Isolation And Identification Of EIL Gene Family And Its Involvement In Plant Immune Response In Cassava

Cassava（Manihot esculenta Crantz）is one of the world’s three major potato crops（potato,sweet potato and cassava）.It is rich in starch and can be ground to make bread.It is also an forage crop and industrial raw material.It is a major source of food and heat for an estimated 500million people in Asia,Africa and elsewhere.cassava bacteria blight（CBB）is the most serious and common disease of cassava.It is very important to study the disease-resistant genes and the molecular mechanism of bacteria blight response in cassava.Ethylene（ET）is an endogenous hormone that is involved not only in physiological processes but also in the immune response to biological and abiotic stress.ETHYLENE INSENSITIVE3-LIKE（EIL）gene family is an important positive regulator of ET signaling pathway.In ET signaling process,disease-related genes is activated and participates in plant immune response to improve plant resistance.The main results are as follows:1.In this experiment,full-length fragments of 6 members of the EILs gene family in cassava were firstly cloned and named MeEIL1,MeEIL2,MeEIL3,MeEIL4,MeEIL5 and MeEIL6.Expression profiles of MeEILs under the stress of Xanthomonas axonopodis pv.manihotis（Xam）were analyzed to screen MeEIL2 as candidate genes for further study.Subcellular localization analysis was performed by transient expression transposition of cassavas protoplasts and it was found that all MeEILs genes were localized on the nucleus.2.Through bioinformatics analysis,the promoter region of the MeEIL2 gene was analyzed for cis-acting elements and found to contain elements such as G-box,W box,and MYC.The induced protein was induced by prokaryotic expression,and the conditions for the induction and purification of MeEIL2 protein were explored,the protein was successfully induced and purified.3.According to the self-activation activity detection experiment,it was found that only MeEIL5 have no self-activation activity.Therefore,the candidate gene MeEIL5 was used as the bait plasmid for yeast two-hybrid screening of interaction proteins.After screening and sequencing,the screened interaction proteins were found in the NCBI database:Manihot esculenta protein SULFUR DEFICIENCY-INDUCED 1-like,Manihot esculenta ketol-acid reductoisomerase,chloroplastic-like,Manihot esculenta probable polygalacturonase.4.The role of MeEILs in cassava was analyzed by using transient overexpression and virus induced gene silencing（VIGS）in cassava.By transiently overexpressing in cassava MeEIL2 and MeEIL5 genes,it was found that the number of bacteria,hydrogen peroxide（H₂O₂）and superoxide anion（O₂^-）content accumulation caused a series of disease resistance reactions.The number of corpus callose deposition obviously than the control group,indicating that transient overexpression of MeEIL2 and MeEIL5 genes increased the resistance to Xam in cassava.In the VIGS system,when the expression of MeEIL2 gene was suppressed,it was found that the number of bacteria,H₂O₂and O₂^-content in the leaves was significantly less than the control group,and the number of callose deposition was less than the control group.This indicated that the resistance of cassava decreased when MeEIL2 gene expression was silenced.In summary,this study identified 6 cassava MeEILs genes,and the MeEIL2 and MeEIL5 genes were further analyzed through expression profile analysis and self-activation detection analysis under Xam stress.Exploring the protein induction and purification conditions of MeEIL2 gene.Screening of MeEIL5 gene interacting proteins.Analyzing the role of MeEILs gene in cassava immune response by transient overexpression system and VIGS system,it was proves that MeEILs gene can positively regulate the resistance to bacteria blight.Providing reference basis for elucidating disease-resistance related genes in cassava and molecular mechanism of response to bacteria blight.