Cloning Of MsCrys Genes Of Magnolia Sieboldii K. Koch And Transformation In Arabidopsis Thaliana

Magnolia sieboldii K.Koch is a national three-level protected tree species with ornamental and medicinal value.Because its seeds have the characteristics of deep physiological dormancy,releasing the seed dormancy of Magnolia sieboldii has become an urgent problem to be solved in seedling raising.Seed dormancy and germination are jointly regulated by seed endogenous hormones and the external environment.Light,as the main environmental factor,plays a role in inhibiting germination in the process of seed germination of most plants.Blue light in natural light mainly inhibits the release of seed dormancy,Cryptochrome（CRY）,as a blue light receptor,makes seeds sense blue light and regulate downstream signals to increase the content of abscisic acid（ABA）in seeds,so as to inhibit seed germination.In order to explore the effects of different light quality on seed dormancy of Magnolia sieboldii,starting with the blue light receptor Cryptochrome protein Crys and referring to the transcriptome data of Magnolia sieboldii seed obtained by flux sequencing,two cryptochrome genes Ms Cry1 and Ms Cry2 were cloned from Magnolia sieboldii,and their structural characteristics,expression characteristics,regulation of seed endogenous hormones and functions in plants were analyzed and identified.The main results of this study are as follows:1.Ms Cry1 and Ms Cry2 sequences were successfully isolated,with a total length of 2103 bp and 1953bp,encoding 700 and 650 amino acids,respectively.Ms Cry1 and Ms Cry2 are two different genes of the crys gene family.The molecular formula of the protein encoded by Ms Cry1 gene is C₃₄₉₆H₅₃₅₃N₉₈₅O₁₀₅₀S₁₇,the relative molecular weight is 78.527 k D,and the p I is 5.44;The molecular formula of the protein encoded by Ms Cry2 gene is C₃₃₁₃H₅₀₇₉N₉₀₉O₉₆₉S₂₃,the relative molecular weight is 73.885 k D,and the p I is5.69.All are unstable proteins.2.Both Ms Cry1 and Ms Cry2 have the phr B superfamily domain,the PHR domain is the N-terminal conserved domain of the blue light receptor Cryptochrome gene,and Ms Cry1 contains the CCE domain.Subcellular localization prediction showed that Ms Cry1 and Ms Cry2 were most likely to be located in chloroplasts.The secondary structure prediction of the protein found that in Ms Cry1 protein,αHelix accounted for 40.29%,irregular curl accounted for 45.14%,and extended chain accounted for 9.86%,βCorner accounts for 4.71%;In the Ms Cry2 protein,αHelix,irregular curl,extended chain andβThe proportions of corners were 36.46%,48.46%,9.69%and 5.38%respectively.The similarity of the tertiary structure of Ms Cry1 and Ms Cry2 coding proteins with CRY1 protein c1u3c A and CRY2 protein c6k8k A as motifs was 69%and 74%respectively,and the confidence interval was 100%.3.Seed imbibition treatment of magnolia can activate Ms Cry1 and Ms Cry2.The expression of Ms Cry1 and Ms Cry2 fluctuated within 0-48 hours of imbibition.At 24 hours of imbibition,the expression of both genes reached the highest,in which the expression of Ms Cry1 was very significant,and the expression of Ms Cry2 was significant.4.Under different light conditions,the relative expression of Ms Cry1 and Ms Cry2 showed a trend of first decreasing and then increasing,and the relative expression was blue light>white light>red light.Therefore,Ms Cry1 and Ms Cry2 are blue light receptor genes in Magnolia sieboldii,which can sense the blue light signal in the environment.The expression of Ms NCED3,a gene related to ABA synthesis,showed an upward trend and the expression of Ms CYP707A1,an ABA metabolic gene,showed a downward trend,indicating that the content of ABA in the seeds increased under the condition of blue light.Therefore,it is speculated that the inhibition of mscrys gene on the seed germination of Magnolia sieboldii is to regulate the expression of ABA synthesis and metabolic genes in the seeds,increase the content of ABA in the seeds,and then regulate the seed dormancy.5.The Ms Crys-p CAMBIA1302 plant expression vector was constructed and transformed into Agrobacterium tumefaciens.The Ms Crys was transferred into Arabidopsis thaliana.The T3 generation transgenic seeds were obtained through resistance screening and molecular identification.The function of Ms Crys was verified.The germination rate of transgenic Arabidopsis thaliana seeds under blue light was measured.The germination rate of cry1 deletion mutant Arabidopsis thaliana was the highest,and the germination rate of mscrys overexpression plants was the lowest,The germination rates of wild-type Arabidopsis thaliana and cry1 deletion mutant Ms Cry1 restored plants were similar,indicating that the Cryptochrome gene Ms Crys of Magnolia sieboldii can specifically respond to blue light signal and reduce the germination rate of Arabidopsis thaliana seeds.