Cloning , Expression And Functional Study Of Gmpm13 Gene In Glycine Max

Caleosin is classified as a calcium regulatory protein because of its EF-hand domain,which plays a very important role in plant development and stress response.In the current study,the researchers found that caleosin can participate in the anchorage combination of monolayer phospholipid layer and oil bodies,which plays a crucial role in increasing the stability of seed oil body.Caleosin contains an N-terminal domain with a single Ca²⁺-binding EF hand motif,and its C-terminal domain contains phosphorylation sites,speculating that caleosin may be involved in signal transduction pathways regulated by calcium ions and has an inevitable relationship with drought stress in plants.In this study,the Gmpm13 gene was cloned from the soybean of Jiyu No.72,and its expression in field experimental materials,drought stress and prokaryotic expression was analyzed.It provides scientific basis for the application of soybean Caleosin in plant genetic engineering,biotechnology and biochemistry.The main results are as follows:1?The soybean Gmpm13 gene was cloned by RT-PCR and a full-length 885 bp cDNA sequence was obtained,which including a 720 bp ORF and?open reading frame?encoding 239amino acids.And the gene sequences were analyzed by online software,to determine that the Gmpm13 gene is was distributed on chromosome 10.The theory of isoelectric point is was 5.15,and there is was no homologous genes and no signal peptide.There is an EF-handed domain at59-228 amino acids,a caleosin domain at 63-92 amino acids,and a transmembrane domain at92-115 amino acids.The full-length 738 bp caleosin was obtained by designing a primer with restriction enzyme sites,which encoded a protein size of 27.1kDa.In addition,it was connected with the prokaryotic expression vector pET-28a,named pET-28a-pm13,and its prokaryotic expression system was optimized.The results showed that the maximum protein expression was reached after the induction at 28?at 1.5 mmol/L IPTG for 12h,accounting for 39.25%in the total bacterial proteins.2?The expression level of Gmpm13 gene in different growth stages?seedling stage,branching stage,flowering stage,pod stage,mature stage?and different organs?roots,leaves,seeds?of the field was determined by qPCR.It was found that with the change of growth period,the expression levels of genes in roots and leaves showed a trend of rising first and then descending.The the highest at podding stage were 0.0631 and 0.3294;the level of gene expression inseeds gradually increased,reaching the maximum value of 0.0378 during the maturation period.Determination of Free Amino Acids and Soluble Proteins were determined by Ultraviolet Spectrophotometry.It was found that the content of free amino acids in the root and leaves showed a trend of rising first and then descending with the change of growth period,and reached the highest at the end of pods,which were 38.3867?g/g and 19.4343mg/g respectively.The variation trend of soluble protein content was the same as that of free amino acids,with the highest value of 0.0358mg/g and 0.0524mg/g respectively.The content of free amino acids and soluble protein in seeds gradually increased.The content of soluble protein in seeds gradually increased,reaching the highest level of 0.8763 mg/g during the ripening period,and the free amino acid content increased first and then decreased.The highest value was 54.3827?g/g.3?The relative expression levels of the Gmpm13 gene in seedling soybean leaves treated with drought stress?The concentrations of PEG6000 were 0,5%,10%,15%,20%and 25%,and the was stress was 1 to 6 days respectively?were measured.It was found that with the increase of stress time and concentration,the gene expression level showed a trend of first increase and then decrease,when the concentration of PEG6000 was 20%for 4 days,reached reaching the maximum value was 17.248.The activity of antioxidant enzymes?SDO,POD,CAT?and the content of membrane peroxide product?MDA?were measured by colorimetry method and UV spectrophotometry.It was found that with the increase of stress time and stress concentration,the activity of POD,CAT and SOD showed a trend of rise decrease after fall firstincrease.The activities of SOD and POD reached the maximum at 3 days when the concentration of stress was 15%,which was 7329.62u/mg?protein?and 2191.43u/?g·min?.The activity of CAT reached the maximum at 4 days when the concentration of stress was 15%,which was 148.347 u/?g·min?.The MDA content increased gradually,and reached the highest value of 116.775 in 6 days at the stress concentration of 25%.The content of osmo-regulatory substances?Pro?were determined by using the Sulfosalicylic Acid method.The results showed that the Pro content gradually increased with time,and then increased first and then decreased with increasing concentration.When the stress concentration was 15%,the maximum value was 316.602ug/g.