Identification And Functional Analysis Of The Interacting Proteins Of JcMADS1 In Jatropha Curcas

As a perennial tropical oil crop belonging to the Euphorbiaceae family,Jatropha curcas has great potential in agriculture,the development of bioenergy and biomedicine due to its drought resistance,strong adaptability to the environment,and high seed oil content.However,strong vegetative growth,a small number of female flowers and low seed yield,greatly limited the production and application of Jatropha.This study is based on the previous study of JcMADS1 gene.Overexpression of JcMADS1 promote flowering in Jatropha.To further explore the mechanism of JcMADS1 regulating the flowering transition in Jatropha at the protein level.One member from our laboratory screened out JcSEUSS and JcASHR2 proteins interacting with JcMADS1 by yeast two hybrid.The strong interaction between JcASHR2 and JcMADS1 was verified by bimolecular fluorescence complementary(BiFC)and GST Pull-down.As for JcSEUSS protein,which has a weak interaction with JcMADS1.In addition,to verify the regions of interaction between JcMADS1 and JcASHR2,we constructed expression vectors with different fragment lengths based on the conserved domains of two proteins,and performed yeast two hybrid experiment with full-length interacting proteins.The results showed that JcMADS1 interacts with JcASHR2 only when MADS-box and K-box coexist,and the SET-domain of JcASHR2 has no effect on the interaction between two proteins.In order to further verify whether JcSEUSS and JcASHR2 directly participate in flower transformation process regulated by JcMADS1 in Jatropha.We used Quantitative Real-time PCR to analyze the expression patterns of JcSEUSS and JcASHR2.The results showed that JcSEUSS was expressed in various tissues in Jatropha,and the relative expression in roots and seeds was higher.JcASHR2 has the highest expression level in the roots,and the expression level in inflorescence buds is significantly higher than that of male and female flowers,and its expression trend in the flower development stage is consistent with JcMADS1.We cloned the JcSEUSS and JcASHR2 gene from cDNA library of Jatropha.Overexpression of JcSEUSS in Jatropha results in the anthocyanin content in young leaves and petioles was up-regulated.However,silencing the JcSEUSS gene using RNAi has no effect on the anthocyanins in the leaves and petioles of transgenic Jatropha compared with wild type.This is contrary to the results of JcMADS1 gene regulating anthocyanin content in leaves and petioles.In addition,overexpression of JcSEUSS also influences the shape of the male flowers and green fruits in Jatropha.The sepals extend outward,and the curling of petals was reduced.In addition,the longitudinal diameter of green fruits grew longer than that of wild type at the same growth period,while the transverse diameter did not change significantly.Furthermore,the number and size of seeds of overexpressed lines became smaller,while RNAi lines were just the opposite,indicating that the JcSEUSS gene may regulate the development of seeds in Jatropha.This subject is mainly dedicated to the identification and functional analysis of the JcMADSl interacting protein.It is found that JcSEUSS and JcMADS1 have opposite functions in regulating the anthocyanin synthesis of the leaf in Jatropha,which is a new function of the JcSEUSS gene that is different from the model plant SEUSS gene.These discoveries provide new clues to the functional research of SEUSS.JcSEUSS may also regulate the development of seeds in Jatropha,by changing the expression of this gene provides the possibility to increase the yield.