Cloning And Expression Of The Ctcaleosin5 Gene From Safflower And Its Effects On Oil Content And Body Size

Carthamus（Carthamus tinctorius L.）,a plant of the genus Carthamus in the asteraceae family.As a new oil crop,Carthamus tinctorius has been widely used in medicine,food and industry,and the plant with the highest content of linoleic acid.As a new type of bioreactor,vegetable oil body expression system has become a means of producing target products.Oleosin,Caleosin and Sterolosin on the surface of the oil body containing a single layer of phospholipid membrane with TAG,can be fused with the foreign target proteins embedded in the oil body protein and the N-terminal or C-terminal of the oil body calcium protein.In this way,medical protein,industrial and agricultural enzymes,animal feed additives,etc.In plant seeds,oils are stored in endosperm,cotyledon and other tissues in the form of oil bodies,and tags stored in oil bodies provide energy for seed germination and plant growth.Under environmental stress,calcium signals are generated in plant cells,and calcium receptor proteins can recognize and transduce calcium signals,causing downstream gene changes and making plants produce stress resistance.Caleosin contains an EF-hand motif that conserved with a single calcium ion.As a calcium receptor protein,the mechanism of action of Caleosin in Ca²⁺signal transduction and stress response remains to be analyzed.In this study,Ctcaleosin5 was selected as a candidate protein by bioinformatics prediction and expression analysis of Ctcaleosin protein family in Safflower.Arabidopsis thaliana and Camelina sativa were obtained by genetic engineering.The lipid changes and oil body size of Arabidopsis thaliana,Arabidopsis thaliana,Arabidopsis thaliana overexpressed,Arabidopsis thaliana and Camelina sativa treated with abiotic stress were compared to explore the regulation effect of Ctcaleosin5 gene on oil synthesis and oil body size.On the one hand,it provides ideas for further study of the role of Ctcaleosin under abiotic stress,on the other hand,it provides support for further study of the mechanism of oil body synthesis.Specific research results are as follows:1.Seven Ctcaleosin genes were screened based on the genome database of Safflower and named as Ctcaleosin1-Ctcaleosin7.The molecular weight of Ctcaleosin proteins ranged from 9.22 k Da to 27.73 k Da by analyzing the physicochemical properties of Ctcaleosin proteins.The theoretical isoelectric point（p I）is between 4.84and 10.03,and the average hydrophilic value（GRAVY）is between-0.576 and-0.226.Both are negative.Through the analysis of the promoter of Ctcaleosin gene family,it was found that the promoter contains biological stress,abiotic stress and hormone type cis-regulatory elements.The phylogenetic tree was constructed by homologous alignment of Ctcaleosin protein sequences with those of castor bean,soybean,brassica napus,Arabidopsis thaliana and sunflower,and it was found that Caleosin was highly conserved among species.2.Quantitative fluorescence（qRT-PCR）was used to detect the expression levels of Ctcaleosin gene family in the roots,stems,leaves and seeds of Safflower.The results showed that the expression levels of Ctcaleosin3 and Ctcaleosin4 were higher in the roots,and the expression levels of Ctcaleosin4 were the highest in the stems.The expression level of Ctcaleosin4 in leaves was the highest,and the expression level of Ctcaleosin5 in seeds was the highest.In this study,Ctcaleosin5 was selected as the target protein for bioinformatics prediction.Ctcaleosin5 is a hydrophilic protein.The secondary structure of Ctcaleosin5 is mainly composed ofα-helix,β-angle and random curling structural elements,and the tertiary structure is mainly composed ofα-helix and random curling.The domain of Ctcaleosin5 protein was predicted,and it was found to contain calcium binding domain,which has the ability to bind with calcium ions.3.Wild-type Safflower seeds were treated with drought,temperature,ababolic acid（ABA）,methyl jasmonate（MeJA）and salt stress to detect the expression of Ctcaleosin5 gene in seeds under abiotic stress,and to determine the grain size and oil content of safflower seeds.The response of Ctcaleosin5 gene was most significant in seeds treated with MeJA.4.Plant expression vectors pOTB-Ctcaleosin5 and pTB-Ctcaleosin5 were constructed and infected with wild-type Arabidopsis thaliana and Camelina sativa by pollen tube channel method.After screening and identification,T3 generation overexpressed transgenic Arabidopsis thaliana and Camelina sativa were obtained.5.Compared with Arabidopsis Thaliana of wild type and Arabidopsis thaliana overexpressing Ctcaleosin5 gene,Arabidopsis thaliana transferred into pOTB-Ctcaleosin5 and pTB-Ctcaleosin5 had larger leaves,higher seed 100-grain weight,1000-grain weight,surface area and germination rate.Compared with wild type Arabidopsis thaliana,the oil content and TAG content increased,and the grain size of overexpressed Arabidopsis thaliana decreased.6.The characters of wild-type Camelina sativa were compared with those that overexpressed Ctcaleosin5 gene.Compared with the wild-type Camelina sativa,the pOTB-Ctcaleosin5 and pTB-Ctcaleosin5 transgenic Camelina sativa had larger leaves,stronger rhizome,100 seed weight,1000 seed weight,surface area and germination rate.Compared with the wild type,the oil content and TAG content were increased,and the particle size of overexpressed Camelina sativa was smaller than that of wild type.The seed germination and growth of wild type Camelina sativa treated with MeJA were inhibited.The seed length,surface area,100 grain weight and 1000 grain weight of pOTB-Ctcaleosin5 and pTB-Ctcaleosin5 overexpressed Camelina sativa were greater than those of wild type Camelina sativa.Meanwhile,the oil content and TAG content of overexpressed Camelina sativa were increased.These results indicated that Ctcaleosin5 gene could maintain seed growth and development in response to MeJA stress.