Identification Of GhFAR Gene Family And Functional Studies Of GhFAR3.1 And GhFAR2 In Gossypium Hirsutum L.

【Objective】 The epidermis of most organs on the ground of terrestrial plants is covered with a layer of wax,which is a hydrophobic barrier formed by various lipid compounds and is an important structure on the surface of plants.Fatty Acyl-Co A Reductase(FAR)can catalyze the formation of fatty alcohols from fatty acids.It is a key enzyme involved in the synthesis of plant wax and plays an important role in the growth and development of plants.At present,FAR genes have been cloned in Arabidopsis and rice successively,but there are few reports on the cloning and functional studies of cotton FAR genes.In this study,Gh FARs genes were identified from the genome analysis of Gossypium hirsutum L,and the functions of Gh FAR3.1 and Gh FAR2 genes were analyzed,in order to further study the molecular regulation mechanism of Gh FARs in cotton anther development and epidermal wax synthesis lay the foundation.【Method】 The homologous comparison method was used to identify and obtain Gh FARs from the upland cotton genome database,and analyze the chromosomal location,gene sequence and expression pattern of FAR genes.According to the results of expression pattern analysis,Gh FARs that are predominantly expressed in leaves and anthers were screened out.The VIGS vector of the target gene was constructed to infect cotton,the gene editing vector was genetically transformed into the upland cotton recipient material "YZ-1",the overexpression vector was transformed into the Arabidopsis mutant ms2,and the function of Gh FARs gene was analyzed by phenotypic identification of the transgenic plants.【Results】1.Ten Gh FARs were identified from the genome of Gossypium hirsutum L,distributed on6 chromosomes,and named Gh FAR2 A,Gh FAR2 D,Gh FAR3.1A,Gh FAR3.1D,Gh FAR3.2A,Gh FAR3.2D,Gh FAR3.3A,Gh FAR3.3D,Gh FAR3.4A,Gh FAR3.4D.These 10 Gh FARs all have NAD-binding-4 and sterile conserved domains specific to plant FAR genes,and belong to the B and C subfamily,respectively.Analysis of expression patterns found that most Gh FARs showed tissue specificity,among which Gh FAR3.1 was predominantly expressed in leaves,and Gh FAR2 expressed the highest in the meiotic and tetrad stages of cotton anther development.2.After using VIGS to silence Gh FAR3.1,under suitable water conditions(control group),compared with WT plants and p TRV2::00 plants,the total wax content and relative water content of the leaves of p TRV2::Gh FAR3.1 plants were reduced respectively over 60% and 13%,silencing Gh FAR3.1 had no effect on the expression levels of the key genes Gh KAS,Gh KCS and Gh LACS in the wax synthesis pathway,but reduced the transcription level of the downstream gene Gh WSD of Gh FAR3.1.Under drought treatment conditions(drought group),the leaf wax content of WT plants and p TRV2::00 plants increased significantly,while the leaf wax content of p TRV2::Gh FAR3.1 plants did not increase significantly,and p TRV2::Gh FAR3.1 The relative water content of the leaves of the plant is reduced,and the leaves of the plant were yellow and wilted.Drought stress caused a significant increase in the expression level of wax synthesis-related genes,but the transcription level of Gh WSD gene in p TRV2::Gh FAR3.1 plants was significantly lower than that in WT plants and p TRV2::00 plants.3.Construct Gh FAR2 A gene editing vector and transform cotton to obtain two Gh FAR2 A gene knockout transgenic plants.Phenotypic analysis showed that there was no pollen in the mature anther of the gene knockout plants and the anthers was shrank abnormally,.When the stigma was pollinated with WT pollen,the plants could boll normally.The function complementation experiment of Arabidopsis ms2 mutant found that compared with Arabidopsis ms2 mutant pollen which is abnormally sensitive to acetolysis,the Arabidopsis ms2 mutant pollen overexpressing Gh FAR2 only showed a certain degree of sensitivity to vinegar hydrolysis,indicating that Gh FAR2,as a homologous gene of Arabidopsis MS2,can restore the phenotype of the Arabidopsis mutant ms2 to a certain extent.【Conclusion】 Ten Gh FARs genes were identified and obtained in upland cotton,and different gene expression patterns are different.Gh FAR3.1,which is highly expressed in leaves,is essential for wax synthesis in cotton leaves,and wax content is very important for plant water retention and drought tolerance.The Gh FAR2 gene predominantly expressed in anthers is related to plant fertility and is mainly involved in the development of the outer wall of pollen.