The Expression And Inheritance Stability Of Bt Insecticidal Gene In Transgenic Insect-resistant Cotton Plants

The application and dissemination of Bt transgenic cotton have brought about great economic profits to the cotton production and alleviated efficiently the pollution made by spraying the chemical insecticidal regent. But there ait two prevalent problems in the production of the Bt transgenic cotton both in home and abroad. The first one is that the efficacy of the Bt transgenic cotton in the early developmental stages is high, then it will decrease thereafter. It needs 3-4 sprays in order to conlrol the insects in the later developmental stages. The second one is that the environmental factors such as the temprature, the light intensity and the moisture influence the efficacy of Bt transgenic cotton plants , and therefore influence the stability of the Bt transgenic cotton production. A series of research works are done in order to resolve these two problems. The results are as follows. There are two HindIII restriction sites at each end of the Bt toxin gene in the vector pGBI 121 S4ABC. Southern blot analysis of the GK 139-20 R3 insect-resistant transgenic cotton plants indicates that the Bt toxin gene introduced by pollen tube pathway has been integrated into the genome of the cotton. There is only one Xhol restriction site in the vector of pGBI 121 S4ABC and the Bt toxin gene. In order to determine the copy number and the inheritance stability of the Bt toxin gene in the transgenic cotton plants, southern blot analysis were done. The results indicate that there are many copies of Bt toxin gene integrated into the genome of cotton. Among them, four copies which are about 17.7, 8, 5.5 and 4.7 kilobases existing in all the tested R3 and R4 materals. This indicates that these four copies can be stably inherited. Furthermore, southern blot analysis of nine R5 transgenic cotton plants DNA digested with Xhol was done in order to prove the above results. The results show that the four copies that possessed by all the R3 and R4 materials exist in the genome of the R5 materials too. It is evident that the Bt toxin gene in the insect-resistant transgenic cotton plant can be stably inherited. lv The bioassay of the insecticidal activity of the Bt transgenic plant at different developmental stages indicates that the phenomen 揟he efficacy of Bt transgenic cotton is high at early developmental stages, and decrease thereafter?exists in the tested GK139-20 insect-resistant Bt trarrsgenic plants too. The results of ELISA display that the expression of the Bt crystal protein is different in different tissues and at different developmental stages. The content of the Bt crystal protein decreased along with the gro~h of the transgenic cotton plants. The results of RNA dot blot analysis indicate that the expression of Bt toxin gene at mRNA level is different in different tissues and at different developmental stages. But the amount of the Bt toxin gene mRNA is not proportional to the content of Bt crystal protein. The result indicates that the decrease of the Bt crystal protein is regulated at post- transcriptional or translation levels. Northern blot analysis of the total RNA extracted at different developmental stages further indicates the decrease of the amount of Bt crystal protein was caused by the decrease of the full-length of Bt toxin gene transcripts. This regulation is at post-transcription level and the gene silencing is developmentally regulated. That explained in theory why the phenomen 搕he efficac...