Function Verification And Weighted Correlation Network Analysis Of The Candidate Gene GhTPR For Cotton Fiber Length

Cotton fiber length is an important indicator that affects fiber quality.Identifying key genes and revealing the molecular mechanism of cotton fiber length is significant in improving fiber length through molecular design breeding.Preliminary work,a candidate gene Gh TPR for the cotton fiber length major QTL（q FL-A12-5）was obtained through fine mapping and gene cloning sequencing analysis;Knocking out this gene using CRISPR/Cas9 in cotton shows that it has a significant impact on fiber elongation.Based on RNA-seq data,Y2H and Bi FC verification,the expression pattern and regulatory factors of Gh TPR gene promoter were analyzed by GUS staining and Y1H,these provided preliminary understanding of the molecular mechanism of Gh TPR regulating cotton fiber elongation.The main research findings are as follows:（1）Overexpression of Gh TPR promotes the development of Arabidopsis root and epidermal hairIn this study,overexpression of the Gh TPR gene in Arabidopsis significantly promoted root elongation,with an average increase of 3.52-6.99 mm;and increased the numbers of stem epidermal hairs.And the promoters with different length of Gh TPR gene were constructed to drive GUS expression vector,and transformed into Arabidopsis.In the stable lines of T₃generation,it can be observed that expressed in the leaf epidermis.（2）Gh TPR positively regulates cotton fiber elongationCRISPR/Cas9 was used to knock out the gene in cotton.In the 6 gene-editing cotton plants,all targets were mutated,the fiber quality testing found that the fiber length was shorter than the control WT,with an average shortening of 1.57-3.26 mm,and the differences were statistically significant in the T₂ and T₃ generations,and some lines reached the level of highly significant difference.In the T₂ of overexpressed plants,the fiber length of 5 lines increased by0.15-0.98 mm compared to the control WT.This indicates that Gh TPR positively regulates cotton fiber elongation.（3）Functional analysis of Gh TPR interaction protein and regulatory transcription factorsThree proteins,Gh KCS2-A,Gh KCS2-D and Gh KCS6-D were screened in the MBI 7747fiber tissue c DNA library by screening yeast library with BD-Gh TPR as bait.After cloning the full-length c DNAs of these genes,the interaction of Gh TPR with Gh KCS2-A,Gh KCS2-D and Gh KCS6-D in vitro and vivo was verified by Y2H and Bi FC,respectively.KCS is the rate-limiting enzyme for the synthesis of very-long-chain fatty acids,which affect the elongation of fiber by affecting the synthesis of VLCFA.Y1H screening library and point-to-point verification showed that it’s regulated by transcription factors Gh MYB212,Gh BZIP50,Gh GATA26,Gh NFYB1.（4）RNA-seq analysis of genes responding to changes in CRISPR/Cas9 plantsA total of 4753 up-regulated genes and 1475 down-regulated genes were identified by transcriptome sequencing using four developmental fiber tissue samples of CRISPR/Cas9transgenic stable lines and control WT.10 genes were differentially expressed in four periods.KEGG analysis of down-regulated genes showed that these genes were mainly enriched in lipids,carbohydrates,energy metabolism pathways,plant hormone signal transduction pathways and plant MAPK signal pathways,indicating that the gene Gh TPR affected the genes in these pathways,thus affecting the development of cotton fiber.（5）Construction of Gh TPR co-expression regulatory network by RNA-seqThe Gh TPR is located in the module with the highest correlation with fiber length traits,the correlation coefficient between modules and fiber length traits is|r|=0.91（p=7.6e-10）,and the gene correlation between modules and traits is 0.89（p<1e-200）,which is the highest compared with other modules;The KEGG pathway was significantly enriched in this module,including fatty acid degradation,fatty acid biosynthesis and fatty acid metabolism pathway.The co-expression regulatory network analysis of gene Gh TPR also showed that there was a regulatory relationship between Gh TPR and enzymes related to lipid metabolism and VLCFA.Based on the above research results,it is speculated that Gh TPR regulation mechanism is regulated by transcription factors such as Gh MYB212,Gh BZIP50 and Gh GATA26,and interacts with 3-ketoacyl Co A synthase to participate in metabolism.It plays an important role in the VLCFA pathway for cotton fiber elongation,and positively regulating fiber elongation.