Expression Analysis Of PHYA And PHYB Homologous Gene In Chrysanthemum Lavandulifolium And C.Vestitum And ClPHYB Functional Verification

Chrysanthemum × morifolium Ramat.is a famous flower originating in China.Its economic value and output are always highest in the world flower industry.Flowering time is an important cultivation characteristic of ornamental plants and it is one of the key factors affecting the industrial production.Chrysanthemum is a typical short-day plant(SDP),and the natural flowering time is mainly concentrated in the autumn,which limits its annual production and gardening applications.So far,the annual production of chrysanthemum mainly utilizes its flowering characteristics in response to short-day exposure,and it is extremely energy-consuming to regulate flowering time by shading and filling light.Therefore,it is an urgent problem to research the mechanism responding to short-day flowering of chrysnathemum can help to solve the urgent problem so as to breed or cultivate new varieties that responds to photoperiod-induced flowering quickly.As an important class of photoreceptors,phytochrome is involved in the regulation of flowering time in higher plants through CO protein stability and FT transcription regulation in the photoperiod pathway.In terms of the integration of the six known flowering pathways,phytochrome is more important.Researches have shown that PHYTOCHROME(PHY)has the function of regulating flowering time in long-day plant Arabidopsis thaliana,and the gene family PHYA,PHYB,PHYC,PH YD and PHYE are independent each other and have functional redundancy.PHYA and PHYB are the earliest branch members of the gene family and are more conservative.In Arabidopsis,PHYA is mainly involved in the shade-avoiding response,while PHYB also plays a role in regulating flowering time,but its function in flowering regulation of short-day plants remains to be studied.In this study,C.lavandulifolium(diploid)and C.vestitum(hexaploid),which are related to the origin of chrysanthemum,were used as experimental materials,and the PHYA and PHYB genes were cloned based on the confirmation of their short-day flowering characteristics.Gene tissue specificity and response to photoperiod expression patterns were analyzed and promoters sequences were cloned and upstream regulatory elements were predicted.Finally,CIPHYB was transformed into wild-type and deletion mutant Arabidopsis and using Agrobacterium-mediated method.Transformation of wild-type and deletion mutants Arabidopsis thaliana and C.lavandulifolium to analyze its function.The main results are as follows:1.The PHYA and PHYB genes were cloned by the 3’ RACE technique based on transcriptome database,named CIPHYA/CIPHYB and CvPHYA/CvPHYB.Both CIPHYA and CvPHYA contain 3366 bp ORF encoding 1121 amino acid residues;CIPHYB and C.vPHYB contain 3394 bp and 3357 bp ORF,respectively.The sequence similarity between CIPHYA and CvPHYA was 99.82%,and the similarity between CIPHYB and CvPHYB was 78.36%.Phylogenetic analysis revealed that the protein was divided into two branches,the PHYA protein and the PHYB protein,and plants of similar families were clustered together.C.lavandulifoliun,C.seficuspe and C.vestitum are gathered together.2.The tissue specificity and photoperiod response characteristics of PHYA and PHYB gene expression in C.lavandulifolium and C.vestitum showed expression levels of ClPHYA/ClPHYB and CvPHYA/CvPHYB were the highest in leaves.ClPHYA/ClPHYB and CvPHYA CvPHYB gene expression were responded to short-day induction and he expression patterns were similar.With the prolongation of short daylight,the expression abundance of ClPHYA and CvPHYA increased gradually,and the expression peak appeared at 16-20 d after short daylight induction,then the expression abundance decreased.The expression abundance of ClPHYB and CvPHYB decreased firstly,and decreased at 12-16 d.It is speculated that the expression and function of this gene may be more conservative in the genus Chrysanthemum.3.The 5’-end 696 bp of the CIPHYA gene and 901 bp of the ClPHYB gene were cloned by chromosome walking.The predictive results of the promoter conserved elements indicated that the ClPHYA promoter includes multiple photoresponsive elements,such as Box 4,P-box,chs-CMA2a;ClPHYB promoter also includes multiple photoresponsive elements,such as ATCT-motif,Box Ⅱ,G-box,GA-motif,I-box,LAMP-element,chs-CMA2b,they also contain multiple hormone-related action elements and other homeopathic elements involved in the drought-induced MYB binding site MBS,It is indicated that the expression of ClPHYA and CIPHYB genes may be affected by photoperiod,development,gibberellin and plant hormones such as auxin,but there are significant differences in the number and type of cis-acting elements.4.In view of the fact that PHYB not only participates in the shade-avoiding reaction,but also plays different roles in regulating flowering time,and phylogenetic analysis showed that ClPHYB is closely related to Arabidopsis thaliana AtPHYB.In order to analyze ClPHYB gene function,pBI1 21m-C IPHYB overexpression vector was transformed into wild-type and PHYB mutant Arabidopsis thaliana and C.lavandulifolium hypocotyl using Agrobacterium-mediated method.Three independent 35S::ClPHYB/Col-0 and two independent 35S::ClPHYB/phyb T1 generation strains were obtained,but no successfully transformed C.lavandulifoliun strain.There was no significant difference in flowering time between Col-0,35S::ClPHYB/Col-0 and 35S::ClPHYB/phyb-1 under long daylight;35S::C1PHYB/Col-0 and wild-type under short daylight was delayed by 15 days while 35S::ClPHYB/phyb-1 had no significant difference in flowering time compared with Col-0.Gene expression analysis revealed that overexpression of ClPHYB down-regulated the expression levels of CO and FT.In addition,the number of 35S::ClPHYB/Col-0 transgenic Arabidopsis rosette leaves,leaf and petiole length reduction,plant dwarfing,leaf dark green,and epidermal hair density increased.In conclusion,the phytoehrome gene expression in the leaves of C.lavandulifolium and C.vestitum was induced by short-day exposure,and their expression patterns between ClPHYA/CIPHYB and CvPHYA/CvPHYB had certain similarities.Also,expression pattern between PHYA and PHYB showed negative correlation,it is speculated that it is more conservative among the short-day genus Chrysanthemum.The transgenic results showed that ClPHYB significantly delayed the flowering time of plants under short days and inhibited the shading response,but had little effect under long daylight.It was speculated that CIPHYB is an inhibitor of short-day flowering.In addition,the genetic transformation system of C.lavandulifolium may be unstable.This study preliminarily analyzed the gene function of CIPHYB and found CIPHYB is a potential target gene that improves the flowering stage by molecularly interfering with the light signal sensing process upstream of the photoperiod pathway.