Genetic Transformation And Resistance Identification Of The Transgenic Maize With ZmPS1 And Cry1Ab13-1 Genes

In the context of climate change and the increase of Maize continuous cropping frequency,drought stress and Asian corn borer（Ostrinia furnacalis）have become major concerns to maize sustainable production.In regards to these multiple adversities,it is of great importance to breed maize varieties with complex biotic and abiotic resistance.The utilization of drought-resistant and insect-resistant genes is an effective way to improve crop stress resistance.In this study,plant expression vectors were constructed by cloning the maize genes viz.Zm PS1（drought-resistant gene）and cry1Ab13-1（insect-resistant gene）.The genetic transformation of maize inbred lines via ultrasonic and pollen tube methods.Bivalent transgenic lines were screened and tested for drought and insect resistance at seedling stage under laboratory and field conditions.The aim of this study was to obtain transgenic maize inbred lines with improved drought and insect resistance,thus lay a foundation for complex resistant varieties crucial for maize breeding programs.The main results are as follows:1.The bivalent plant expression vectors of Zm PS1 and cry1Ab13-1 genes were constructed using seamless cloning method.The inserted fragment was accurately confirmed via blue-white spot screening,double enzyme digestion verification of the recombinant vector,PCR detection of the target gene and vector sequencing,and the recombinant vector was successfully constructed2.Two maize inbred lines W55 and W107 were transformed by the ultrasonic-mediated method and pollen tube channel method,and T₁transgenic plants were obtained after screening for glyphosate resistance and PCR identification.The T₂and T₃generation transgenic plants were randomly selected for Southern blot verification.The results showed that cry1Ab13-1 gene was integrated into maize genome in the form of single copy with differential integration sites among maize inbred lines.Meanwhile,q RT-PCR was performed using roots,stems and leaves of the T₂and T₃transgenic plants.The results showed that Zm PS1 and cry1Ab13-1 displayed significantly（P<0.01）different expression levels in the different lines and organs.The highest relative expression level was obtained from the leaves.Finally,OE-1 of W55 transgenic line was obtained by ultrasonic-mediated transgenic method,and OE-2 of W107 transgenic line was obtained by pollen-tube transgenic method.3.Drought-resistance screening of T₃transgenic plants at seedling stage was performed using the indoor water cut-off method.The results showed that under 3,7,11 and 15 days of drought stress,the proline contents of OE-1 were respectively,5.81μg/g,7.04g/g,16.24μg/g and 20.35μg/g,which were significantly（P<0.01）higher than those of the receptor control by1.58,1.28,1.95 and 1.82 folds.The proline contents of OE-2 were respectively,4.38μg/g,5.42μg/g,10.18μg/g and 14.80μg/g,significantly（P<0.01）higher than those of the receptor control by 1.90,1.53,1.45 and 1.49 folds.This was confirmed by the significant expression of ZMPS1 in the transgenic plants detected by quantitative PCR.In addition,dry matter weight,relative water content,SOD activity and POD activity of transgenic plants were significantly（P<0.01）higher as compared to the wild type control after 15 days of stress.Moreover,MDA content was significantly（P<0.01）lower than that of the receptor control.Thus,the overexpression of ZMPS1 caused excessive accumulation of proline in the transgenic plants,which resulted in significantly improved droμght resistance for the transgenic plants.4.The indoor biological activity trial results showed that after 6 days of feeding,the larval mortality in the transgenic corn group was statistically（P<0.01）1.77 folds higher than that in the receptor control group,while the swallowed maize leaves biomass in the receptor control group was significant（P<0.01）1.42 folds higher than that in the transgenic corn group.The field bioactivity test results showed that after 14 and 21 days of inoculation,the leaf feeding level of the transgenic plants was significantly（P<0.01）lower than that of the receptor control,and the resistance level of the transgenic plants to the corn borer increased from moderate to high.Thus,transgenic cry1Ab13-1 gene can improve the resistance of maize to the Asian corn borer.