兼抗虫、除草剂、干旱转基因玉米的获得和鉴定

doi:10.3864/j.issn.0578-1752.2015.02.02

摘要/Abstract

摘要： 【目的】培育抗玉米螟、抗草甘膦、抗旱的转基因复合性状玉米种质。【方法】通过农杆菌介导的玉米茎尖遗传转化法，把重组到同一植物表达载体的cry1AcM、epsps、GAT和ZmPIS转入玉米骨干自交系9801和齐319（Q319），获得转基因植株。通过逐代除草剂筛选、分子检测和抗虫性鉴定，从大量转基因株系中优选出6个优良玉米株系。在此基础上，以非转基因自交系9801、Q319为对照，在严格控制条件下对6个转基因株系进行抗虫、抗除草剂和抗旱性检测。由于不同生长时期植株对玉米螟危害的敏感程度有差异，在抗虫性检测试验中，对不同生长阶段的转基因玉米的抗虫性，分别进行了田间和室内玉米螟接种试验，并以灌浆期玉米的籽粒和苞叶饲喂玉米螟幼虫检测其杀虫性。在草甘膦抗性鉴定试验中，对6叶期田间玉米植株喷洒大田除草用量的草甘膦溶液评估其应用价值；采用3倍应用剂量的草甘膦溶液喷施3叶期玉米植株测试其草甘膦耐受力。在抗旱性鉴定试验中，对10叶期玉米植株进行干旱胁迫处理，观测转基因植株的表型变化并测定光合作用和叶绿素荧光等参数。【结果】选择出6个遗传稳定的兼抗亚洲玉米螟、抗草甘膦和抗旱性提高的转基因玉米株系，其中株系L1—L3来自自交系9801，Q1—Q3来自自交系Q319。通过RT-PCR检测4个转基因的转录强度，证明它们在转基因植株中有效表达；利用Western blot方法检测cry1Ac蛋白的表达水平，确定其在玉米植株中稳定表达。植株抗虫性鉴定结果显示这6个株系在玉米营养生长期和灌浆期的抗虫能力均显著高于未转基因自交系。在草甘膦抗性测试中，转基因植株表现出明显高于未转基因自交系的草甘膦抗性。在干旱控水期间，转基因植株能维持较强的光合能力和光系统Ⅱ活性，对干旱胁迫的抗性显著高于对照植株。【结论】将cry1AcM、epsps、GAT、ZmPIS一同转入玉米，赋予了植株抗玉米螟、抗除草剂草甘膦特性，提高了植株的抗旱性，达到生产中应用标准。培育出具有优良复合性状的转基因玉米新材料。

关键词: 转基因玉米, 复合性状, 抗玉米螟, 抗草甘膦, 抗旱

Abstract:

【Objective】 The objective of this experiment is to study the production of transgenic maize with triple resistance of insect, glyphosate and drought stress. 【Method】In this study, an expression vector harboring four target genes (cry1AcM, epsps, GAT and ZmPIS）was used to transform to maize elite inbred lines 9801 and Qi 319 (Q319) mediated by Agrobacterium, and produced transgenic maize plants with multiple modified traits. Based on herbicide resistance, PCR analysis, transcriptional analysis, western blot detection and insect resistance assay, six stable transgenic maize lines were selected from a large number of transgenic lines. Subsequently, the inbred lines 9801 and Q319 were used as the nontransgenic control to analyze the resistance of transgenic plants to Ostrinia furnacalis In view of different resistances to O. furnacalis of maize plants at different developmental stages, the resistance to O. furnacalis of transgenic plants at different developmental periods was tested indoor and in the fields by innoculating the worm of O. furnacalis, and kernels and the bracts of ears from transgenic plants at filling stage were used to feed the worm. In the herbicide resistance trials in fields, transgenic plants at the six-leaf stage were sprayed with 0.84 kg·hm^-2 glyphosate (commercial application dosage) to evaluate the resistance of transgenic plants. A spraying experiment with triple dose of glyphosate on the plants at three-leaf stage was also performed to evaluate the glyphosate-resistant levels of transgenic lines. To evaluate the resistance of drought stress, the morphological changes of transgenic plants were observed and their photosynthesis and chlorophyll fluorescence were measured on plants at the 10-leaf stage in a drought stress treatment. 【Result】Among six stable transgenic maize lines, L1-L3 come from inbred line 9801 and line Q1-Q3 come from inbred line Q319. The stable expression of the four transgenic genes in the transgenic lines was confirmed by using RT-PCR assay. The levels of cry1Ac protein in the different transgenic lines were determined by Western blot, which showed reliable expression in various organs of the transgenic lines. Six lines with higher resistance to the corn borer compared with wild type were selected through the corn borer resistance experiment in the plants at vegetative stage and filling stage. For herbicide resistance trials, the glyphosate resistance of transgenic plants showed more tolerant than that of wild type control. During drought stress, the transgenic plants had more strong photosynthetic capacity and photosystem II activity than that of the nontransgenic control plants under drought stress conditions. 【Conclusion】The introduction of the cry1Ac-M, epsps, GAT, and ZmPIS genes into maize elite inbred lines increased the insect-resistance, glyphosate-tolerance, and improved the drought-resistance of transgenic plants, and the transgenic plant are up to the level of extensive utilization in corn production. Six new corn transgenic lines with excellent complex traits were obtained in this study., glyphosate and drought under strictly controlled experimental conditions.

Key words: resistance genetically modified maize, complex traits; insect resistance, herbicide tolerance, drought

孙越，刘秀霞，李丽莉，官赟赟，张举仁. 兼抗虫、除草剂、干旱转基因玉米的获得和鉴定[J]. 中国农业科学, 2015, 48(2): 215-228.

SUN Yue, LIU Xiu-xia, LI Li-li, GUAN Yun-yun, ZHANG Ju-ren. Production of Transgenic Maize Germplasm with Multi-Traits of Insect-resistance, Glyphosate-Resistance and Drought-Tolerance[J]. Scientia Agricultura Sinica, 2015, 48(2): 215-228.

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