Isolation And Validation Of Protein Interacted With GhLFAE

Cotton, one of the most important agricultural and commercial crops, mainly produces cotton fibers treated as significant textile raw materials. Improving the yield and quality of lint is the essential breeding objectives of cotton because the market value and the quality of cotton products are directly related to fiber quality. It is well known that the cell membrane penneability plays an important role on the maintenance of turgor pressure during the development of fiber. As a crucial component of membrane lipid, PUFA can lead to changes in both membrane fluidity and permeability characteristics. So in order to understand the molecular mechanism of fiber development to enhance yield and quality, it is very important to study the features of genes related to PUFA synthesis.GhLFAE (Gpssypium hirsutum Long chain Fatty Acid Elongation enzyme) which is homologous to KCS of Arabidopsis was cloned from upland cotton by our group. The following research results demonstrated that a polyunsaturated fatty acid(PUFA), namely (z)-5,11,14,17-eicosateraenoate exhibited in the transgenic tobacco via overexpression of GhLFAE and the permeability of plasmalemma was enhanced.In order to further analyze the regulation of GhLFAE during the cotton fiber cell development, and also reveal the molecular mechanism influencing the cell membrane permeability, Yeast Two-hybrid System was employed to screen certain protein interacting with GhLFAE. Then, the expression profile of candidate genes were created using RT-PCR in cotton. Finally, the certain protein interacting with GhLFAE was verified through bimolecular fluorescence complementation (BIFC).The main results are listed as follows:1. The autoactivation and toxicity testing of bait proteinThe Fusion expression vector pGBKT-GhLFAE was successfully established. The autoactivation and toxicity testing results indicated that GhLFAE as a bait protein could not activate the reporter gene expression in Y2H GOLD system alone. In addition, the following Yeast Two-hybrid System experiment could be carried out as GhLFAE was safe and non-toxic to yeast cells.2. The Screened by Yeast Two-hybrid System and verification of certain protein interacting with GhLFAEThrough setting up the cDNA library has containing the eight DPA cotton fiber cDNA mating with the pGBKT-GhLFAE in the yeast, after repeated screening of different reporter gene and one-to-one co-transformed verification in yeast, eight kinds of positive clones were got by several selection medium. Consequences gained from BLAST comparison demonstrated that those positive clones’sequences were respectively GhBCP2, GhSUT2A, GhPR2and several unknown functional protein genes.3. Created and analysis the candidate genes expression profile.For the analysis the expression profile of the selected protein genes in cotton, the expression of candidate genes in different parts of cotton and different growth periods was analyzed by means of RT-PCR method. Research results showed that GhBCP2has the most similar expression mode with GhLFAE, has a high value in six to eight DPA Fibers. So GhBCP2was taken into the following analysis.4. The verification of interaction between GhBCP2and GhLFAE utilizing BIFC techniqueWith the aim of further verifying the interaction of GhBCP2and GhLFAE in plant cells and locating the interaction. Co-expression the agrobacterium containing of GhLFAE-YFPN and GhBCP2-YFPc into Nicotiana benthamiana leaves, after36hours later, yellow fluorescent light could be observed under laser scanning confocal microscope. It is could be seen the proteins GhLFAE and GhBCP2interact with each other in cell membrane of tobacco leaves.