Analyses Of WRKY Family And Its Responses To Abiotic Stress In Ginkgo Biloba L.

Ginkgo biloba L.is one of the world famous relic species which has high application values such as leaf use,timber use,oramental value,medicinal value and so on.G.biloba prefers a warm and cool climatic environment,and abiotic stresses such as high temperature,salt and drought are important limiting factors in cultivation.The WRKY proteins is one of the largest transcription factor superfamilies in plant,playing important regulatory roles in responses to abiotic stresses.To further explore the effect of WRKY genes in G.biloba under abiotic stress,we identified WRKY gene family members in G.biloba and analyze the characteristics of family members.Based on the above,we investigated expression pattern of WRKY gene in G.biloba under different abiotic stresses thoroughly,and selected the candidate genes induced by multiple abiotic stresses.These candidate genes were then being cloned and constructed overexpression vector for subcellular localization analysis and genetic transformation of poplar to explore the functions of these genes under abiotic stress.The main results were as follows:(1)A total of 37 WRKY transcription factors were identified on G.biloba genome using the Hidden Markov Mode.These genes were named as GbWRKY1?GbWRKY37 according to their position on the chromosomes.The chromosomal localization results showed that 36 ginkgo WRKY genes were unevenly distributed on 9 chromosomes,and 1 gene(Gb WRKY3 7)was located in scaffold 1055.Gene structure analysis showed that the length of GbWRKY genes ranged from 474 bp to 3123 bp,and the number of exons ranged from 2 to 17.Based on the analysis of the replication gene events among the members of G.biloba WRKY family,it was found that 20 genes were distributed replication,8 genes were tandem replication,8 genes were proximal replication,and 1 gene was singleton replication.The phylogenetic tree showed that the WRKY gene family number of G.biloba was similar to that of P.abies,which was significantly lower than that of A.thaliana and O.sativa WRKY gene family.(2)Based on NCBI annotation information,GO and KEGG enrichment of potential target genes of G.biloba WRKY family were analyzed.GO enrichment found that these genes were mainly concentrated in the functions of cellular components,cellular functions,catalytic activities and binding functions of molecular functions,as well as metabolic processes and cellular processes involved in biological processes.In addition,385 target genes were enriched to stress response and 83 target genes were enriched to signal transduction.A total of 20 pathways were identified by KEGG pathway analysis,among which the main enrichment pathways were plant hormone signal transduction,purine metabolism,Mrna monitoring pathway,biosynthesis of phenylalanine,tyrosine and tryptophan,and photosynthesis.(3)The expression of WRKY genes in G.biloba was significantly different in different tissues.Most of the WRKY genes of G.biloba had higher expression abundance in roots.Additionally,there were also significant differences in organizational expression in each branch.The expression abundance of Group I gene in each tissue was generally higher than that of other branches while the expression abundance of Group ?c gene in each tissue was generally lower than that of other branches.The quantitative PCR results were consistent with transcriptome data.(4)The expression of WRKY genes in G.biloba under different stresses indicated that multiple genes of the WRKY family could respond to abiotic stress.Under drought treatment,7 differentially expressed WRKY genes were screened,of which 4 were down-regulated and 3 were up-regulated.5 differentially expressed genes were screened under salt treatment,of which 1 was up-regulated and the remaining 4 were down-regulated.Under high temperature treatment,16 differentially expressed genes were screened,of which 3 were up-regulated and the rest 13 were down-regulated.Under Uv-B treatment,7 differentially expressed genes were screened,among which 3 were up-regulated and 4 were down-regulated.The results of quantitative PCR further confirmed the expression trend of these genes in abiotic stress.(5)37 family members were compared with WRKY genes related to known abiotic stress genes.The results showed that Gb WRKY15,GbWRKY18 and GbWRKY37 were closely related to extreme temperature.In addition,Gb WRKY15 and GbWRKY13 were closely related to salt and drought stress while the whole WRKY family of G.biloba had a relatively distant relationship with known genes related to Uv-B stress.Combined with transcriptomic data and fluorescence quantitative results,we speculated that GbWRKY6 and Gb WRKY23 genes in G.biloba WRKY family play an important role in responding to Uv-B stress,and Gb WRKY13,GbWRKY15,GbWRKY18 and GbWRKY37 genes played key roles in responding to abiotic stress such as extreme temperature,salt and drought stress.(6)We further carried out stress treatment experiments to explore the expression patterns of identified key genes.The results showed that GbWRKY6 and GbWRKY23 could significantly respond to Uv-B treatment,and the expression of GbWRKY6 was significantly up-regulated,while that of Gb WRKY23 was significantly down-regulated at 3 h.In addition,GbWRKY13,GbWRKY15,GbWRKY18 and GbWRKY37 can rapidly respond to multiple abiotic stresses such as extreme temperature,drought and salt stress.The expression of Gb WRKYl3 was significantly down-regulated in both high and low temperature treatment for 1 h while up-regulated in mild drought and salt stress treatment for 2 h.The expression of GbWRKYl5 was significantly down-regulated under high temperature,low temperature and salt treatment while up-regulated under mild drough.The expressions of GbWRKYl8 and Gb WRKY37 were significantly down-regulated under high temperature treatment for 4h and low temperature treatment for 1 h,and significantly up-regulated under salt stress treatment for 2 h.(7)GbWRKY13,GbWRKY15,GbWRKY37 were successfully been cloned and constructed overexpression carriers.The results of subcellular localization showed that GbWRKYl3 and GbWRKY37 only had localization in the nucleus and both had strong fluorescence,while GbWRKYl5 had localization and very strong fluorescence both in the nucleus and cell membrane.After the transformation of poplar(84K),18,14 and 16 positive seedling lines were obtained respectively.Compared with the wild-type tissue culture seedlings cultured in the same time,there was no significant difference in the aboveground part of transgenic plants,but their roots were more developed,and the length and number of fibrous roots increased significantly.However,the resistance to abiotic stress of transgenic lines will be further studied.