Identification,expression Analyse Of ARF,AUX/IAA Family Genes,and Their Relationship With Rhythmic Genes In Longan(Dimocarpus Longan Lour)

Longan(Dimocarpus longan L.)is an important subtropical fruit tree of Sapindaceae family,which has great economic value.The flowering mechanism and the regulation are still as the one of difficulties and hotspots among fruit tree cultivation and breeding research.Flowering in plants is a complex process that requires the interaction and regulation of various hormones.Among them,auxin play an important role in regulating plant flower bud differentiation.Aux/IAA and ARF act as the important transcription factors involved in auxin signal transduction.In this study,two auxin response factors Dl ARF6 and Dl ARF11 and one gene Dl IAA1 acting as a suppressor of auxin induction were screened out by analyzing the transcriptome and the real-time quantitative q RT-PCR experiments;the interaction between transcription factor and these three genes were verified by yeast one-hybrid.Verified the activation effect of transcription factors on promoters Dl ARF11 and Dl IAA1 by establishing a dual-luciferase transient expression system;confirmed the interaction of transcription factors by establishing a yeast two-hybrid system;the main research contents of this study are as follows :1.The putative functions of ARF and IAA genes in longan have been identified by analyzing the bioinformatics of the ARF family and the AUX / IAA family.17 Dl ARF genes and 18 Dl IAA genes were identified,by analyzing the classification and evolutionary relationship,domain and protein structure of Dl ARF and Dl IAA have retained their conservation in evolution compared with other models such as Arabidopsis,but there are independent branches different from herbaceous plants in classification.Dl ARF13 is an independent branch,and Dl IAA2 also is an independent branch.By analyzing the expression of Dl ARF and Dl IAA Gene in longan organization,the results showed that D1ARF-1,-2,-6,-8,-9,-11,-16 may have the role in regulating the flower bud differentiation,and Dl IAA-1,-10,-15,-16,-17 show a highest expression in the flower bud;2.There are obvious changes on expression leves of Dl ARF2,Dl ARF6,Dl ARF11 and Dl IAA1 during physiological differentiation stage of flower bud differentiation and the stage of flower bud differentiation the morphological differentiation analyzed by real-time quantitative q RT-PCR experiments.It showed that these four genes might be involved in regulating the longan flower bud differentiation.Furthermore,according to the longan genome datebase,we cloned promoter fragments of Dl ARF6,Dl ARF11 and Dl IAA1 gene,which in length of 1672 bp,1849bp,and 1553 bp respectively.There were some cis-acting elements in the promoter sequences of these three genes,which related to the light response,low-temperature response of LTR,circadian biological rhythm regulation,and auxin response;3.Constructed one-yeast hybrid interaction system to study the interaction between Dl ARF6、Dl ARF11、Dl IAA1 promotor and Dl GI、Dl FKF1、Dl ELF4 genes.The test results show that promotor of Dl ARF6 does not interact with transcription factors,Dl ARF11 gene promotor interacts with Dl FKF1,and Dl IAA1 gene promotor interacts with Dl ELF4-1,Dl ELF4-2,Dl FKF1,and Dl GI.Therefore,it speculated that Dl ARF mainly participates as an upstream regulatory factor in the auxin synthesis transduction pathway,and AUX/IAA may act as a downstream gene,which involved in the auxin synthesis and transduction pathway of plants under the regulation of upstream transcription factors.The results of the transient expression experiments of double luciferase showed an obviously activation when Dl FKF1 acts on Dl ARF11 gene promotor,all gene showed a significant activation effect on Dl ARF11 gene promoter except Dl ELF4-1,especially,it show the highest activation when Dl ELF4-2and Dl GI worked together on Dl ARF11 gene promotor,we guess that the rhythm genes Dl ELF4-2 and Dl GI indirectly regulate the expression of the Dl ARF11 promoter by regulating other genes;for the auxin inhibitor Dl IAA1,when Dl ELF4-1and Dl FKF1 work together on Dl IAA1 gene promoter,there is a significant inhibitory effect.4.The results of yeast two-hybrid experiments showed that Dl GI interacted with Dl ELF4-2,which further verified that Dl GI and Dl ELF4-2 form a dimer of protein and activate the expression of Dl ARF11.Due to the self-activation of the bait vectors of Dl ELF4-1,Dl ELF4-2,and Dl FKF1,the interactions between Dl ELF4-1 and Dl ELF4-2,Dl ELF4-1 and Dl FKF1 need to be further verified.In this study,17 Dl ARF and 18 Dl IAA genes were identified,and initially screened four genes that may be involved in regulating longan flower bud differentiation(D1ARF-2,-6,-11,Dl IAA1).It’s beneficial to further study the relationships between auxin and the flowering pathway,and it will provide the basis for exploring the molecular mechanism of auxin and circadian genes in regulating longan flower bud differentiation according to the result of the interactions between the important genes involved in the auxin synthesis pathway Dl ARF6,Dl ARF11,Dl IAA1 gene promoters and flowering rhythm genes Dl ELF4-1,Dl ELF4-2,Dl FKF1,Dl GI.