Cloning And Expression Analysis Of GbTCP5 And GbTCP10 Gene In Gossypium Barbadense

Cotton is an important agricultural cash crop in China,which plays a key role in the global economy.It not only provides most of the natural fibers in the world,but also is an important renewable resource.Cotton fiber is an important raw material in textile industry,but the fiber quality of our country is single,and many external factors such as low temperature,drought,salt alkali,insect pests and so on have greatly inhibited the growth and development of cotton,thus affecting the yield and quality of cotton fiber,can not meet domestic demand for high-quality cotton fibers.At different stages of fiber development,a large number of genes are involved in the regulation of fibre cells development.It is helps to reveal the molecular mechanism of cotton fiber development and provide candidate gene resources for improving fiber quality by genetic engineering.TCP protein is involved in the regulation of plant growth and development.It is a unique transcription factor in plants.Its encoded protein contains a conserved TCP domain,a helix-loop-helix(b HLH)containing 59 amino acid residues(AA).It can bind to DNA or interact with proteins.The TCP protein is evolutionarily conserved in many plant species and is involved in the process from seed germination to vegetative growth until flower and fruit formation.The TCP transcription factor is an important growth regulator that can convert many endogenous and environmental signals into growth regulators.In this study,the full-length c DNA of Gb TCP5 and Gb TCP10 genes were cloned from the cotton fiber of "Xinhai 21" 5 DPA.The expression patterns of two genes were analyzed by fluorescence quantitative PCR.The transcriptional activation activity and binding specificity of Gb TCP5 and Gb TCP10 were analyzed by yeast system,and the biological functions of the two genes were further analyzed by transforming Arabidopsis thaliana.The main results were as follows:1.The Gb TCP5 gene was cloned from the cotton fiber of sea-island cotton "Xinhai 21" 5 DPA with an open reading frame(ORF)of 945 bp.The gene encodes 314 amino acids with a protein molecular weight of 34.95 k D and an isoelectric point of 8.48.Contains a highly conserved TCP domain.Quantitative PCR analysis showed that Gb TCP5 gene expression was higher in cotton fibers for 35 days,suggesting that it may be involved in the secondary synthesis of cotton fibers.In different tissues of cotton,the expression level of Gb TCP5 gene was higher in flower buds.Transcriptional activation experim ents showed that Gb TCP5 is transcriptionally active and the transcriptional activation region is between 139-202 aa.The binding specificity experiments revealed that Gb TCP5 binds to TCP elements and has a high binding specificity to component TCPII.The MYB-related genes Gh MYB(lrp)and Gh MYB(6286)did not bind to the MBS element in the Gb TCP5 promoter.The Gb TCP5 gene was transformed into Arabidopsis thaliana.Phenotypic observat ions revealed that the Gb TCP5 transgenic Arabidopsis line had significantly increased hair roots,leaf epidermal hair density,and stalk epidermal hairs compared to wild-type Arabidopsis thaliana.The results of semi-quantitative RT-PCR showed that Gb TCP5-1 gene up-regulated the expression of GL1,ETC2,and ETC3 genes in the transgenic plans and down-regulated the expression of GL2,GL3,and TCL1 genes.Gb TCP5 binds to TCP elements in the CPC promoter of epidermal hair-related genes in Arabidopsis thaliana.The plant expression vector ami RTCP5-p CAMBIA3301 containing the ami RTCP5 precursor was constructed using artificial mi RNA technology and transformed into cotton.2.The Gb TCP10 gene was cloned from the cotton fiber of "Xinhai 21" 5 DPA.Its ORF is 1386 bp.The gene encodes 461 amino acids,the molecular weight of protein is 49.93 KD and the isoelectric point is6.74.The sequence contains a highly conserved TCP domain.Quantitative PCR analysis showed that Gb TCP10 gene was highly expressed in cotton fiber for 5 days,suggesting that Gb TCP10 gene might be involved in the development of cotton fiber.The expression of Gb TCP10 gene was the highest in different cotton tissues.Transcriptional activation assay shows that Gb TCP10 does not have Trascriptional activity combined with specific experiments,it was found that the upstream promoter region of Gb TCP10 and TCP element binding.Gb TCP10 contains TCP element,and Gb TCP10 binds to TCP element,suggesting that Gb TCP10 transcription factor may regulate its own transcription.MYB-related gene Gh MYB(lrp)and Gh MYB(6286)bind to AC-I element in Gb TCP10 promoter.Gb TCP10 gene was transformed into Arabidopsis thaliana.Phenotypic observation showed that Gb TCP10 significantly increased root hair,leaf surface fur density and stem surface fur of Arabidopsis thaliana.Semi-quantitative RT-PCR results show that transgenic genes compared with wild type,Gb TCP10-2 up-regulated the expression of GL1,ETC2,TCL1,TTG1,and down-regulated the expression of GL2,CPC,TRY,ETC3.Gb TCP10 could bind to TCP elements in CPC and TTG1 promoter of Arabidopsis thaliana epidermal genes.The plant expression vector ami RTCP10-p CAMBIA3301 containing ami RTCP10 precursor was constructed by artificial mi RNA technique and transformed into cotton.