Isolating And Function Identification Of Cysteine Proteinase Inhibitor Gene From Populus Hopeiersis

Gene control was one of the core technologies of the forest integrated pest management (IPM) tactics, the key and basis was found autonomous inntellectual property functional gene, cysteine proteinase inhibitors (CPI) are kinds of super-family that widely distributed in the animal, plant and microbe, CPI could effectively inhibit hydrolyzation of insect intestine cysteine proteinase and produced the death of insect, CPI has been applied to resisting pest gene engineer. Longhorned beetles were major forest insect pests and damaged very serious. Longhorned beetles belonged to coleoptera insect, they digested mainly depending on the cysteine proteinase of digestive tract. Separating and cloning phytocystatins gene and applying to resisting pest engineer were an effective measure to control longhorned beetles. This paper took the Chinese native species populus hopeiersis as resisting pest gene resource, isolating and cloning CPI gene from secondary cambium by RT-PCR method at first in china, according to eukaryotic expressing vector construction and inducing expression, recombined protein was acquired by using affinity chromatography and Western detection. At last, the functional identification of the gene was carried on by insect feeding experiment. Main research results were as follows:1. Code region of CPI from secondary cambium of populus hopeiersis was isolated and cloned by applying to RT-PCR technology. The sequence analysis showed that code region of gene was 710bp and included a total open reading frame (ORF) and coded 143 amino acid residues. This sequence had a typical consensus region of phytocystatins, and got the 759 bp 3' end CPI cDNA of populus hopeiersis by RT-PCR method.2. Related characters of coding protein were analyzed and predicted from CPI cDNA...