Gene Cloning And Expression Analysis Of Marop1 In Banana, And Its Antibody Preparation

Banana was a kind of typical respiration climacteric fruit, its ripening was consid-ered a signal transduction process that endogenous and exogenous ethylene participate in. Rop proteins were vital regulators of signal transduction in plants. ROPs potentially in-teracted with cell surface–associated signal perception apparatus for regulating many cellular process.such as cell movement, morphogenesis, host defense, cell division and gene expression. Our laboratory had already obtained the SSH fragment of ROP gene by constructing the SSH library of banana fruit and found it might be associated with fruit ripening by the chip analysis. All of these indicated it was necessary to further research. Designed the PCR primers on the base of the SSH fragment and cloned a 720bp fragment named Marop1 by the technology of RACE-PCR from banana fruit double strand cDNA library. The results of Blastx showed that Marop1 shared a high degree of sequence similarity with ROP of other plant species,and the analysis of full length of amino acids deduced from cDNA indicated that it had a highly conserved domain,which was the typical structure of the ROP proteins. The result of Genscan indicated that Ma-rop1 included an integrated ORF and there were 196 amino acids. The result of Southern blot showed that the copy number of Marop1 was low in the banana genome. In order to research the expression of Marop1,we choose 0rd,2rd,6rd,10rd,12rd and 14rd as materials for expression according to the quantity of ethylene release. at the same time ,we selected the root,rhizome,flower,leaf and fruit as materials for expression of specific organise. And then extracted the RNA from all the materials and reverse tran-scripted them to cDNA as the templates for expression.The result of RT-PCR of specific organise expression showed that Marop1 expressed in all of organises and the most in rhizome. The result of RT-PCR of every ripening stage revealed that the expression of 0rd was higher and 12rd was the highest. But the quantity of the ethylene was the most in 14rd. By the result of expression,we could predict that its function may be associated with the synthesis of ethylene and hurt stimuli. Ethylene was closed associated with fruit ripening, which had been manifested by many researches. So we could conclude that Marop1 was relevant to banana fruit ripening. Forthermore, we constructed pET-30a-Marop1 expression plastid, then transformed it into E.coli BL21(DE3). After inducing the expression of recombinant bacterium, the recombinant protein was isolated in 12% SDS-PAGE. Cut it in solution of 0.25M KCl and grinded it in Freund's adjuvant. The completely emulsified recombinant protein was injected in rabbit. The antibody was verified by ELISA and Western blot. The value of antibody was very high(>10000) and the specificity was very good(no noise stain). All of these are the groundwork of deeply researching Marop1's function, especially in banana fruit ripening.