Cloning And Functional Analysis Of Pigment Related MYB Transcription Factor Genes In Brown Cotton

Naturally colored cotton is an ideal textile raw material for the environmental protection and human health.In order to explore the formation of fiber pigment and further understand its molecular mechanism,the experiments were conducted with cotton near isogenic lines of white cotton cultivar RT-white and brown cotton cultivar Brown 1-61.The research on fiber development and differentiation,as well as,the distribution of pigment deposition were conducted by the method of morphology.The expression pattern of the structure genes in flavonoid biosynthesis and the related transcription factor in different tissues was analyzed by real-time fluorescent quantitative PCR.Research on reciprocal action mode between MYB and the structure genes in flavonoid biosynthesis was carried on.The influence of MeJA on the expression level of the structure genes in flavonoid biosynthesis and the related transcription factors was analyzed in ovule culture in vitro.The influence of MeJA on cotton fiber color and quality was analyzed in the field.The main conclusions were as follows:1.Comparison of fiber development and differentiation of RT-white and Brown 1-61The epidermal cell protuberances of Brown 1-61 ovule appeared earlier and developed faster than RT-white.There was black dense material deep dyed in the edge of Brown 1-61 fiber cell wall from10dpa.With the secondary wall thickening and cell dehydration,the black dense material was gradually close to the fiber cell cavity,and finally deposited in the fiber cell cavity.So the black dense material may be the pigment of Brown 1-61.2.Analysis of the expression level of structural genes and the related transcription factors in the flavonoid biosynthesis pathway of brown cottonThe expression of structural genes in flavonoid biosynthesis was not tissue-specific both in RT-white and Brown 1-61.GhUFGT in brown 1-61 was still very high at 25dpa and 30 dpa,which was obviously different from the other structural genes,and the expression of GhUFGT was significantly higher than that of RT-white.A positive gene GhTT2 and two negative genes GhMYB1 and GhMYB6were screened through this experiment,which may regulate the pigment synthesis of brown cotton fiber.3.Clone of MYB family members GhTT2,GhMYB1 and GhMYB6There was no difference in cloned sequences of GhTT2,GhMYB1 and GhMYB6 between RT-white and Brown 1-61,which indicated that the formation of brown 1-61 color may be because of the expression regulation function of these three genes rather than the sequence mutation.4.The relationship between the MYB transcription factor genes and the structural genes in flavonoid biosynthesis pathway by yeast one hybridThe result of yeast one hybrid showed that GhMYB1 interacted with GhANS,and that GhMYB6interacted with GhUFGT,inferring that GhMYB1 and GhMYB6 may respectively regulate the expression of GhANS and GhUFGT,which further affected the pigment synthesis of brown cotton fiber.5.Subcellular localization of GhMYB1 and GhMYB6The subcellular localization of GhMYB1 and GhMYB6 genes in tobacco cells was mediated by Agrobacterium.The results showed that both proteins encoded by GhMYB1 and GhMYB6 were localized in the nucleus,which suggested that GhMYB1 and GhMYB6 play a role of regulation in the nucleus.6.The ovule culture in vitro of brown cottonThe ovule culture in vitro of color cotton was conducted with the treatment of 40.0?mol·L^-1 MeJA.After the treatment of 40.0?mol·L^-1 MeJA,the expression level of GhUFGT showed a hundred times increase in both RT-white and Brown 1-61,and the expression of transcription factor GhMYB6 gene also showed an increasing trend,which increased up to about 15 times at 30DPC in Brown 1-61.Based on the result of yeast one-hybrid experiment,it was speculated that GhMYB6 could regulate the expression of GhUFGT,which may be induced by MeJA,leading to a greater change range of GhUFGT gene expression.7.The spraying of MeJA in the fieldThe chromatic aberration of RT-white reached maximum at 40?mol·L^-1MeJA,while that of Brown1-61 reached maximum at 400?mol·L^-1MeJA.The treatment of MeJA could lead to low fiber quality of RT-white and high fiber quality of Brown 1-61.And the fiber quality of Brown 1-61 was best at 40?mol·L^-1MeJA.