Molecular Mechanism Of Light On The Regulation Of Brown Cotton Fiber Pigment In Vitro

Brown cotton belongs to upland cotton and exhibits natural color,which is the result of pigment deposition in the fiber.It is the natural color of brown cotton that lead to the subsequent processing without dyeing,which not only saves the production cost,but also reduces environmental pollution.However,due to the short brown cotton lint,pigment uneven distribution,genetic instability and other defects,The production and application of brown cotton is seriously limited.The structural genes in the synthetic pathway of proanthocyanidins were cloned from brown cotton fiber(F3H、DFR、CHI,ANS、ANR etc),identification results of chemical properties of brown cotton fiber pigment.It is concluded that the synthetic precursor of brown cotton fiber pigment is Proanthocyanidins.The fiber color of brown cotton was formed in the middle and later stage of fiber development.Research shows that the appearance the color of the fiber is related to light.But,it is not clear whether there is light regulation in the process of fiber pigment synthesis,the direct relationship between fiber pigment synthesis,transport,and light still needs further study.With the development of high-throughput sequencing technology and the completion of the genome sequencing,application of new research means to study the regulation of brown cotton pigment synthesis pathway has important theoretical and practical significance.In this work light brown cotton was used for in vitro ovule fiber processing.The content determination,structural of gene expression,and effects of light on the synthesis of brown cotton fiber were analyzed.With the aid of high-throughput sequencing and bioinformatics technique,the light from the transcription of the molecular mechanisms of brown cotton pigm ent synthesis approach to regulation was parsed.The main results are as follows:1.Using brown cotton as material,the in vitro culture system of ovule was optimized.The results showed that high survival rate of ovules 1D after flowering.The suitable disinfection method was 75%ethanol of 10 min+0.1%mercuric chloride 5 min.Brown cotton ovule in vitro culture of appropriate ratio of hormones GA3 1.0μmol·L-1+IAA 1.0μmol·L-1+ZR 1.0 μmol·L-1.The content of soluble PAs and insoluble PAs in brown cotton fiber reached peak value at 20DAC,the content of insoluble PAs was always higher than that of soluble PAs,the content of soluble PAs and insoluble PAs in white cotton fiber changed little in 10-30 DAC.2.The in vitro culture of the ovule of brown cotton 1D was carried out by light and dark culture.Based on the analysis of the related physiological and biochemical indexes during the development of fiber and the expression of pigment synthesis related genes.The effect of light on pigment accumulation of brown cotton was studied.Results showed that the contents of proanthocyanidins,flavonoids,and soluble sugar were higher in prophase.With the increase of incubation time,its content was gradually decreased.In the early stage of fiber development,the content of PAs in the fiber of the light treated ovule was significantly higher than that of the dark treated ovule.In the late stage of fiber development,the content of PAs increased significantly in the dark treated ovule fibers.There was a significant decrease in PAs content in the fibers of the ovule treated with light at the same time.The key enzymes of pigment synthesis were expressed only in the early and middle stages of fiber development(GhCHS、GhF3H、GhDFR、GhANS、GhANR),the expression of 5DAC was highest and the expression was decreased,the expression of fiber growth and development may be low.3.By using high-throughput sequencing technology,the differentially expressed genes were isolated from the light and dark culture fibers.In the GO function annotation results,the main function of gene molecular exercise binding,catalytic activity,transporter activity of these differentially expressed,mainly involved in metabolic process,biological process,single cell process,in the metabolism of pigment synthesis and transport during cell active,so as to ensure the synthesis and transport of fiber cell pigment quickly.The expression products of the differentially expressed genes were more than that in the cell and cell membrane.In the KEGG annotation results,7211 different genes than to KEGG database were involved in 122 metabolic pathways.These metabolic pathways were enriched to phenylalanine metabolism,metabolic pathways of ABC transporters,nitrogen metabolism,carbohydrate metabolism pathways,including glycolysis/dysplasia sugar,starch and sucrose metabolism,pentose phosphate pathway and pentose and glucuronic acid ester tautomerism.4.There were 1350 differentially expressed genes in the light and dark culture by transcriptome sequencing.805 of the expression increased,and 545 of the expression decreased.By comparing the brown pigment synthesis pathway,a total of three genes related to anthocyanin transport were cloned.Named as GhTT10-1,GhTT12-1,and GhTT19-1,respectively.NCBI login number was KX782337,KY484835,KX782336.qRT-PCR analysis was consistent with the sequencing results.According to the prediction of transmembrane domain and subcellular localization,GhTT10-1 is a membrane protein,which may be mainly responsible for the oxidation of proanthocyanidins in the wall space.GhTT12-1 is a kind of membrane protein,which may be mainly responsible for the translocation of procyanidins.GhTT19-1 may be related to the transport of Procyanidins from the cytoplasm to the vacuole.5.By using bioinformatics methods,ARF and LAV subfamily members of the B3 super family of upland cotton were characterized by sequence,chromosome location,conserved motifs,gene structure,phylogenetic evolution,and expression patterns.ARF and LAV subfamily members of the B3 super family of upland cotton were screened and identified 49 GhARF and 8 GhLAV in the genome of Upland Cotton.Through the expression pattern analysis,GhARF17,GhARF18,GhARF20,GhARF21,GhARF43,GhARF44,GhARF45,GhARF47,GhARF49,GhLAV1,GhLAV3,and GhLAV6 represented the expression pattern of the fiber at different developmental stages,which was consistent with the change trend of the content of proanthocyanidins in the fiber,and presumably involved in the process of pigment synthesis.