| Colored cotton is a type of cotton with naturally colored fibers.Compared with conventional white cotton fibers,its fibers do not need bleaching and dyeing during the processing into fabrics,which is conducive to saving resources and protecting the environment.The processed fabric does not contain harmful substances and is conducive to human health.Therefore,the research on colored cotton has huge development prospects.Green cotton is a main application type of colored cotton,charactered by its green fiber property controlled by the gene Lg.which encodes an R2R3-MYB protein regulating the suberin pathways to make the cotton fibers colored.By up-regulating expression of Lg during secondary wall accumulation in fibers of white cotton J14(Jimian No.14),our group successfully obtained three FLg transgenic lines(FLg-1,FLg-16,FLg-19)in which the fiber turned green.On the basis,this thesis compared the yield and quality traits of mature fibers of three transgenic lines,white cotton Null lines and naturally green cotton RIL-51 line;analyzed the relationships among the content of suberin and cellulose in fibers,fiber secondary wall development,fiber yield and fiber quality;and demonstrated the possible transcriptional binding sites of Lg in the metabolic pathways of suberin.The main results are as follows:1.Up-regulation of Lg in fibers of the secondary wall thickening stage makes yield and quality of mature fibers worseThe fiber quality and yield of the three transgenic lines were compared with the white cotton Null line and the RIL-51 line by randomized block experiment design,variance analysis and multiple comparisons,the results showed that there was significant heterogeneity between transgenic green fibers and white fibers of Null line,while there was no substantial difference between transgenic green fibers and naturally green fibers of RIL-51 line.Based on this,the quality and yield data of mature fibers of three transgenic lines,white cotton Null line and RIL-51 line were further analyzed.It was found that the quality characters of green fibers(transgenic green fibers and naturally green fibers of RIL-51 line)were significantly worse than those of white fibers of Null line,except for the upper half average length and fiber uniformity index,furthermore,the yield characters of green fibers(transgenic green fibers and naturally green fibers of RIL-51 line)were also significantly worse from those of white fibers of Null line,and the differences reached a very significant level,which indicated that the up-regulated Lg gene was the main reason for the decline of quality and yield of mature green fibers.2.Characteristics of suberin deposition in transgenic green fibersFirstly,using paraffin section technology to study the physiological structure changes of transgenic green fibers and it was found that the cell wall thickness of mature fibers of the transgenic lines was significantly smaller than that of the null lines.Further,the transmission electron microscope was used to observe the cross-sections of fibers of25 DPA,35 DPA,and 45 DPA of FLg line and Null line,and it was showed that there were typical suberin lamellae on the inner side of the secondary wall in fibers of the FLg line,and the secondary wall thickness of FLg line fibers was significantly thinner than that of Null line fibers at all stages,but no suberin lamellar structure was observed in the Null line fibers in all periods.Furthermore,to analyze the suberin accumulation characteristics in fibers of FLg line and Null line by using GC-MS technology based on regarding ω-hydroxydocosanoic acid as the standard,the results showed that the mature fibers of FLg line contained a large amount of suberin,but the content of suberin in the mature fibers of Null line was zero.To analyze the suberin content in 15 DPA,20 DPA,25 DPA,30 DPA,35 DPA,40 DPA,45 DPA fibers of FLg line and Null line,it was showed that there was no significant difference in the content of suberin between FLg line and Null line at 15 DPA,and the content was 0.But,the suberin content in FLg line gradually increased with days passing by after 15 DPA,while we did not detect suberin in fibers of Null line at all stages.The above results indicated that the transegenic green fibers contained a large amount of suberin after 15 DPA,but the fibers of Null line did not contained suberin in all periods,and the accumulation of suberin in transgenic green fibers was a gradual process.3.The deposition of suberin in the fiber affects the development of secondary wall and the fiber quality and the fiber yieldThe results of paraffin section assay and ultramicrostructure analysis of the fibers showed that the deposition of suberin in the fibers had great negative effects on the development of secondary wall.Cellulose is the main component in secondary wall of cotton fiber,and suberin may affect the development of fiber secondary wall by affecting the accumulation of cellulose in the fiber secondary wall.To test this hypothesis,fistly,the cellulose content of FLg line fibers and Null line fibers at 15 DPA,20 DPA,25 DPA,30 DPA,35 DPA,40 DPA,45 DPA were measured,it was found that the increase of cellulose content of FLg line fibers decreased significantly after 20 DPA compared to that of Null line fibers.The total fiber weight per seed mainly composes the fiber yield,the cellulose content of the total fiber on a single seed can reflect the thickening level of fiber secondary wall to a certain extent.Thus,we further measured the total fiber weight and the cellulose content of the total fiber on a single seed in FLg line and Null line at 15 DPA,20 DPA,25 DPA,30 DPA,35 DPA,40 DPA,45 DPA,it was found that the total fiber weight per seed of FLg line was significantly lower than that of Null line in all periods except for 15 DPA and 25 DPA,and the cellulose content of the total fiber on a single seed of FLg line was similar to that of total fiber weight per seed at all stages.Based on above results and combined with the results of suberin content measurement,it was fully proved that the suberin gradually accumulating inside the secondary wall of cotton fiber would decrease the cellulose content in the fiber,which would affect the development of fiber secondary wall and the fiber quality and the fiber yield.4.Lg can regulate the metabolic pathways of suberin in fibers by binding to ACCTAC siteThere were a large number of differentially expressed genes related to the suberin metabolic pathway in plant tissues where Lg was differentially expressed.15 representative genes with up-regulated expression were selected from these differentially expressed genes to verify the activation effect of Lg.The results showed that,except for Gh＿A08G1366,Lg had significant transcriptional activation effect on the other 14 genes selected,indicating that Lg had the ability to regulate related gene expression in suberin metabolic pathway.Based on this result,the Gh＿D13G0494 gene with the largest activation effect activated by Lg was selected,and the dual luciferase reporter assay was used to study the DNA binding elements of Lg,the results showed that Lg had an obvious transcriptional activation effect on the 23 bp segement containing ACCTAC site from S20-5 to S20-6.Further,mutational treatment of the ACCTAC site in the 23 bp segement revealed that when ACC or TAC sequences were changed,Lg lost the transcriptional activation effect on the 23 bp segement,indicating that ACCTAC is an important transcriptional core binding element for Lg to regulate the expression of Gh＿D13G0494.Besides,the results of yeast one-hybrid experiments showed that Lg had a direct binding effect on sequences containing ACCTAC sites,indicating that Lg could regulate Gh＿D13G0494 expression by directly binding ACCTAC sites.In summary,Lg can directly bind to the ACCTAC site to regulate related genes expression in suberin metabolic pathway,to promote the accumulation of suberin on the inner side of the secondary wall of fiber cells and endow the fiber green character at the meantime,and leading to the decrease of cellulose content in fibers,which affecting the development of secondary wall of fiber cells and declining the fiber quality and fiber yield. |
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