Comprehensive Profiling Of Tubby-like Proteins In Soybean And Functional Identification Of GmTLP8 Gene In Response To Drought And Salt Stresses

Soybean（Glycine max L.）is an important oil economic crop in China.It is widely planted in China and even the world,and is the main source of high quality protein and oil.However,soybean is very sensitive to abiotic stresses such as drought and high salinity,which seriously affect its yield.Therefore,it is of great significance to explore soybean resistance candidate genes,analyze their mechanism and apply it to genetic improvement,it is of great significance to improve soybean resistance and yield and promote industrial development.Tubby-like proteins（TLPs）are transcription factors that are widely present in eukaryotes and generally participate in growth and developmental processes.In this study,the GmTLPs gene family was comprehensively identified,including phylogenetic analysis,chromosome location,gene structure analysis,cis-element analysis,tissue expression analysis and so on.Based on the results of expression pattern analysis,GmTLP8 gene was selected and transferred into soybean hairy roots for stress resistance identification.The main research results are as follows:1.Identification of GmTLP family members and genetic analysis.22 GmTLP genes were found in soybean gene annotation files by HMMER 3.0 module using Hidden Markov Model.Phylogenetic tree were constructed using Arabidopsis,maize,rice,wheat,tomato,apple,poplar,cotton and newly identified soybean TLP family members.According to the homology of proteins,GmTLP family members were divided into five groups（I-V）.According to chromosome localization,GmTLP genes were named as GmTLP1 to GmTLP22.Gene structure showed that each GmTLP member contained introns and exons.Protein domain analysis revealed that GmTLPs included two key domains,F-box and Tub domains.Tissue expression profile analysis showed that the expression of GmTLP family members in soybean had great temporal and spatial differences.Analysis of cis-acting elements showed that most of the cis-acting elements in the promoter of GmTLP family members were involved in hormone or abiotic stress responses,suggesting that they were involved in plant stress resistance responses.2.Expression pattern and subcellular localization analysis.Through the transcriptome data analysis of ABA,drought,and salt stresses in soybean,combined with real-time quantitative PCR（q RT-PCR）verification,it was found that GmTLP8 was up-regulated under various stress conditions.So GmTLP8 was chosen for further study.Subcellular localization analysis indicates that GmTLP8 protein was located in the nucleus and cytoplasm of Arabidopsis protoplasts.DAPI staining confirmed its staining in the nucleus,which may play a role as a transcription factor in the nucleus.3.Functional identification.To further verify the function of GmTLP8,Agrobacterium-mediated transformation of soybean hairy roots was used.Agrobacterium rhizogenes containing overexpression of GmTLP8（GmTLP8-OE）,RNA interference of GmTLP8（GmTLP8-RNAi）and empty vector（EV-Control）were used for genetic transformation of soybean hairy roots to obtain transgenic composite plants with transient expression of soybean hairy roots.The resistance of transgenic soybean hairy root composite plants under drought and salt stresses were analyzed.The results showed that GmTLP8-OE enhanced the resistance of soybean to drought and salt stresses,while GmTLP8-RNAi showed the opposite phenotype.Under drought and salt stresses,compared with EV-Control,GmTLP8-OE increased the contents of chlorophyll and proline（Pro）,and decreased the contents of oxygen free radical（O₂^-）,hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）.While GmTLP8-RNAi is opposite to EV-Control.The above results indicated that overexpression of GmTLP8 could significantly enhance the drought and salt resistance of transgenic soybean hairy root composite plants.4.Mechanism analysis.To further analyze the molecular mechanism of GmTLP8in plant stress response,the expression patterns of stress-related genes（Gm DREB1,Gm DREB2,Gm NAC11,Gm NCED3,Gm SOS1 and Gm WRKY27）were systematically analyzed by using GmTLP8 transgenic soybean hairy root composite plants.The results showed that these six stress-responsive genes were up-regulated in GmTLP8 transgenic soybean hairy root composite plants induced by drought and salt stresses.The results showed that under stress conditions,GmTLP8 gene may participate in the molecular biological process of plant response to stress by regulating the expression of downstream stress response genes,and improve the resistance of soybean.