Establishment Of Transient Expression System And Identification,Analysis And Functional Study Of AP2/ERF Gene Family From Caragana Intermedia

Caragana intermedia is a perennial and deciduous shrub of Caragana Fabr,Leguminosae,which has broad ecological adaptability and strong resistance to abiotic stress such as severe cold,drought,salinity and inferiority.In this study,the transient expression system of C.intermedia was established and the types and concentration of the surfactants,as well as Agrobacterium strains used in this system were optimized.Moreover,the AP2/ERF transcription factor family genes were screened from transcriptome sequencing database of C.intermedia,and the phylogenetic relationships,physico-chemical properties,protein structure and expression pattern were analyzed at length.Two genes encoding AP2/ERF transcription factor were isolated to investigate the function in abiotic stress tolerance in Arabidopsis thaliana and C.intermedia.What's more,one gene encoding CiABR17 was cloned based on the dehydration-induced suppressive subtractive hybridizations(SSH)cDNA library,and its function was characterized in A.thaliana.The following are the detailed research results:1.The Agrobacterium-mediated transient expression system of C.intermedia was developed for the first time,and the system was optimized as well:the optimal surfactant was Silwet L-77,the optimal concentration was 0.001%,and the optimal A.tumefaciens strain was GV3101.2.A total of 22 AP2/ERF transcription factors with complete open reading frame(ORF)were identified based on the transcriptome database of C.intermedia,which including 3 of DREB A1 group,2 of DREB A2 group,2 of DREB A4 group,4 of DREB A5 group,1 of DREB A6,2 of ERF B1 group,7 of ERF B3 group and 1 of RAV group.3.The expression patterns of all AP2/ERF transcription factors were detected by quantitative real-time PCR(qRT-PCR),and the results showed that most of them showed tissue-specific expression,and all of them could also be induced by cold,heat,salt and mannitol stress treatments at different degree.4.The cDNA of CiDREBIC gene in the AP2/ERF transcription factor family was cloned.The length of cDNA sequence of CiDREB1C was 1437 bp and the ORF was 609 bp,which encodes 203 amino acids.The CiDREB1C has the highest homology with MtERF021(Medtr1g060910)of Medicago truncatula(the similarity was 64.2%),and AtERF030(At4g25470)of A.thaliana(the similarity was 43%).The pCanG-CiDREB1C-HA plant heterologous expression vector was constructed and the function of transgenic A.thaliana was studies.It was found that heterologous expression of CiDREB1C reduced the seed germination rate of the transgenic A.thaliana,slowed growth and development of the transgenic A.thaliana,enhanced the tolerance of osmotic and cold stress,and promoted capacity to withstand dark-induced leaf senescence.Furthermore,transient expression of CiDREB1C enhanced tolerance of the transgenic C.intermedia to drought,salt and ABA treatments.5.The cDNA of CiDREB3 gene in the AP2/ERF transcription factor family was also cloned.The length of cDNA sequence of CiDREB3 was 903 bp and the ORF was 666 bp,which encodes 222 amino acids.The CiDREB3 has the highest homology with MtERF025(Medtr2g101340)of M.truncatula(the similarity was 61.1%),and AtERF041(At5g11590)of A.thaliana(the similarity was 48%).The pCan G-CiDREB3-HA plant expression vector was constructed and the function of transgenic A.thaliana was studied.The results suggested that heterologous expression of CiDREB3 reduced the seed germination rate,slowed growth and development of the transgenic A.thaliana,and improved its tolerance of drought stress.6.The EST sequence of CiABR17 gene was screened from the SSH cDNA library and the complete ORF sequence of CiABR17 was amplified by RACE(rapid amplification of cDNA ends).The length of CiABR17 sequence was 594 bp and the ORF was 471 bp,which encodes 157 amino acids.CiABR17 shows a high similarity with ABR17 in M.truncatula,Cicer arietinum and Pisum sativum.The transcription of CiABR17 could be differently induced by salt and heat stress,especially by dehydration.The fresh weight,dry weight and rosette diameter of CiABR17 over-expressed A.thaliana lines were significantly increased when compared with the wild type.