Cloning And Functional Analysis Of Transcription Factor GbTCP44 In Gossypium Barbadense

Cotton is an important agricultural cash crop in the world,and the cotton industry is of strategic significance for socio-economic development.Supply-side structural reform of agricultural products,so that cotton production from the pursuit of high yield to focus on improving quality.Cotton fiber quality is also one of the key indicators to measure cotton quality.Improving cotton fiber quality has also become one of the important goals of current cotton genetic breeding.However,cotton fiber development is a complex process that is regulated by a large number of genes and ultimately affects fiber quality and yield.Therefore,cloning and functional research of cotton fiber development-related genes can provide a theoretical basis for improving cotton fiber quality.Simultaneously,Gossypium barbadense has excellent fiber quality,and its inherent excellent genes have also become the object of research on cotton quality improvement.In addition,TCP transcription factors are widely involved in plant growth and development,and in cotton,several TCP genes have been cloned and shown to be involved in cotton fiber and epidermal hair development.Therefore,in this study,a full-length cDNA of the TCP family transcription factor GbTCP44 was cloned from the Gossypium barbadensse,the gene structure was analyzed,the expression patterns of the gene was analyzed by qRT-PCR,the yeast system was used to analyze the transcriptional activation activity of the gene,the prokaryotic expression analysis was used to explore the optimal conditions for its protein induction,and the biological function of the gene was analyzed by VIGS technology,in order to provide a scientific theoretical basis for the improvement of cotton fiber quality.The main findings are as follows:1.The cDNA sequence of the GbTCP44 gene of Gossypium barbadensse "Xinhai 21" was cloned using RT-PCR technology and analyzed by bioinformatics,the GbTCP44 gene has 1035 bp open reading frame,encoding 344 amino acids,with the predicted molecular formula C1618H2538N484O525S13,a molecular weight of 37.59 KD and an isoelectric point of 8.83.Its sequence(N-terminal)has a highly conserved TCP functional structure domain,and belongs to the Class I member of the TCP transcription factor family.The secondary structure of the gene consists of 12 alpha-helixes,4 beta-sheets,and random coils;The amino acid sequence is free of signal peptides;The protein does not have a transmembrane region,is a more hydrophilic protein and may be subject to protein phosphorylation.2.Subcellular localization shows that the GbTCP44 gene was localized in the nucleus and was a nuclear expression protein;Transcriptional activation activity analysis demonstrated that the GbTCP44 protein is transcriptionally activated;Primary expression analysis showed that GbTCP44 protein was efficiently expressed under the conditions of IPTG concentration of 0.8 mmoL/L,37℃,and 6 h.3.Expression pattern analysis revealed that the expression of the GbTCP44 gene was significantly higher in the reproductive than in the nutritional organs;The gene was most expressed at 20 d of cotton fiber development;When cotton fibers developed to 25 and 30 d,the expression of this gene was lower in long-fiber varieties than in short-fiber varieties,with significant differences(P<0.05),indicating that the GbTCP44 gene may be involved in the development of fibroblast elongation in cotton fibers.4.VIGS technology silencing the cotton GbTCP44 gene showed successful construction of pTRV2-GbTCP44 silencing vectors using tobacco brittle virus plasmids and obtained cotton plants with higher(>50%)silencing efficiency of the GbTCP44 gene.In contrast to the control,(1)This silent plant showed asymmetric growth,folds,and even deformities of the leaves from early growth and development to the flower bud stage,suggesting that the GbTCP44 gene may be involved in the regulation of leaf shape development.(2)The gene was silenced in the land cotton variety "Ben-6-2 Hairiness" and the number of stalk epidermal hairs was found to be reduced by 59%in silenced cotton plants compared to control plants.It is speculated that the GbTCP44 gene may be involved in regulating the development of plant epidermal hair.(3)qRT-PCR analysis of cotton fiber development-related gene expression in silenced GbTCP44 gene plants showed that GhSK13,GhNAC1,GhCAM7 and GhCPC gene expressions were significantly upregulated compared with the control;The expression of GhPRP5,GhSUSAI,and GhFLA genes was significantly reduced,revealing that the GbTCP44 gene may have a direct or indirect role in the regulation of cotton fiber development.