Identification And Functional Analysis Of The U-box Gene Family In Tea Plan

U-box protein,as the main E3 ubiquitin ligase,widely exists in plants and plays an important role in regulating the growth and development of plants,biological stress and abiotic stress.Studies have shown that U-box protein plays an important role in protein degradation pathway related to cell cycle,signal transduction,stress response,transcription regulation,DNA repair,apoptosis,morphogenesis,secretion pathway and other processes.Tea tree(Camellia sinensis(L.)O.Ktze)often suffers from abiotic stresses such as high temperature,drought and low temperature,which seriously affects the yield and quality of tea.However,at present,there is no research on the adversity of tea U-box family and its genes in tea trees.This study focuses on the research of tea U-box gene family,and analyzes the basic biological information of tea U-box gene family members by bioinformatics(including phylogenetic tree,conserved domains,protein motifs,chromosome location,abiotic stress expression pattern analysis,etc.).By selecting genes with high expression in gene family,designing gene primers,and performing real-time fluorescence quantitative analysis to verify whether the results are related to the predicted results of genome database.The CsU-box39 gene was cloned and genetically transformed into tobacco.The expression level and related physiological indexes of transgenic tobacco under abiotic stress were measured and analyzed,so as to understand the way of stress regulation of U-box gene family related genes in tea plants,and to provide reference for further revealing the role of CsU-box in the growth and development of tea plants and stress response.The main research results are as follows:(1)The functional domain of U-box in Pfam database(Pfam04564)was obtained by consulting literature,and the members of tea U-box gene family were determined by bioinformatics method,and the physical and chemical properties of family members were predicted.The U-box gene family of tea plants was analyzed by bioinformatics methods,including phylogenetic tree,conserved domain,protein motif,chromosome location,expression pattern analysis of abiotic stress,etc.The results showed that 92 tea CsU-box genes were divided into 11 groups according to their domains,among which the first group had the largest number of members,including 41 CsU-box members,and the domain was a single U-box domain,while the eighth to tenth groups had only one CsU-box member.The analysis of gene structure and conserved motif shows that most genes are highly conserved and have great similarities with each other.From the analysis of the expression patterns of eight tissues of tea tree,it was found that 32%CsU-box was expressed in all tissues.The expression levels of CsU-box27,CsU-box70 and CsU-box91 genes in roots are much higher than those in other tissues.The overall expression level of CsU-box39 is higher,and the expression level in flowers and old leaves is higher than that in other tissues.Different expression patterns of abiotic stress show that CsU-box genes of tea plants are involved in the regulation of tea plants under abiotic stress,and have different responses under different abiotic stresses.(2)The expression patterns of seven genes(CsU-box27,CsU-box28,CsU-box39,CsU-box46,CsU-box63,CsU-box70 and CsU-box91)in tea plants under drought stress and high temperature stress were analyzed by qRT-PCR.Under drought stress for 0～72 h,seven genes were significantly up-regulated at different time points,while CsU-box63 and CsU-box70 were slightly down-regulated at 12 h and 24 h respectively.After high temperature treatment,the expression of all CsU-box genes was significantly up-regulated at 24 h,except CsU-box63 at 12 h.The results showed that the expression patterns of seven CsU-box genes were similar to those of abiotic stress in transcriptome database.(3)Bioinformatics analysis(including promoter cis-acting elements,conserved domains and interacting proteins)of CsU-box39 with high overall expression,The promoter sequence of CsU-box39 gene contains four elements related to light regulation(ACE,ATC-motif,GATA-motif,Sp1),four elements related to hormone regulation(CGTCA-motif,GARE-motif,TATC-box,TGACG-motif)and four elements related to other regulation(CAAT-box,GATA-motif).The amino acid sequence of the conserved domain of CsU-box39 shows that the amino acid sequence of CsU-box39 gene at positions 250 to 313 constitutes a U-box domain with RING type modification and no zinc chelate.Compared with the protein sequences of other homologous genes Arabidopsis thaliana and Oryza sativa,the results showed that the U-box domain was highly conserved.The protein name of CsU-box39 gene is A0A4S4D5K4,and there are 10 proteins interacting with it.Among them,A0A4S4EW14(containing LRNT＿2 domain)and A0A4S4DV48(protein containing protein kinase domain;It belongs to protein kinase superfamily.Serine/threonine protein kinase family)are involved in plant MAPK pathway(map04016)and plant signal transduction pathway(MAP04075).A0A4S4EPK3 and A0A4S4D4H5 contain TCP domains,which are involved in inflorescence development.A0A4S4D5N6 and A0A4V3WK02 are involved in the biosynthesis of unsaturated fatty acids and Rab protein signal transduction.(4)Obtaining transgenic tobacco plants.In order to identify the function of CsU-box39 gene in plants,the constructed overexpression vector pBWA(V)KS-CsU-box39 was transformed into tobacco leaves by Agrobacterium-mediated transformation,and the transgenic tobacco plants were identified by GUS histochemical staining and DNA extraction.The results showed that GUS activity was not detected in wild tobacco plants,GUS activity was detected in transgenic tobacco plants,no target band was detected in wild tobacco plants,but the expected target band was detected in transgenic plants,and finally CsU-box39transgenic tobacco plants were obtained.(5)The drought stress treatment of wild tobacco plants and CsU-box39 transgenic tobacco plants showed that the relative expression of CsU-box39 transgenic tobacco plants under drought stress was higher than that of wild tobacco plants.The results of measuring the activities of antioxidant enzymes in wild tobacco plants and CsU-box39 transgenic tobacco plants after 21 days of drought stress showed that the activities of related enzymes in leaves of CsU-box39 transgenic tobacco plants were significantly higher than those of wild tobacco plants after 21 days of drought stress.The determination of chlorophyll content showed that the chlorophyll content of wild type and transgenic tobacco decreased after 21 d treatment,but the chlorophyll content of transgenic tobacco was still significantly higher than that of wild type.At the same time,after stress treatment,MDA content in wild tobacco leaves was significantly higher than that of transgenic tobacco.The results showed that CsU-box39 transgenic tobacco had a certain resistance to drought stress compared with wild tobacco.