| Cotton is one of the world's most important economic crops.China is the world's largest producer and consumer of textiles.With the continuous development of society,people need goods with sustainable development and environmental health.Natural colored cotton is the new cotton varieties created using modern scientific technology.Natural colored cotton meet the requirements of the public for health,environmental protection and non pollution,because there is no need for chemical dyeing and printing,no pollution,and contain natural antioxidants,and on the skin without any stimulation and other characteristics,so a lot less harm to human health and the environment.At the same time,natural colored cotton fiber in the production of textile products to reduce a large number of bleaching,dyeing and other chemical treatment.So it can meet the requirements of natural,harmonious and environmental protection,and reduce the pollution to the ecological environment.Anthocyanin as an important secondary metabolite belongs to the flavonoid compound and is a pigment widely exists in green plants.Color cotton fiber is production of accumulation of anthocyanin and its derivatives in cotton fiber cavity,therefore,Study on the expression of key genes is crucial in the metabolic pathway of anthocyanin in cotton.Purple mutation of upland cotton purple mutant(HS2)is caused mutation by exogenous T-DNA into the key enzyme genes of downstream metabolic pathway of anthocyanin,so that the whole plant is purple.This study found related genes of regulation of anthocyanin synthesis and accumulation in the cotton anthocyanin metabolism pathway by the cotton genome database,used bioinformatics analysis method with upland cotton purple mutant(HS2)rich in anthocyanins and proanthocyanidins as the material.Based on bioinformatics analysis of related genes,we found that there are three key enzyme genes in the anthocyanin metabolic pathway:CHS,ANS and LAR correspond to Charles ketone synthase,anthocyanidin synthase and leucocyanidin reductase gene respectively.In different developmental stages of cotton fiber,the transcription levels change of identify genes was examined and relative expression levels of related genes in the anthocyanin metabolic pathway were analyzed using real time polymerase chain reaction(qRT-PCR),meanwhile cryptochrome genes affected photosynthesis were quantitatively analyzed.Finally,the total content of anthocyanins and proanthocyanidins from different cross combinations at different developmental stages was also measured,and the correlation between them was preliminarily analyzed.The results obtained are as follows:1.Cotton purpled mutant HS2 and multiple color fiber varieties were used to made a number of cross combinations,were hybridization of HS2 and brown No.1,hybridization of HS2 and green No.l,hybridization of HS2 and new color No.1,hybridization of HS2 and light brown No.1-52,hybridization of HS2 and deep brown No.1-61,hybridization of HS2 and new color No.5,hybridization of HS2 and new color No.7,hybridization of HS2 and new color No.7586,respectively.F2 generation character segregation population was obtained by the self-bred of F1 generation hybrid combinations.In order to determine the color of cotton and important agronomic traits,advantage combination is selected in the above combination.The physiological and biochemical analysis of photosynthesis in leaves of F1 cross combination and different combinations of F2 generation were carried out respectively.At the same time,the comparative analysis for the cotton boll,cotton fiber length and cotton seed phenotype of F1 hybrid offspring of different lines of cotton is also carried out.2.To determine the total content of anthocyanins and proanthocyanidins in different types of cotton leaf of natural colored cotton and its hybrid combinations.The total content of anthocyanin in cotton leaves of different lines was determined by pH differential method using UV spectrophotometer,and determination of total content of anthocyanins in different kinds of cotton leaves by DMACA chemical reaction method.The purple mutant HS2 and colored cotton hybrid parents change the content of anthocyanin in leaves of cotton hybrid offspring,so as to explore the effects of changes of anthocyanin metabolism in different types of cotton and selection of offspring traits by using the purple mutant.3.To measure expression of cryptochrome gene cry1-2,cry1-4,cry2-1 and cry2-4 in the mutant seedlings by using qRT-PCR at different illumination time of different light source,and focus on the expression of cry1-2 and cry2-1 gene in blue light,red light and natural light.To explore the influence of cryptochrome genes on anthocyanin and polymer accumulation in cotton leaves and fibers under different illumination conditions.4.To detect the expression of three genes CHS,ANS and LAR in cotton leaves of different colored cotton hybrid combinations and different developmental period of cotton fiber.Gene sequences of LAR,ANS and CHS in cotton were analyzed by NCBI.Primer Express 5.0 software provided by ABI company designed related gene expression and analysis of PCR primers.To measure the expression of three genes CHS,ANS and LAR in different colored hybrid cotton leaves and different developmental stages by using real-time fluorescence quantitative PCR instrument.And correlation analysis between the expression of three genes CHS,ANS and LAR and the total content of anthocyanins and proanthocyanidins in different developmental stages of cotton fiber.In order to provide a basis for further clarifying the effect of anthocyanin on metabolic pathway and a preliminary study on color fiber modification by using the purple mutant,we explored expression differences of the key gene CHS,ANS and LAR from anthocyanin metabolic pathway of different strains of cotton hybrid combinations.5.A comprehensive analysis of the relative expression between the content changes of three key genes LAR,ANS and CHS in purple mutant,different combinations of colored cotton leaves and cotton fiber at different developmental stages.It showed that the abnormal metabolism of anthocyanins in the purple mutant could obviously affect the accumulation of anthocyanins in the fiber,and laid a solid foundation for further genetic improvement of the color fiber.This article studied cryptochrome genes and key genes of anthocyanin metabolic pathway,explored the effect of CHS,ANS,and LAR gene on anthocyanin accumulation at different developmental stages of cotton fiber from different strains under the different light conditions.for later use cryptochromes and key genes regulating anthocyanin,provide a scientific basis for proanthocyanidin metabolism,and lay theoretical basis of genetic engineering to improve the color of fiber.The significance of this study is to provide a scientific basis for later use of cryptochromes and key genes regulating metabolism of anthocyanin and original anthocyanin,and lay a theoretical foundation for improvement color fiber by genetic engineering. |
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