Quantification Of Cry1Ac Protein In Bt-Cpti Rice And Its Resistant Efficacy Chilo Suppressalis (Walker)

In this paper, expression of Bt insecticidal crystal proteins and their resistant effects of transgenic rice (Oryza sativa L ) on striped stem borer( Chilo svppressalis Walker) were explored by ELISA, Electron microscope observation and bioassay methods. Some important results were obtained as follow.In order to replacing the rice plants with the artificial diet or semi-artificial diet to raising striped stem borer C. suppressalis a series research were conducted. It was found that cane shoot and chufa can be used to raise striped stem borer C. suppressalis successfully. The results from the ELISA showed that Cry1 Ac protein could be detected in all the organs along the developmental process of cry1Ac/CpTI transgenic rice. The contents of the Cry1 Ac proteins varied obviously in different tissues at different stages. Generally, the contents of Cry1 Ac proteins was higher in earlier stage than that in later stage. The highest levels of the Cry1 Ac proteins in terminal leave of main stem were found at jointing stage, since then, it decreased at booting stage, and the lowest level was detected at flowering to filling stage, while increased a little at maturing stage. Higher levels of Cry1 Ac proteins in main stem were found at seedling stage, jointing stage and flowering stage than at other stages. There were relative lower level of Cry1 Ac proteins in seed. The results from laboratory bioassay showed that there were high resistance of the terminal leave of main stem along the developmental process to the newly hatched larvae Chilo suppressalis. The corrected mortalities of C. suppressalis on the 4th day after feeding on the detached leaves of cry1Ac/CpTI transgenic rice were up to 90% before filling stage , while decreased nearly down to 60% after filling stage. It was showed that the results of whole plant bioassay were quite coincident with those of the detached leaf bioassay. The tendency of larval moralities in the detached -leaf bioassay and whole plant bioassay were consistent with that of Cry1 Ac proteins contents in different growing periods/organs.By detached -leaf bioassay method, the resistant levels of 3 type of transgenic rice lines (single cry1A transgenic lines, single CpTI transgenic lines and cry1Ac/CpTI transgenic lines respectively) to C. suppressalis were investigated .It was found that MSB( cry1Ac/CpTI transgenic line) show high and stable levels of resistance to infestation of striped stem borer larvae. Moreover, the corrected mortalities of striped stem borer larvae infested on MSB was obviously higher than those of the other transgenic rice. The corrected mortalities of C. suppressalis on the 4th day after feeding on the detached leaves of MSB were up to 100% in seedling stage, stooling stage and stooling stage. However, those corresponding value for 81AS1 (single CpTI transgenic line), 86AS2 (single CpTI transgenic line), 86AB1 (single cry1A transgenic line) and 86AB2 ( single cry1A transgenic line ) were about 30% before flowering stage. The corrected mortalities of C. suppressalisfeeding on detached leaves of 81AS1,86AS2,86ABland 86AB2 in booting stag were 44.681%, 32.653%, 63.265% and 52.041% respectively. It was indicated that transferring double gene could enhance resistant levels of rice plants effectively.Under electron-microscop, the midgut tissues of the C. suppressalis larvae feeding on transgenic rice plant were observed. It was show that midgut tissues of the 3d larvae feeding on transgenic rices were physiologically and pathologically changed. The main changes were in the midgut microvilli, endoplasmic, recticulum, mitochondria and the cell nucleolus. After larvae fed on the leaves of MSB, the midgut microvilli swelled and then fell off. The intersoace of the endoplasmic recticulum expanded and then hollow formed. The mitochondria swelled ,and then cristae mitochondriales were absent. The cell nucleolus was distorted ,and the chromatin got together. When larvae were injured badly, the membrane of the cell and nucleolus would break off. However the pathological changes induced by 86AS1 and 86AB2 in midgut tissues of C. suppressalis larvae were much more slightly than those induced by MSB. It was indicated that transferring multi-gene could obviously enhance the resistance of transgenic rice to C. suppressalis.