Cloning And Functional Analysis Of The Auxin-Induced Gene CgPCNT115 In Citrus Grandis(L.) Osbeck. Cv. 'Hongmianmiyou'

Citrus grandis?L.?Osbeck.cv.‘Hongmianmiyou'is mutated from‘Guanximiyou'which shows a light red spongy layer.Poncirus trifoliata has been widely used as the rootstock of‘Guanximiyou',whereas‘Hongmianmiyou'is incompatible with available P.trifoliata rootstock.Differential gene analysis based on the transcriptome data shows that CgPCNT115 gene may play an important role in seedling etiolation.Although auxin induced protein PCNT115 has been identified in many plants,the related functions and mechanisms remain unclear.In this study,we cloned and analyzed the sequences of CgPCNT115 gene and promoter in Citrus grandis cv.'Hongmianmiyou'and'Guanximiyou'.The expression characteristics of CgPCNT115 during leaf development stages and different tissues among graft combinations were analyzed by real-time PCR.The response characteristics of CgPCNT115 gene in leaves were induced by different concentrations of IAA and TIBA treatment.Furthermore,its function was studied by transient overexpression in the blade.The main results are listed as follows.?i?Cloning of CgPCNT115 gene The CgPCNT115 gene and its promoter were cloned from the gDNA of'Hongmianmiyou'and'Guanximiyou'.The nucleotide of full length and the promoter sequences are 1772 and 2122 bp,respectively.There are some single-base variations in gene sequences,mainly located in the intron region.Nucleotide alignment showed that two promoter sequences are completely consistent and have a transcriptional initiation site and core promoter region in the promoter sequence,which contains some special cis-acting elements,such as thermal stress response elements,hormone response elements,MYB binding element,etc.Genetic structure analysis showed that CgPCNT115 gene belongs to the aldehyde ketone reductase family?AKRs?,including two transmembrane structural regions.CgPCNT115 exhibited a close relationship with CsPCNT115,followed by CcPCNT115 by alignment and phylogenetic analyses.?ii?Expression characteristics of CgPCNT115 Gene expression analysis showed that CgPCNT115 revealed a rise trend during leaf development stages among different graft combinations.In the Phase?,CgPCNT115 gene expression quantity was significantly lower than two control checks.As in the tissues,the most abundant transcripts were detected in flower,followed by stem segment and leaf.?iii?Response Characteristics of CgPCNT115 50,100 and 150 mg·L^-11 of exogenous IAA and TIBA were treated on the young leaves of'Hongmianmiyou'.Semi-quantitative and real-time quantitative PCR showed that the expression of CgPCNT115 has a rapid response process within 8 hours,revealing an increase and then a decrease change.The expression of CgPCNT115 most sensitively responsed by 50 mg·L-1 of IAA and TIBA treatment,with the faster response by IAA than TIBA treatment.Therefore,CgPCNT115 has a sharply up-regulated expression for IAA and TIBA treatment within short time,which shows a sensitive response mechanism to changes in IAA content.Compared with IAA treatment,the response is later by TIBA treatment.?iv?Gene function of CgPCNT115 The leaves of'Hongmianmiyou'started to etiolate after five days by transient overexpression of CgPCNT115 gene.Both total chlorophyll and carotenoid contents decreased significantly at five days after injection.The endogenous hormones,GA₃,was significantly higher than the control group from three to seven days after injection.The expression levels of key genes,CgARF,CgGH3 and CgSAUR involved in IAA response and signal transduction were also determined,which were significantly down-regulated except for CgAUX/IAA.All of them were?extremely?significantly negatively correlated.Thus,CgPCNT115 gene may have positive regulatory effects on chlorophyll and carotenoid content and GA₃ synthesis,while have negative regulatory effects on the IAA response pathway.The above results indicated that CgPCNT115 promoter has hormone response elements,and CgPCNT115 gene may play an important role in the hormone response process.Based on the response characteristics of CgPCNT115 to exogenous IAA and TIBA treatments,CgPCNT115 may play an important role in IAA response and signal transduction pathway.The overexpression on C.grandis leaves showed that CgPCNT115 gene may participate in the response and signal transduction of IAA and the interaction between IAA and GA₃.As a result,we suggest that CgPCNT115 might promote IAA content and affect graft compatibility.