棉花学报   Cotton Science  2006，18(1)：19～22

Study on the Quantitative Method and the Degradation Rule of Bt protein in Bt Cottonseeds
XU Bao-liang^1，2，SU Ning²，YUAN Fe²，WANG Yin-xia¹，CHEN Ying²，WU Ya-jun²，ZHANG Qing-wen¹
(1. China Agricultural University，Beijing 100061，China；2. Chinese Academy of Inspection and Quarantine，Beijing 100025，China)

Abstract：Cottonseeds，cottonseed kernels and cottonseed shells of 99B and 33B were stored in a dry and dark place at room temperature. Samples were taken every month and grounded. Extractions of cottonseeds，kernels and shells were prepared using the method described in the instructions of the kits. The extractions were serially diluted with the extraction buffers at the ratio of 1∶1 (volume to volume). Bt protein contents of cottonseed kernels were determined using Envirologix kit followed the instruction of the kit. Optical densities of the extraction and the diluted extractions from cottonseeds，kernels and shells at 450 nm and 630 nm were determined using Envirologix kit and Agdia kit respectively followed the instructions of the kits.
    The Bt protein contents in 99B and 33B cottonseed kernels were 4376.4 ng·g^-1and 4321.7 ng·g^-1 respectively. The OD₄₅₀ of series of dilutions of the cottonseed kernel extracts measured using the Envirologix kit was linearly correlated with the OD₆₃₀ detected using Agdia kit，and the correlation coefficient was almost 0.99. Furthermore，the OD₆₃₀ of the dilutions was also linearly correlative with the contents of Bt protein，and the correlation coefficient was larger than 0.99. Thus the degradation rule of Bt protein in 99B and 33B cottonseeds，kernels and shells in the process of 12 months storage were determined using Agdia kit，and the Bt protein contents in the cottonseeds and kernels were half after the storage，while the contents in the shells were one fourth，both in 99B and 33B.
    Envirologix kit with Bt protein reference standards could be used to determine the Bt protein quantitatively. Agdia kit with high sensitivity was able to detect the Bt protein in cottonseed，kernels and shells qualitatively. The quantitative method with high sensitivity for the determination of Bt protein was established in this study based on the characteristics of Envirologix kit and Agdia kit，and the degradation rules of Bt protein in 99B and 33B cottonseeds， kernels and shells in the process of storage were described in Bt amounts. The contents of Bt protein in cottonseed shells decreased rapidly in the process of storage，and the contents of Bt protein in cottonseeds and kernels decreased more rapidly in the storage. When cottonseeds， kernels and shells were used as materials in the study related to the Bt protein，the degradation of Bt protein in the storage process of those materials should be taken into account. When the Bt protein contents in cottonseeds and kernels were determined using Envirologix kit and Agdia kit，the Bt protein contents were not linearly correlated with net optical density at low diluted times，but were linearly correlated with the net optical density at high diluted times. As such， for the determination of Bt protein the extractions of cottonseeds and kernels should be diluted until the Bt protein concentrations were linearly correlated with the net optical density measured using the kits. The extraction should be diluted about 100 times，and thus the Bt protein contents measured using the kits were real，otherwise the content of Bt protein in cottonseeds and kernels determined using the kits would be lower than that normal.
Key words：Bt cottonseed；Bt protein content；degradation rule
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