Cloning Of Auxin Response Factors CmARF2,3,4, From Chrysanthemum And CmARF3 Functional Identification

Chrysanthemum morifolium is perennial herb of the genus Chrysanthemum in the composite family.Chrysanthemum originated from China,is one of the top ten traditional famous flowers of China and four major cut flowers of the world.Due to its excellent ornamental traits,chrysanthemum has been grown worldwidely and become one of the four most popular cut flowers,ranks the second place in the consumption of fresh cut flowers,which determines its high economic value and important position in the modern flower industry.Chrysanthemum has complex flower types and multiple petal types,which are the important embodiment of its ornamental value.However,the molecular mechanism of chrysanthemum growth and development are still poorly understood.Auxin is one of the most important plant hormones,and are closely related to plant growth and development.Plant auxin signal transduction and mechanism of action has been a hot spot,in which auxin response factor(ARF)plays a key role in auxin signal transduction process.In present study,chrysanthemum CmARF2,CmARF3 and CmARF4 were cloned,and its expression characteristics,subcellular localization,transcriptional activity were attempted.We successfully generated the CmARF3 overexpressing chrysanthemum and illucidated its functions.The main findings are as follows:1.We cloned CmARF2,CmARF3 and CmARF4 genes from chrysanthemum.CmARF2 has total length of 2842bp,2364bp ORF encoding 787 amino acids,which shows 62.19%sequence identity with that from Sesamum indicum.CmARF3 has length of 2115 bp,1866bp ORF encoding 621 amino acids,the deduced amino acid sequence with 39.64%sequence identity with that from Theobroma cacao.CmARF4 has length of 3109bp,2322bp ORF encoding 773 amino acids,the deduced amino acid sequence with 50.24%sequence identity with that from Vitis vinifera.2.The Real-time PCR results show that the CmARF2,CmARF3 and CmARF4 genes are expressed at root,stem and leaf 'Jinba'.Expression level of from the highest to the lowest is in leaves,roots and stems.Expression level of CmARF3 from the highest to the lowest is in stems,leaves and roots.Expression level of CmARF4 from the highest to the lowest is in the order of stems,leaves and roots.3.We constructed the green fluorescent protein(GFP)fusion expression vector of pMDC43-GFP-CmARF2,pMDC43-GFP-CmARF3 and pMDC43-GFP-CmARF4,and transformed it to onion epidermal cells by bombardment.Subcellular localization analysis showed that CmARF2,CmARF3 and CmARF4 proteins were localized in the nucleus.4.Yeast expression vector of pGBKT7-CmARF2,pGBKT7-CmARF3 and pGBKT7-CmARF4 was generated,and transformed into yeast strain Y2H,which was selected in a single defect medium SD/-Trp and then filtered into the culture medium on double default SD/-His-Ade.The results showed that CmARF2,CmARF3 and CmARF4 were not of transcriptional activation activity.5.Plant overexpression vector pMDC32-CmARF3 was constructed,and transformed into chrysanthemums 'Jinba' using Agrobacteria mediated leaf disc infection.Hygromycin resistance filtering,PCR detection and expression level of CmARF3 by qRT-PCR were conducted.Finally,two OX-CmARF3 overexpressing lines were obtained.Studies on the obtained overexpression lines showed that CmARF3 had transcriptional inhibitory effects on CmIPT5 and CmWUS.CmARF3 gene mainly regulate adventitious bud regeneration,leaf length,angel between leaf and stem and plant height,but not root development.