Genome Wide Identification Of WRKYs From Caragana Korshinskii And Function Analysis Of Four Group Ⅱ Members

Caragana korshinskii belongs to Caragana leguminosae,distributed in desert and semi desert areas in northwest China,and has strong resistance and adaptability.WRKY transcription factor is one of the largest transcription factor families.It has a variety of biological functions in plants and plays an important role in plant stress response and development.However,there is little research on the WRKYs of C.korshinskii and its homologous species C.intermedia.On the basis of genome sequencing of C.korshinskii and transcriptome sequencing of its homologous species,this study analyzed the members of the WRKY gene family of C.korshinskii through bioinformatics methods,and preliminarily studied the functions of some of the WRKY genes.The specific results were as follows:1.A total of 86 WRKYs genes were identified from the C.korshinskii genome and C.intermedia transcriptome data,81 of which were mapped to eight chromatin regions,while the remaining five genes were not mapped to chromatin regions.These genes can be divided into 3 groups,including 16 members of group I,56 members of group II,and14 member of group III,among them,the number of group II members accounted for65%,and the evolutionary analysis of 28 species also showed that group II WRKYs had been dominant in number since its emergenced from terrestrial plants.This study focused on the analysis and functional research of the genes of this subgroup.2.RT-qPCR was used to detect expression patterns of selected four group II CiWRKYs.The results showed that CiWRKY17,CiWRKY48 and CiWRKY23 were expressed differently in different organs at different stages;CiWRKY17,CiWRKY36,CiWRKY48 and CiWRKY23 are induced by multiple phytohormones.3.Four CiWRKYs overexpression vectors 35S:HA-CiWRKY17、35S:HACiWRKY36、35S:HA-CiWRKY48 and 35S:CiWRKY2-GFP were constructed to study the function of transgenic Arabidopsis thaliana.CiWRKY17 played a regulatory role in senescence and other developmental pathways in A.thaliana.Under dark treatment,CiWRKY36 regulated the elongation of hypocotyl in A.thaliana.CiWRKY48 and CiWRKY23 maked plants short by affecting the development of in A.thaliana,and its homologous genes At WRKY48 and At WRKY23 also played a regulatory role in the development signal pathway.4.Proper reference genes are essential for accurate and reliable results of RT-qPCR analysis.In this study,ACT,GAPDH,EF1α,UBQ,TUA,CAP,TUB,TUB3,SKIP1,SKIP5-1,SKIP5-2 were selected as candidate reference genes from different tissues and organs of C.intermedia at different development stages.The stability of these genes was evaluated by using ge Norm,Norm Finder,Best Keeper and Ref Finder software.The results showed that EF1α was the best reference gene in all samples,SKIP1 is the best reference gene in the above ground part;SKIP5-2/SKIP5-1 were the most stable reference gene combinations in field samples;CAP/ACT were the most stable reference gene combination in greenhouse samples.