Genetic Transformation And Insect Resistance Identification Of Cry1Ab13-1 Insect Resistance Gene In Maize

The maize is one of the most important food crops and industrial raw materials in China,and China's demand for maize is also increasing.Therefore,increasing maize production is an urgent task,but the problem of maize pests has always been an important factor restricting the increase of maize production.It will have a huge impact on China's maize production every year,especially the damage caused by maize borer.The use of chemical agents to control pests has the disadvantages of polluting the environment and destroying ecosystems,and cannot solve the problem of insect pests fundamentally.Therefore,it is very important to use transgenic technology to create new insect-resistant maize inbred lines and breed new varieties.In this experiment,a Cry1Ab13-1 prokaryotic expression vector was used to construct a eukaryotic plant expression vector PCAMBIA3301-Cry1Ab13-1 with the herbicide resistance Bar gene as a selection marker through seamless cloning technology.349049,90493,S3002,MJ-1,H299,W107,W108,Bao A,Bao B,Bao C,Bao D,Bao E and Zheng 58 are the recipient inbred lines,using the pollen tube channel method and ultrasonic pollen-mediated method The plasmid containing the eukaryotic plant expression vector PCAMBIA3301-Cry1Ab13-1 was introduced into the maize receptor,and the T₁transgenic plants were subjected to preliminary PCR detection.Positive fruit ears were harvested.T₂plants were planted in the second year and conventional PCR,Southern blot,Quantitative real-time PCR detection and indoor and outdoor insect resistance identification,further breeding a new insect-resistant maize inbred line,the test results are as follows:1.The Cry1Ab13-1 gene was cloned from the prokaryotic expression vector of Cry1Ab13-1and the Cry1Ab13-1 gene was connected to the eukaryotic plant expression vector PCAMBIA3301-Cry1Ab13-1 by seamless cloning technology.The herbicide resistance gene Bar was successfully constructed.The gene is a selection marker eukaryotic plant expression vector PCAMBIA3301-Cry1Ab13-1.2.The eukaryotic plant expression vector PCAMBIA3301-Cry1Ab13-1 was introduced into 16 recipient maize inbred lines using the pollen tube channel method and ultrasonic pollen-mediated method.It was planted in the second year,and the T₁generation was screened with herbicides.About 28,800 plants were tested,and a total of 159 plants were selected with a survival rate of 0.552%.T₁generation transgenic positive plants were obtained by pollen tube channel method with a transformation rate of 7.5%,and 4 T₁generation transgenic positive plants were obtained by ultrasonic pollen-mediated method with a transformation rate of 5%.3.Using the Cry1Ab13-1 gene as a probe,Southern blot tests were performed on T₂positive strains.Eight strains had positive bands.Among them,LMA-1-1 and LMA-6-2 were multiple copies,and the rest were single copies.Irregular distribution.It was proved that the Cry1Ab13-1 gene has been successfully integrated in different parts of the maize genome.4.The quantitative fluorescent detection was performed on the root,stem and leaf parts of8 strains that were positive for Southern blot.The Cry1Ab13-1 gene expression was the highest in the roots.The strain is LMA-1-2,and the strain with the highest expression in leaves is LMA-6-4.Different positive strains have different expression levels of foreign genes in the same part,and different parts of the same strain have different expression levels.5.The maize borer resistance identification results in laboratory showed that the survival rate and weight growth rate of maize borer fed with LMA-1-1 and LMA-1-2 strains were significantly lower than those of the control group LMA-1-CK;The survival rate of maize borer fed with LMA-6-1,LMA-6-2,LMA-6-3 and LMA-6-4 maize leaves was significantly lower than that of LMA-6-CK;Except for LMA-6-2 group,the weight gain rate of the other 3 groups of maize borer was significantly lower than that of the control group,of which LMA-6-1 and LMA-6-4 The weight of maize borer in the group was basically unchanged.It was proved that the six lines of maize transgenic with Cry1Ab13-1 gene were resistant to maize borer.Maize borer growth was inhibited and even died after feeding Cry1Ab13-1 gene transgenic maize.6.The identification results in laboratory resistance to Mythimna separata showed that:no Mythimna separata death was found in the LMA-6-4 group,the LMA-9-1 group and the LMA-12-1 group,but the average speed of weight gain of each test group was significantly lower than that of the control group within 48 hours.It was proved that the LMA-6-4,LMA-9-1and LMA-12-1 lines transgenic with Cry1Ab13-1 gene were resistant to Mythimna separata,and the Mythimna separata was obviously reluctant to eat the maize transgenic with Cry1Ab13-1gene.7.The identification results in field resistance to maize borer showed that the resistance level of strain LMA-1-1 was resistance,the resistance level of LMA-1-2 was high resistance,and the resistance level of control group LMA-1-CK was medium resistance;Strains LMA-6-1,LMA-6-2,LMA-6-3,LMA-6-4,,except for the resistance level of the strain LMA-6-3,all others reached high resistance,and the control group with a high resistance level of LMA-6-CK was intermediate resistance.The resistance of each transformed strain to maize borer was significantly improved compared with the control group,and the resistance of each transformed line was different,and the identification results of indoor resistance to maize borer were basically consistent with the identification results of field resistance to maize borer.It was proved that the insect resistance of maize transgenic with Cry1Ab13-1 gene was significantly improved and had good insect resistance.8.The eight maize lines transgenic with the Cry1Ab13-1 gene LMA-1-1,LMA-1-2,LMA-6-1,LMA-6-2,LMA-6-3,LMA-6-4,LMA-9-1,and LMA-12-1 all significantly improved their insect resistance and showed excellent insect resistance.