Cloning And Functional Analysis Of The Promoters Of The Floral Development Regulating Genes CyfaAG1 And CyfaAG2 From Cymbidium Faberi Rolfe

Cymbidium faberi Rolfe is a Chinese traditional flower with elegant leaves and attractive fragrance.As a potted plant with the strongest fragrance,C.faberi Rolfe has been cultivated for centuries.Many famous varieties with various flower traits including their color,number and the form of the perianth have been widely planted for appreciation as orchid culture.Flowers are one of the most ornamental organs of garden plants.Exploration of the molecular mechanism of flower organ morphogenesis is benefit to improve atrractive traits of flower-phenotype and develop novel cultivars.Previous studies suggested that function of AGAMOUS(AG)homologs in regulating pistil and stamen development are highly conserved,and sequence,expression pattern or function changes of AG homologs resulted in double flowers.Promoter contains important cis-acting elements regulating gene expression.In this study,CyfaAG1 and CyfaAG2 promoters were isolated to explore the function of CyfaAG1/2 gene promoter regulating the floral organ morphogenesis of C.faberi Rolfe.Activity analysis of truncated deletion promoters and the expression pattern of the both corresponding gene of C.faberi Rolfe were measured during floral bud differentiation.Our results provide potential clue for biotechnical engineering to create novel floral phenotypes and germplasm in ornamental cultivars of C.faberi Rolfe.The main results are as follows:(1)Cloning and structural analysis of CyfaAG1/2 genes promoter in C.faberi Rolfe.The 1392 bpCyfaAG1 promoter(pCyfaAG1I)(-1207/+185)and a 1660 bpCyfaAG2 promoter(pCyfaAG2)(-1473/+187)were isolated by using genome walking mehtod,respectively.Further prediction of cis-acting elements in promoter region suggested that both pCyfaAG1 and pCyfaAG2 contain CAr G-box(MADS-box transcription factor specific binding site)and AACAAA-/TTTGTTmotif(AP2-like transcription factor specific binding site),which are usually located at promoter region of AG homologous genes.Meanwhile,pCyfaAG1 and pCyfaAG2 contain multiple anther specific expression elements,such as POLLEN1LELAT52 and GTGANTG10,suggesting that the both promoters may drive corresponding genes to regulate the anther cap and gynandrium development of C.faberi Rolfe.Furthermore,the recognition and binding sites of CONSTANS transcription factors that promote flowering is only found in pCyfaAG1 region,suggesting that pCyfaAG1 may drive CyfaAG1 to promote flowering of C.faberi Rolfe.(2)Expression activity analysis of pCyfaAG1/2The both promoters pCyfaAG1(-1207/+185)and pCyfaAG2(-1473/+187)can drive the GUS gene to express in transgenic Arabidopsis inflorescences,sepals,filaments,pistils of flower buds at the stage 12,as well as sepals,anthers,filaments,top of pistils,and stigmatic papillae of matute flower,indicating that the promoters pCyfaAG1 and pCyfaAG2 have certain redundancy in regulating gene expression,and both promoters can regulate genes involved in flower organ development.The GUS gene was not expressed in the anthers of the early flower buds,but the expression activity of the GUS gene was higher in the anthers of the mature flowers.According to the different expression zones and expression activities of the GUS gene driven by different deletions in transgenic Arabidopsis,combined with the cis-regulatory elements in the promoter,it is speculated that the core functional region of the promoter pCyfaAG1 is-1203 ～+185bp,and the core functional region of the promoter pCyfaAG2 is-1058 ～ +185bp.(3)Expression pattern analysis of CyfaAG1/2Tissue-specific expression analysis indicated that CyfaAG1 was mainly expressed in the roots,young leaves,anther cap,gynostemium and ovary of C.faberi Rolfe,but no transcription signal was detected in sepals,petals and lips.In addition,gynandrium has the highest expression level of CyfaAG1,which was significantly higher than that of the other tissues(LSD,P<0.05),and the expression level followed by that in anther cap.While CyfaAG2 was mainly expressed in anther cap,gynandrium,and ovary of C.faberi Rolfe,but not in other tissues.The anther cap has the highest expression level of CyfaAG2,which was significantly higher than that in other tissues(LSD,P<0.05),and this expression level followed by that in gynandrium.The expression analysis of both genes in the same tissue showed that the expression level of CyfaAG2 was significantly higher than that of CyfaAG1 in anther cap or gynandrium(LSD,P<0.05).We further explored the expression change of these two genes during flower bud differentiation.The results showed that CyfaAG1 and CyfaAG2 were obviously expressed in flower buds with the differentiation of flower buds and the development of anther cap of C.faberi Rolfe in August(S1stage).With the temperature decreased in November,flower buds began seasonal dormancy(S2stage),and the expression levels of these two genes decreased to the lowest level.When the temperature arose at the end of February,the flower buds broke dormancy and sprouted again.And the anther microspore mother cells began meiosis(S3 stage),their expression level increased significantly.The expression of CyfaAG1 peaked at the mononuclear microspore stage(S6 stage),and maintained a high expression level until the formation of 2-cell mature pollen(OF)in the microspore tetrad on flowering day.While the expression level of CyfaAG2 was the highest when2-cell mature pollen was formed in the microspore tetrad on flowering day,and the second highest when the microspore mother cell started meiosis(S3 stage).The expression difference analysis of the two genes in the same period showed that the expression level of CyfaAG2 was significantly higher than that of CyfaAG1 at the beginning of meiosis(S3 stage)and on the day of flowering(LSD,P<0.05).In conclusion,the functions of pCyfaAG1 and pCyfaAG2 regulating corresponding genes involved in flower development of C.faberi Rolfe showed overlap and difference.Both CyfaAG1 and CyfaAG2 are involved in the development of anther cap,gynandrium and ovary in C.faberi Rolfe.However,CyfaAG1 is mainly involved in the gynandrium development of C.faberi Rolfe and determines the differentiation of flower buds,while CyfaAG2 is mainly involved in the anther caps development of C.faberi Rolfe.