Cloning And Preliminary Functional Analysis Of Cytokinin Response Regulator Family Genes In Chinese Hickory (Carya Cathayensis Sarg.) During Grafting

Chinese hickory(Carya cathayensis Sarg.)is not only a woody oil tree species belonged to Carya in Juglandaceae(Juglandales)which is distributed in Zhejiang and Anhui provinces of China,but also an important economic species which is selected for ‘one mu of mountain can make ten thousand yuan’project in Zhejiang province because of special flavour and high nutritive value in its fruits.However,several issues in seedings of Chinese hickory,such as long juvenile stage,hard to harvest because of large tree height,low levels of improvement have restricted the development of Chinese hickory industry.As an important way of horticulture technique,grafting is an effective way to solve the issues in Chinese hickory industry.Many phytohormones involved in successful reunion process between the rootstocks and scions,among which cytokinin plays an important role in wound healing and tissue redifferentiation.In cytokinin signaling pathway,response regulators functions in receiving the cytokinin signal from upstream pathway,activating the transcription of downstream functional genes and regulating the network of phytohormone.However,the functions of Response Regulator genes are unknown in the successful grafting process of Chinese hickory.In this study,using Chinese hickory as the main material,CcRR family genes were identified and cloned;the features of CcRRs were analyzed;the dynamic changes of trans zeatin riboside and the expression patterns of CcRRs were measured during grafting;the promoters of CcRRs were cloned,the cis-acting elements on CcRRs promoters were detected and the expression changes of CcRRs in response to phytohormones were determined;finally,the preliminary functions of CcRRs were explored in terms of subcellular localizations and overexpression in Arabidopsis.The main results are as follows:(1)The full CDS sequences of 17 CcRR genes were cloned,including 8 type-A,8 type-B and 1type-C RRs,all of which encoded hydropathicity proteins.All CcRR subfamilies contained the typical receiver domain,while type-B CcRR subfamily also contained MYB-like DNA binding domain(expect for CcRR11).(2)During grafting of Chinese hickory,the content of trans zeatin riboside,the main form of endogenous cytokinin,displayed the trend of increasing 3 days after grafting,maintaining the same level between 3 days 7 days of grafting,decreasing 14 days after grafting,and recovering to normal 30 days after grafting.Expression analysis of CcRRs by transcriptome indicated that compared with the data of grafting after 0 day,more than 50% of the Type-A RRs and Type-B RRs were identified as differentially expressed genes(DEGs)after 7 and 14 days of grafting in rootstocks and scions(except for 14 days after grafting in scions),while the proportions of DEGs ranged from 0 to 4.35% in rootstocks and scions compare 7 with 14 days of grafting.CcRR2,CcRR4,CcRR10,CcRR22 had a relatively higher expression in pericarp,embryo,root,testa,respectively,while CcRR5 and CcRR11 had low expression in shoot.Verification of expression changes during grafting indicated that,compared with the corresponding expression levels of 0 d after grafting,12 CcRRs were up-regulated in scions 3 d after grafting;6 and 11 CcRRs were up-regulated in rootstocks and scions 7 d after grafting,respectively;9 and 4 CcRRs were up-regulated in rootstocks and scions 14 d after grafting,respectively;the expression levels of 9 CcRRs recovered to normal(the levels of 0 d after grafting).Across the whole grafting process,CcRR5 maintained high levels of expression;the expressions of CcRR4 and CcRR22 were restrained;while CcRR8 displayed high expression levels 7 d and 14 day after grafting.(3)A number of phytohormone-related cis-acting elements,including auxin,CTK,GA,ABA,ETH,SA,Me JA related elements were detected on the promoters of CcRRs.Expressions of CcRRs were induced by different kinds of phytohormones to varying degrees.For example,the relative expression of CcRR4,whose promoter contained auxin-responsive cis-element,was up-regulated after IAA treatment.(4)Type-A CcRRs were sub-localized in nucleus or nucleus and cytomembrane;type-B CcRRs were sub-localized in nucleus;Type-C CcRR was sub-localized in nucleus and cytomembrane.3,15,7,4,16 transgenic Arabidopsis homozygote lines were identified for CcRR1,CcRR5,CcRR10,CcRR12 a,CcRR12 b,respectively.