| The persistent outbreak of bollworm had a devastating loss to the cotton production.so how to control the pests effectively had became a large problem to the improvement of the cotton’s production. In1990s, people constructed the appropriate plant expression vector with insect resistance genes by the method of biological engineer. Then they transformed the vector into cotton and got the transgenic cotton later. Such as Guo san dui research group who had got univalent resistance cotton of Bt and bivalent resistance of Bt and cpti in Chinese Academy of Agricultural Biotechnology Research institute. Not only reduced the use of pesticide, improved the economic benefits of famers,but also improve the insect resistance of cotton, what is imporment,it is no pollution to the environment. So it has been widely promoted in the production.However, with the large-scale cultivation of cotton, regardless of univalent or bivalent, a certain years after planting, pests will have the appropriate resistance or tolerance to the transgene cotton which made the agricultural biotechnology facing a severe test once again. In view of this situation.someone plus the signal peptide to the both ends of the exogenous genes, by the directed transport of foreign genes in plants then improve the accumulation of foreign genes in plants. In1996. Alexander Schouten etal increased a endoplasmic reticulum retention signal peptide(KDEL)to the C-terminal of a single-chain antibody(scFv). The results showed that with the existence of the signal peptide gene. the protein expression was increased nearly1percent. Based on this situation. The main rusults of this research are as follows:1. Based on the bivalent gene in our lab, we increased a soybean trypsin inhibitor(SKTI) signal peptide sequence in the3’ end of Cpti gene and a endoplasmic reticulum retention signal peptide(KDEL)sequence in its5’end, at the same time, we increased a chloroplast targeting peptide sequence in the5’end of Bt gene. Finally. We constructed a bivalent expression vector which contains the signal peptide sequence:2. By the southern hybridization, we analysis the copies of exogenous gene in the receptors plants. The results showed that exogenous genes had integrated into the recipient plant with different copies:3. The accumulation of foreign genes in receptor plants were analyzed by ELISA. The results showed that the accumulation of Bt protein increased by1-8times, while the accumulation of CpTI protein was about I times, we guess this may be caused by the expression of cpti itself and the binding capacity of antigen-antibody or may be the operation process of ELISA.4. We analysis the insect resistance of the transgenic plants by the insect receiving experiments. The results showed that the leaf of the Bivalent transferred gene plants have a lower damage and the bollworm has lethal; but the control’s leaf has a severely damaged and the bollworm was still alive, also the volume increased significantly. The leaf of the Bt transgenic plants was also moresevere damaged and the bollworm was death after four days. The serious injury of Bt transgenic plants shows that bollworm does have a certain amount of resistance, the modified bivalent plant’s resistance is significantly enhanced. This shows that the add of the signal peptide in the high-performance plant expression vector insurely improved the insect resistance to transgenic cotton.In this research, we constructed a biovalent expression vector which contains endoplasmic reticulum retention signal peptide sequence, the signal peptide sequence of soybean tryspin inhibitor and chloroplast targeting peptide sequence, then we transformed the vector with restore line Y18as the receptor. When we got the transgene plant,we had the southern hybridization, ELISA and insect resistance test on this plant. This research provide a new method for the obtain of a higher resistance cotton of bivalent as well as the accumulation of plant protein in other plant of genetic engineering. |
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