Expression Characterization And Antiserum Preparation Of Cotton (Gossypium Hirsutum L.) GhMAP1-LC3 Gene

Cotton (Gossypium hirsutum L.)Gh MAP1-LC3 gene was cloned in our laboratory previously. Amino acid sequence of the gene is identic to the conservation region of the light chain 3(LC3) of microtubule-associated protein 1A(MAP1A) and 1B(MAP1B). So it seems the gene is the counterpart of MAP1-LC3 gene in cotton.The expression characteristic of GhMAP1-LC3 gene in Shiyuan 321 (Gossypiwn hirsutum. L) was analyzed by real-time quantitative PCR(RQ-PCR) and antiserum against MAP1-LC3 was prepared. The results were as follows:1. The CTAB-PVP method was adopted to extract RNA of cotton tissues and high quality RNA was obtained.2. The expression characteristic of GhMAP1-LC3 gene in cotton tissues was compared by RQ-PCR. The expression of the gene was not detected in hypocotyl, -3DPA ovule, 30DPA fiber and 35DPA fiber while it was detected in radicle, leaf, petal, anther, ODPA ovule, 5DPA ovule, 10DPA fiber, 15DPA fiber, 20DPA fiber and 25DPA fiber, the highest expression detected was in 20DPA fiber.3. The CTAB-PVP method was adopted to extract genome DNA of cotton leaf, polymerase chain reaction was followed by gene-specific primer. The PCR product of genome DNA and 20DPA fiber cDNA shows identic molecular weight, suggesting the absent of intron in the gene.4. Having incorporated the open reading frame of the gene into pET30a vector, we transferred the recombined vector into JM109(DE3) E. coli Strain. After inducing, the expression product of GhMAP1-LC3 gene is 2% of total protein of induced E. coli.5. Purified protein was obtained by affinity column chromatography and SDS-PAGE recovery method.6. Antiserum against purified protein was prepared by immunization of rats and it can specifically recognize MAP1-LC3 in western blotting.