Cloning And Functional Analysis Of CmCRY1 And CmCRY2 In Chrysanthemum Morifolium

Chrysanthemum morifolium,one of the traditionally famous flowers in China and one of the four best-known cut flowers worldwide,was an important cash crop used for ornamental purposes and also in tea and medicine.C.morifolium is a typical short-day flowering plant,and its flowering is mainly regulated by the photoperiod pathway.Cryptochrome(CRY)is one of the three photoreceptors in plants,it can sense blue and near-ultraviolet light signals in the external environment.In different stage of plant growth,especially in photoperiod flowering pathway,CRYs plays important regulatory role.The functions of CRYs were well studied in the long-day model plant Arabidopsis thaliana,and the similar regulation roles were also proved in the short-day model plant rice.In Chrysanthemum lavandulifolium,another plant of the genus of chrysanthemum,CRY has proved regulated flowering time by interactive with circadian clock genes and regulated the expression of COL gene.However,in Chrysanthemum,how CRYs responds to photoperiod,and how they participated into the complex molecular regulatory network of flowering time regulation,is still not very clear.In this study,four CRY genes in early-flowering variety ’Fen Xuanqiu’(C92)and late-flowering variety ’Fen Lian’(C30),were cloned and their expression patern and function were invested.The main results are as follows:1.Cloning and analysis of CRY1 and CRY2 genes in chrysanthemum variety ’Fen Lian’ and Ten Xuanqiu’(1)Four CRY genes were isolated from leaves of’Fenlian’ and ’Fenxuanqiu’ using nest-PCR,and named them as C30-CmCRYl,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 respectively.CRY1 and CRY2 genes are highly conservative between the two varieties:C30-CmCRY1 and C92-CmCRY1 are 2108 bp in length,and the lengths of their open reading frame(ORF)are 2025 bp,encoding 674 Amino acids.The relative molecular weight of the protein encoded by C30-CmCRY1 is 76799.29 Da,and the theoretical isoelectric point is 5.21;the relative molecular weight of the protein encoded by C92-CmCRY1 gene is 76767.23 Da,and the theoretical isoelectric point is 5.21.C30-CmCRY2 and C92-CmCRY2 genes are 1839 bp in length and encode 612 amino acids;the relative molecular weight of the protein encoded by C30-CmCRY2 is 69692.18 Da;the theoretical isoelectric point is 5.44,and the relative molecular weight of the protein encoded by C92-CmCRY2 is 69790.33 Da;Theoretical isoelectric point is 5.95.(2)Phylogenetic tree of different species based on CRY protein was constructed,and CRY1 and CRY2 proteins from different plants are perfectly divided into two branches.In CYR1 branch C30-CmCRY1 and C92-CmCRY1 colsely clustered together with other CRY1 proteins from compositeae plants such as Artemisia annua,Chrysanthemum boreale,and then together with other plants;In CRY2 branch C30-CmCRY2 and C92-CmCRY2 aslo firstly had a convergence with other Compositae plants such as Helianthus annuus and Artemisia annua,and then groupded together with CRY2 from other plants.2.Analysis of Expression Patterns of CmCRY1 and CmCRY2 Genes in Chrysanthemum morifolium(1)The expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 in different organs of chrysanthemum was detected,and the results showed that CmCRY1 and CmCRY2 are expressed in Chrysanthemum roots,stems and leaves,which indicated that they are constitutive expression genes.The expression level of CmCRY1 and CmCRY2 in leaves were significantly higher than that in roots and stems,indicated they may mainly functions in leaves.This is consistent with the conclusion that leaf is the main light receptor in plants.(2)The expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 at different stage within a single short-day light cycle(24 h)was examined,and all of the four CRY genes showed certain periodic changes,the expression of CmCRY2 differs greatly between the two varieties,compared with CmCRY2,the CmCRY1 gene shows stronger rhythm in both varieties.(3)To detect the expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 in the leaves of different periods(1,4,7,10,13,16 and 19 d)of continuous short day treatment,the results showed that the expression patterns of CRYs were very similar in the same variety.Two expression peaks in the expression curve of C30-CmCRY1 and C30-CmCRY2 and appears at 4 d and 13 d under short day respectively.Expression peaks of C92-CmCRY1 and C92-CmCRY2 appear at 16th d of short day treatment.3.Function studies of CmCRY1 and CmCRY2 genes in Chrysanthemum morifoliumOver expression vectors of CmCRY1 and CmCRY2 from early-flowering Chrysanthemum variety’Fenlian’(C30)and late-flowering Chrysanthemum variety ’Fenxuanqiu’(C92)were successfully constructed respectively and named as pMD-C85-C30-CmCRY1,pMD-C85-C92-CmCRY1,pMD-C85-C30-CmCRY2 and pMD-C85-C92-CmCRY2.The CYRs were transformed into Arabidopsis thaliana by Agrobacterium-mediated method.The transgenic plants were selected by Hygromycin B,and CmCRYs were proved expression at transcription level by RT-PCR method.The phenotype analysis of the transgenic plants showed that all the CmCRYs of Chrysanthemum can promote flowering in Arabidopsis.However,the transgenic plants of CRYs isolated from early-flowering Chrysanthemum variety ’Fenlian’(C30)showed much ealer flowering than that with CRYs from late-flowering Chrysanthemum indicated that CRYs are one of the reasons lead to different flowering time between’Fenlian’ and ’Fenxuanqiu’.