Cloning Of The Key Enzyme Gene Of Biology Synthesis Of Ethylene In Chewing Cane And Study On Its Expression

Ethylene is a kind of plant hormones which play an important part in the whole life circle of plant. It is not only has great influences on seed germination,plant developing,fruit ripening, sex differentiation ,but also can make response reaction to stresses such as Mechanical damage, cold damage, Waterlogging and Pathogen invasion.etc. So, to improve Resistance of plant and it also participate in plant cell's programmed death. Biology synthesis of ethylene delays on the catalysis of 1-aminocyclopropane-q-carboxylate(ACC) oxidase and ACC synthase. The activity of the two enzymes decides ethylene'output directly. In order to to further regulate plant's growth, evelopment, maturation and senescence, physiological stress, feedback regulation and antisense expression technique were used to control ethylene'biology synthesis and genetic engineering technique was used to clone the key enzymes. In this study, using chewing cane as experimental materials, cloned ACO gene by homology cloning techniques and made some other researches on it.1. Design specific primer pairs of gene of ACO,the gene ACO was amplified by RT-PCR from two aspects of DNA and RNA separately, the result of sequencing showed: the sequence of DNA is 1156bp,the cDNA is 1001bp accordingly, this gene contains intron about 155bp. To compare that of chewing cane and rice, the homology of this gene was 98%(AY521566.1), and rice was 89%(AP005559.3) by Homology search.2. The copies of ACO on 13 different chewing cane materials were analyzed by means of Southern–blot, the result declared: this gene has two copies in Badila, Waigandan, Fengchengzip, Baishan, Baxin, Dudu, Fuan and Ningde, have one copy in the remaning materials.3. ACO gene was induced by ethylene, the results showed that: in the beginning, the expression of this gene was inhibited,but after 9 hours, the expression recovered normol level,12 hours later,the expression overstep normol level and increase obviously.Then, the expression started to decline,after 72 hours,recovered to the original level. This pHenomenon showed:Under the inducing of ethylene,the expression of this gene emerged"S"tendency.4. Eukaryotic expression vector of ACO and Prokaryotic expression vector of ACO were con- structed. They were transformed into E.coli BL21(DE3) and induced to express. A 40KDa fusion protein product was detected by SDS-PAGE which is in the same size as expected, the result showed that it was expressed in Escherichia coli BL21 successfully.