and wild-type tobacco plants are significantly lower than those of transgenic tobacco plants with SikCDPK1 gene.GhCDPK1 transgenic tobacco plants have fewer leaves but larger leaf area,oblong shape and thick stems.Stomatal observation revealed that the number of stomata in the leaves of the two transgenic tobacco plants per unit area was significantly higher than that of the wild-type tobacco plants.4.The two transgenic tobacco plants in the same growth period were subjected to drought and low temperature stress at the same time,and it was found that the GhCDPK1gene transgenic tobacco plants had stronger drought resistance than the SikCDPK1 gene transgenic tobacco plants,and the SikCDPK1 gene transgenic tobacco plants had stronger cold resistance than the GhCDPK1 gene transgenic tobacco plants.Tobacco plants quickly recovered and showed signs of flowering after rewatering under drought stress.Although the two transgenic tobacco plants have certain differences in resisting drought and low temperature stress,their cold resistance and drought resistance are significantly stronger than wild-type tobacco plants.The test results of related physiological indicators of stress resistance show that transgenic tobacco plants can reduce cell membrane damage under adversity stress,and a large increase in osmotic adjustment substances is used to maintain the intracellular osmotic balance to further improve its stress resistance.