| The transgenic lines of apple that carring cowpea trypsin inhibitor (CpTI) gene were used to study the exogenous gene expression by using Isoelectric Focusing Electrophoresis (IEF) and Capillary Zone Electrophoresis CZE, and feeding the Helicoverpa armigera larvae with the leaves. The transgenic lines were Gala, Orin, Jonagold, and Fuji cultivars, and obtained via Agrobacterium-mediated gene transformation. They were numbered Gala 1 to 46, Orin 47 to 55, Jonagold 56 to 58, and Fuji 59 and Fuji 60. The survival rate and the changes of the midgut chymotrypsin-like enzyme activity of the larvae were recorded. The main results were as follows:1. The optimal protocol for IEF in apple were that: extracting the protein form leave by using improved ace-sediment method;choosing the concentration of the gel as 3.66%;choosing the carrier ampholytes" pH of 3.5-9 and its concentration of 2%;and loading 33.8μg protein for each tube. The main proceeding was: 100V, 10min;500V, 10min;750V, 3h 30min. The Coomassie blue-stained was used for the gel staining.2. The result of IEF showed that twelve clones in Gala cultivar, which were 2, 8, 11, 12, 14, 19, 27, 31, 32, 33, 34, and 36, had found specific protein brands between the pH ranges of 6.18-6.30. Four lines in Orin, which were 47, 50, 51, and 55 had found specific protein brand between the pH ranges of 6.18-6.30. It was also found a specific protein brand in Fuji 59 clones. But there was no specific protein brand founded in Jonagold cultivar.3. The optimal protocol for CZE was that: extracting the protein form leave by using improved ace-sediment method;Setting the voltage as 20kV and the temperature as 25 ℉;and setting the isoelectric focusing time as 17 minutes and the wavelength as 280nm. The result of CZE showed that at the 7.65 minutes there were exceptional protein brands founded in the lines of 12,19, 32, and 39 in Gala cultivar.4. The feeding experiment showed that some lines had the effect on restricting the larvae growth or killing. The line 32 and 51 had the strongest affection by killing the larvae of 100%. There were twenty eight lines showed positive value of the modified death rate, and that were line 2, 4, 8,9, 10, 11, 12, 13, 16, 17, 19,21,24, 27,31,32,33,34,36,39,43,47,50,51, 55, 56, 58, and 59. And the modified death rate of the line 2, 12, 19, 27, 32, 31, 34, 39, 50, 51 and 56 were higher than or equal to 50%. That indicated that those lines have the ability torestrict the growth of the larvae or kill the larvae. The weight of the larvae that fed by the leave of the line 27, 31, 32, and 51 showed significant difference with the control.5. The activity of the midgut chymotrypsin-like enzyme of the larvae after fed by the leave of the transgenic lines was varied from the lines. The activity was significant difference from the control when the larvae were fed by the leave collected from the line 4, 10, 12, 32, 39, 43, 47, 50, and 56. That indicated those lines had certain capacity of restraining the growth and develop of the insect.
|
PDF Full Text Request