Functional Identification Of Soybean Insect Resistance-related Gene GmERF54 And Candidate Genes Of Wild Soybean Insect Resistance Locus QWS11

Soybean[Glycine Max（L.）Merr.]is an important grain and oil crop in the world,containing a lot of plant protein and fat that are beneficial to the human body.Soybean are usually negatively affected by a variety of insect pests during the entire growth period,resulting in a serious reduction in soybean yield and quality.The common cutworm[CCW,Spodoptera litura（Fabricius）]is one of the main leaf-eating insect pests in soybean production areas in southern China.The larvae enter the binge-eating period to eat soybean tissues violently,causing huge economic losses to the soybean industry during the outbreak.Although the application of chemical pesticides can reduce the harm of insect pests,it brings problems such as environmental pollution,food safety,and insect resistance.Therefore,discovering insect resistance genes and cultivating new varieties of crop insect resistance are of great significance for the sustainable development of agriculture.This study unearthed soybean resistance-related genes from the cultivated soybean transcriptome data and wild soybean natural population association analysis positioning interval,through CCW induced expression analysis,tissue expression analysis,bioinformatics analysis,promoter analysis,subcellular localization,yeast two-hybrid,soybean hairy root transformation system,CRISPR/Cas9 technology analyze and identify the function of candidate genes.The main research content and research results of this study are as follows:1.Ethylene-responsive transcription factor（ERF）plays a vital role in soybean molecular regulatory network in response to stress.In this study,a gene encoding soybean ERF transcription factor GmERF54 induced by CCW was screened from transcriptome data.After RT-PCR verification,it was found that this gene response mode in resistant and susceptible lines induced by CCW were different.In the resistant line（KF）,the gene expression level of GmERF54 at 1 h to 48 h is always do wn-regulated.In the susceptible line（NN 1138-2）,the gene expression level of GmERF54 at 1 h and 6 h didn’t change much,and down-regulated after 12 h to 48 h.After verification of CCW induced expression,the gene GmERF54 was cloned from the Qinyang soybean cDNA sample.The coding sequence is 1221 bp in length and encodes 406 amino acids.It is predicted that the protein contains an AP2 conserved protein domain and belongs to the soybean AP2/ERF transcription factor family member.Tissue expression analysis exhibited that GmERF54 had extremely high expression levels in KF root,pod,seed and NN 1138-2 root,and relatively low expression levels in stem,leave and flower of the two lines.GmERF54 is located in the nucleus.Agrobacterium rhizogenes K599-mediated soybean hairy roots overexpressing with this gene and RNA interference,the insect resistance of soybean hairy roots overexpressing the GmERF54 gene was reduced,while the insect resistance of hairy roots with RNA interference down-regulated gene expression was improved,indicating that GmERF54 negatively regulates soybean resistance to CCW.2.Transcription activation activity analysis showed that GmERF54 has transcription activation activity,and its transcription activation domain exists in the C-terminal 131 aa interval.Using yeast two-hybrid technology,four interaction proteins were screened from the soybean root cDNA library,including Glyma.02g087800,Glyma.01 g099700,Glyma.05g123500,Glyma.12g056300.After turning verification,GmERF54 interacted with the proteins encoded by the four genes.Nine interacting proteins were screened from soybean leaf cDNA library.The promoter sequence of GmERF54 gene was further cloned from resistant and susceptible lines.The analysis found that the promoter contained multiple cisresponse elements such as hormones,light,and adversity.The resistant line promoter had four more cis-response elements than the susceptible line,include AT-rich element,Box 4,ERE,and L-box.The hairy root GUS staining experiment confirmed that the response patterns of GmERF54 promoter induced by CCW,MeJA and IAA were different in resistant and susceptible lines.In order to use this gene to improve soybean resistance to insects,this study constructed a GmERF54 knockout vector based on CRISPR/Cas9 technology.3.Wild soybean[Glycine soja Sieb.＆Zucc.]is a wild relatives of cultivated soybean,which retains many types of excellent genetic variation lost during domestication of cultivated soybean,and is an ideal germplasm resource for mining resistance genes.In early,a natural population of wild soybeans was used to GWAS,and a locus qWS11 that was significantly associated with larval weight and resistance level was located on chromosome 11.This study conducted a functional analysis of 6 candidate genes in the LD interval of this locus.Through genetic transformation soybean hairy roots with overexpression and RNA interference and feed to CCW.The results showed that when feeding on GsRbohAl overexpression hairy roots for 4 d,the larval weight of CCW was significantly less than control group.Overexpression of Glysoja₀04805 gene expression level,the larval weight of CCW was significantly lower than the control group on the 3 d after feeding.Overexpression of Glysoja₀04823 gene expression level,the larval weight of experimental group was significantly higher than the control group on the 6 d after feeding.Indicating that GsRbohAl、Glysoja₀04805、Glysoja₀04823 has a regulation effect on wild soybean insect resistance.After overexpressing or RNA interfering with Glysoja₀04807,Glysoja₀04810,Glysoja₀04814 gene expression levels in soybean hairy roots,the larval weight of CCW didn’t show significant changes,indicating that they weren’t involved in regulating wild soybean resistance defense to CCW.