The Establishment Of Genetic Transformation Systems And Preliminary Study On The Function Of GhPCBER1 In Upland Cotton

Cotton is one of the most important economic crops in the world,Cotton fiber is the most important natural fiber in textile industry,the quality and yield of cotton fiber is an effective driving force for economic development in the cotton textile industry.The quality and benefit of cotton textiles are directly related to the quality of cotton fiber.Therefore,How to improve the quality of cotton fiber is an important problem to be solved in cotton breeding.The quality of cotton fiber is formed during the development of cotton fiber,the presence of the phenylpropane metabolic pathway in the development of cotton fiber is the second most important metabolic pathway in the fiber after cellulose metabolism.The secondary metabolites produced by this pathway may have important influence on the development and quality of cotton fiber.Cloning and molecular mechanism of cotton fiber development related genes,which laid a theoretical foundation for improving the quality of cotton fiber by genetic engineering.The proteins encoded by the PIP subfamily genes are important enzymes for the synthesis of secondary metabolites such as lignans,flavonoids and isoflavones in phenylpropane metabolism.Among them,phenylcoumaran benzylic ether reductase(PCBER))is the key enzyme in the process of lignan synthesis of phenylpropane,and the PCBER gene is expressed in the roots,stems,leaves and flowers of cotton,What kind of biological function does PCBER play in the development of cotton fiber? In this study,6 PIP subfamilies were cloned from cotton fiber using bioinformatics and molecular cloning techniques.Objective to study the expression of PIP subfamily in different tissues of cotton,the PIP subfamily gene GhPCBER1 was overexpressed into cotton and Arabidopsis thaliana to analyze the preliminary function of this gene.The results are as follows:Using the protein sequence of PCBER as a probe,six of genes showing high sequence homology were obtained from a genome database of Gossypium hirsutum L.tetraploid cotton with Blastp search.The full-length cDNA sequences of these six genes were cloned from Gossypium hirsutum L.by RT-PCR,and named GhPCBER1,GhPCBER2,GhPCB3,GhIFR,GhPLR1 and GhPLR2,respectively,according to the six gene sequences,bioinformatics analysis showed that the six genes encoded 306 ~ 313 amino acids,the molecular weight of the protein is 33 854.9 ~ 35 199.6 Da.The protein has a strong hydrophilic and no signal peptide,is a non-secreted protein,subcellular localization predicted PIP protein located in the cytoplasm.All six proteins contain all conserved motifs and active residues of the PIP-type protein,belonging to the PIP subfamily.Real-time quantitative PCR showed that the expression of PIP subfamily genes was higher in the fibroblast growth wall.According to the expression characteristics of the gene,it wasspeculated that the PIP subfamily might play an important role in the development of cotton fiber.In this study,GhPCBER1 of PIP subfamily was expressed in cotton fiber,which may have an important effect on cotton fiber development.2.we successfully build pET-28a-GhPCBER1 protein expression vector,The size of the strip was determined by enzyme digestion and sequenced correctly,and then transformed into E.coli BL21(DE3).Induction of IPTG at a concentration of 1 mmol/L was performed at different time gradients,Identification of expressed product by SDS-PAGE.The results show that the maximum amount of 4h protein expression induced by 37℃;the relative molecular weight of expressed protein was about 34.7 K D,pET-28a-GhPCBER1 is constructed and expressed successfully,which lays the foundation for further research of the gene function.3.In order to explore the function of GhPCBER1 gene,plant expression vector /inhibited expression vector was constructed by Gateway,through agrobacterium mediated floral dip method over expression of Arabidopsis thaliana,the resistant plants were verified by PCR,the harvest plant seeds continue to use antibiotic screening,to obtain homozygote,extraction of Arabidopsis RNA reverse transcription cDNA,qPCR analysis was carried out to select the high expression of GhPCBER1 lines for further study.4.Overexpression/inhibits expression of GhPCBER1 transformating of Gossypium hirsutum L.Varieties ‘YZ-1’.The infected cotton hypocotyl tissue culture and resistance screening,have induced embryogenic callus differentiation into seedlings,PCR tested positive cotton plants,laid the foundation for further research on the function of GhPCBER1 gene in cotton...