| Maize is the first food crop in the world, which has been widely used in food, feed, industrial processing, new energy development and other aspects. Fluctuations in corn production will bring a series of negative effects in the worldwide. To ensure high and stable yields, and the quality of corn, to strengthen the prevention and control of pests and diseases and natural disasters is very important. In the past20years, people have successfully used genetic engineering for breeding new crop germplasm which have many excellent traits such as insect-resistant, disease-resistant, stress-resistant and herbicide-tolerant. Transgenic maize with insect-resistant and herbicide-tolerant has been widely planted, which effectively improve the maize yields and decrease production cost. At present, three factors of insect pests, weeds and drought, they seriously affect the corn yields. Therefore, it is a task with strategic significance to cultivate insect-resistant, herbicide-tolerant and drought-resistant transgenic maize. To develop transgenic maize with composite traits by multi-genes transgenic technique shows the highly competitive advantages, and is a worthy project for study.In the study a crylAc-M gene was used for the control of the heaving damage of corn by Ostrinia furnacalis, which has a strong insecticidal activity to Lepidoptera. CrylAc-M gene has been modified by optimization of codon and encoding frame length without changing the amino acid sequence of cry1Ac, so that the gene can be efficiently expressed in monocots. In order to improve glyphosate resistance of maize plants, we introduced both the epsps gene and GAT gene into maize. The epsps gene was cloned from a bacterial Halomonas Variabilis which isolated from extremely glyphosate-polluted soil and has the intellectual property of our country. The GAT acetylate herbicide glyphosate in the plants to relieve glyphosate toxicity. Two herbicide-tolerant genes cooperate to improve the ability of glyphosate-resistace effectively. The ZmPIS gene, cloned from maize in our laboratory, its expression was induced by a variety of stresses. Overexpression of the ZmPIS gene has significantly improved the drought resistance of maize plants.In this study, the plant expression vector with the four genes (crylAc-M, epsps, GAT and ZmPIS) was transformed into maize inbred lines9801and Qi319(Q319) mediated by Agrobacterium respectively, to obtain transgenic plants. The presence of the target genes was determined by the method of PCR detection in the transformed plants. And the stable transcription of the four target transgenes in the transgenic plants was confirmed using RT-PCR assay. In addition, we used real time PCR and Western blot to detect and confirm the stable expression of cry1Ac in the transgenic plants. After generational herbicide screening, PCR detection and insect resistance assay of plants in the fields, six genetically stable transgenic maize lines from a large number of transgenic plants were selected. Among them lines L1-L3come from9801inbred line, and lines Q1-Q3from Q319inbred line. Subsequently, inbred lines9801and Q319as the non-transgenic control to analysis the resistance of plants to Ostrinia furnacalis, glyphosate and drought under strictly controlled experimental conditions.Resistance to Ostrinia furnacalis of transgenic plantsBased on the different resistance to Ostrinia furnacalis of maize plants at different developmental stages, in this study we tested the resistance to Ostrinia furnacalis of transgenic plants at different developmental periods indoor and in the fields by innoculating the worm of Ostrinia furnacalis. The observations about the insect-resistace of maize plants showed that high resistance lines only had a small amount of wormholes on the initial infestation leaves, and no signs of further harm. These lines showed much better resistance to the corn borer, compared with the control plants, highly significant differences at the0.01level arrived. Examination of transgenic corn kernels and bracts of ears at filling stage to the insect toxity concluded that the mortality of corn borer fed with transgenic kernels and bracts of ears was significantly higher than that of control (non-transgenic). Thses results indicated that the transgenic maize have a good effect on preventing the damage from corm borer and arrived to the level of extensive utilization in corn production.Glyphosate resistance analysis of transgenic plantsIn this study, the epsps gene was combined with GAT gene to improve the glyphosate-resistance of transgenic plants. We sprayed1.05kg ae ha-1glyphosate as a selection pressure at maize three-leaf stage to screen the resistant corn lines. In the herbicide resistance trials in the fields, transgenic plants were sprayed with0.84kg ae ha-1Roundup at the six-leaf stage to evaluate the resistance of transgenic plants (the commercial application dosage is0.84kg ae ha-1). Transgenic plants showed good resistance, which has meeted the criterion of actual application. In addition, we also performed the high concentration herbicide spraying experiment, choosing twice the dose of glyphosate to value glyphosate-resistant levels of transgenic plants. The transgenic plants appeared slightly damage symptom with latter effects at this dosage, but all non-transgenic plants died.Drought resistance analysis of transgenic plantsMaize plants at the10-leaf stage were used to carry out drought stress experiments, and the soil moisture content was maintained at17-18%for7days before restoring watering the plants. During drought stress, both transgenic plant morphology and photosynthesis rate showed much better compared to the WT, such as more expaned, leaves and lesser extent of wilt. After the restoration of watering transgenic plants quickly recovered normal growth. Photosynthesis and chlorophyll fluorescene measurements indicated that transgenic plants had more strong photosynthetic capacity and photosystem II activity than that of the non-transgenic control plants under drought stress conditions,.The transgenic plants were less damage by drought stress,and showed improved drought-resistance.In summary, the introduction of the crylAc-M, epsps, GAT, ZmPIS gene into maize elite inbred lines increased the insect-resistance, glyphosate-tolerance of transgenic plants and improved the drought-resistance of transgenic plants. This study created six new com transgenic lines with excellent complex traits, which provided new germplasms for the breeding of transgenic multi-trait stacking corn varieties. |
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