Preliminary Analysis Of Functional Diversity Of RALF Peptide Family In Arabidopsis Thaliana

Small secreted peptides are gaining importance as signalling molecules in plants.The Rapid Alkalinization Factor(RALF),which exists as a gene family,is a kind of conservative peptide molecule in green plant.There are at least 35 RALF genes in model plant Arabidopsis thaliana.Previous studies show that certain members of RALF family,such as RALF1 and RALF 23 ?RALF4 and RALF19 function as the ligand of Cr RLK1 L members FERONIA(FER)and BUPS1/2 respectively,thus regulate cell elongation,plant immune response and double fertilization,RALF34 as the ligand of Cr RLK1 L member THESEUS1(THE1)participate in module fine-tunes lateral root initiation.However,whether other RALF members have biological activity and whether distinct RALF have varied functions are still unclear.Previous studies found that the members of the RALF family of peptides,some involved in the growth inhibition of plant cells,some involved in the double fertilization,at the same time,the members of the family of RALF also plays a very important role in plant immunity.To further verify the biological function of other RALFs,this work we want study the root growth inhibition,hypocotyl elongation inhibition,root hair inhibition,flg22-induced ROS release and MAPK phosphorylation level experiments to assays the influence of different RALF relative system,preliminary explore the regulation function in these areas.Based on this,we carried out a series of experiments.The specific research results of this study are as follows:(1)The coding sequence of 19 mature RALF peptides from RALF family members in Arabidopsis thaliana were successfully constructed into pET32 a prokaryotic expression vector,and Trx-6*His-S-RALFs recombinant proteins fusing Trx-6*His tag were efficiently expressed using E.coli prokaryotic expression system.The molecule of purified recombinant protein were consistent with the predicted protein massa molecular.(2)In this study,RALFs treatment of the main roots and root hairs of Arabidopsis thaliana WT seedlings showed that 19 RALF could inhibit the growth of wild type(Col-0)main roots,but the inhibition rate of main root growth was different;The root hair suppression experiment results show that different RALF can promote root hair growth,but the degree of promotion is different;(3)In this study,RALFs treatment of hypocotyls of Arabidopsis wild-type plant seedlings revealed that different RALFs were involved in functional differences in hypocotyl elongation inhibition,and RALF10 significantly promoted hypocotyl elongation.The rest inhibit the hypocotyl elongation;(4)ROS release and MAPK results showed that there were functional differences in MAPK phosphorylation induced by different RALF.RALF1,RALF3,RALF9,RALF10,RALF14,RALF18,RALF23,RALF30,RALF32,RALF33,RALF34 could rapidly induce MAPK phosphorylation.However,RALF17 had no significant effect on the phosphorylation of MAPK;further analysis of the release of ROS by 19 RALF on flg22,16 inhibited the release of ROS induced by flg22 to varying degrees,while RALF3,RLAF17,RLAF32 Promotes the release of ROS induced by flg22.In conclusion,the results of this study showed that different RALF have both functional redundancy and differences.This study complements and improves the understanding of the complexity and diversity of RALF functions.