| Maize is one of the most important crop. Corn borer is one of main pests. If the pest occur, maize yield decreased by 10%-30%, and even more. Research and application of transgenic insect-resistant maize has become one of treatment to prevent corn borer all over the world. Currently, Bt crystal protein gene(cry) is used in transgenic insect-resistant maize widely, especially cry1 gene. And pest resistance has occurred with cry extensive used. To obtain a new cry gene through modified cry gene is an ideal way to solve this problem. And modified cry gene can expansion of cry gene insect resistant spectrum., increasing expression of gene in maize, increasing the insect-resistant ability of maize, And then get a new insect-resistant maize with high insect resistance.A new cry gene cryFLIa has obtain by domain swapping and codon optimization in early stage of laboratory work. In this study cryFLIa gene was transformed into HiII by agrobacterium mediated tansformation. Molecular biology identification, biological active identificantion and agronomic traits analysis were carried out on the progeny of transgenic plants. To explore the insect-resistant ability of cryFLIa gene in maize, and created a new maize germplasm with genetic stability, resistance to corn borer. And provide a theoretical reference to cry gene modified by protein engineering,then provide a new material resource to insect-resistant genentic maize. The result were as follows:cryFLIa has transformed into maize by agrobacterium mediated tansformation, and obtain T0 cryFLIa maize. The result of immune strip test show that CryFLIa protein can normally expressed in cryFLIa maize.To analysis enetic and expressed stability of cryFLIa maize. PCR, immune strip test and Southern blot anaylsis were performed to 3 self-generation, exhibiting that cryFLIa can genetic and expressed stability with one copy.RT-PCR and Real-time PCR were performed to leaves of development each stage of cryFLIa maize for cryFLIa transcript analysis The results showed that different cryFLIa expression quantity in leaves in different period. From high to low is filling stage, eight-leaf stage, tasselling and flowering stage, five-leaf stage. The results show that cryFLIa can transcript in each stage ELISA was performed to each stage and each organization for cryFLIa translation quantity analysis.The expression quantity of CryFLIa have different in each stage and each organization of cryFLIa maize.The expression quantity of Cry FLIa of leaves were higher than other organization in same stage. Expression quantity of CryFLIa in eight-leaf stage and filling stage were relative higher than other stage. CryFLIa expression quantity in each organization of filling stage was higher than same organization of other stage The results show that CryFLIa protein in each stage and each organization can express, and the expression quantity were different.Evaluation of cryFLIa maize resistant ability to corn borer through laboratory and field identification of insect resistance. Insect resistance identification in laboratory showed that mortality rate of corn borer in cryFLIa maize was 78%, which was higher than control group. Insect resistance identification in field showed that the resistance level of cryFLIa maize is resistant. The wormhole number was lower than non-transgenic plant. Insect tunnel length, ear damaged site number, seeds damage number were lower than non-transgenic plant significantly.The results show that cryFLIa maize has resistance to corn borer.The results of agronomic traits analysis show that cryFLIa maize lower than non-transgenic plant in height, and the difference was significant. The ear height, ear length, ear diameter, rows number, and kernels per row of cryFLIa maize were lower than non-transgenic plant, and the difference were not significant. The 100-kernel weight of cryFLIa maize was higher than non-transgenic plant, and the difference was not significant. |
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