Isolation And Identification Of Cotton Fiber Lignans And Preliminary Analysis Of The Function Of Synthetic Related Gene GhPCBER

Cotton is an important natural fiber crop in the world.The quality and output of cotton fiber is the main driving force for the economic development of the cotton textile industry.China's cotton consumption is in the forefront of the world,but the quality of cotton fiber is poor,and there is still much room for improvement in fiber fineness,strength and length.Cotton fiber is mainly composed of cellulose and hemicellulose.In recent years,it has been found that there are a large number of phenylpropane compounds in the development of cotton fiber.The phenylpropane metabolic pathway is considered to be the second largest metabolic pathway after cellulose synthesis.Phenylpropanoid metabolism may have an important impact on cotton fiber development and quality formation.The secondary metabolites produced by the phenylpropane metabolic pathway include various substances such as lignin,lignans,and flavonoids.The lignans are a class of phytoestrogens that are polymerized from small molecules of phenylpropanoid.It is found in xylem,roots,stems and leaves that it contains lignan compounds,which not only have certain cytotoxicity and phytotoxicity,but also promote plant defense against pests and diseases.PCBER is the key enzyme gene for the synthesis of lignan.It is mainly found in lignified plant cells and is expressed in various tissues and organs of cotton.The specific form of lignans present in cotton is still unclear.In this study,the lignan compounds in cotton fibers were isolated and identified to analyze the form of lignans present in cotton fibers.At the same time,the GhPCBER gene was transformed into Arabidopsis thaliana and cotton.To study the effect of changes in GhPCBER gene expression levels on cotton phenylpropanoid content,excavate the role of phenylpropanoids in the development of cotton fibers.The results are as follows:1.White cotton fiber as material,using the principle of like dissolves like,extracting organic compounds in cotton fiber with organic solvent ethanol as extract.Combined extract then rotary evaporation to obtain concrete;The obtained extract is roughly divided into four components of A1,A2,A3 and A4 by silica gel column chromatography and thin layer chromatography;after TLC analysis,the A2 component containing more lignan compounds was selected.Further separation of 5 compounds by purification preparative chromatography,nuclear magnetic spectroscopy revealed that all five compounds contained certain impurities.Further separation is required to identify its structure.2.The overexpression vector pGWB17-GhPCBRE of GhPCBER gene was successfully constructed,and Arabidopsis thaliana was digested by silk flower method.Six overexpressing transgenic lines were obtained by PCR.qRT-PCR analysis showed that the expression level of GhPCBER gene was higher in 1,2,and 3 lines,and the expression is obvious in stem and leaf tissues.Compared with wild-type plants,the content of lignin,lignan and flavonoids in stems and leaves of transgenic Arabidopsis thaliana was significantly reduced;qRT-PCR analysis of related genes in phenylpropanoid metabolic pathway revealed that overexpression of GhPCBRE gene resulted in the phenylpropanoid metabolic pathway is redirected.3.The gene silencing vector pTRV2-GhPCBER was constructed by VIGS technology.GhPCBER gene silencing cotton plants were obtained by injection-impregnated cotton seedlings.qRT-PCR analysis showed that compared with wild type,the expression levels of silenced cotton plants decreased by 12% and 26%,respectively.Which indicate that the tobacco rattle virus(TRV)system successfully inhibited the expression of GhPCBER gene in cotton.Compared with wild-type plants,The lignin and lignans content in the stems,leaves and the flavonoids content in the leavesof the silenced cotton plants were significantly reduced.Histochemical staining showed that the color of stems of silent cotton plants was significantly lighter,which indicate the lignin content in the stem of the silenced cotton plant is reduced.qRT-PCR analysis of related genes in the phenylpropanoid metabolic pathway revealed that the inhibition of GhPCBRE gene expression also led to the reorientation of the phenylpropanoid metabolic pathway.Due to gene silencing,the content of cotton secondary metabolites changed,resulting in a decrease in disease resistance of silencing cotton plants,and the disease index increased to 80.13 compared with the control plants(69.06).4.The GhPCBER gene was edited by CRISPR/Cas9 gene editing technology,and the Cas9-GhPCBER editing vector was successfully constructed.The Agrobacterium-mediated cotton genetic transformation technology was used to transform it into Xinluzao 7 to obtain the genetically edited cotton plant.For further development,exploring the role of the GhPCBER gene in the synthesis of secondary metabolites in cotton fibers provides mutant materials.