摘要
将通过Error-prone PCR方法获得的表达水稻EPSP酶突变基因epsp102和未经修饰的水稻epsp基因分别导入籼稻恢复系明恢86,获得转化克隆84个和109个。对T1、T2代转基因水稻三叶期和分蘖期喷施草甘膦,各选出4个对草甘膦高抗性的T3代材料,进行分子鉴定。对其中的3个株系进行农艺性状考察,并各选1个株系作为父本与其他优良恢复系进行杂交,进一步考察epsp和epsp102基因在不同遗传背景下的抗性和农艺性状表现。Southern结果显示:epsp的整合拷贝数为2～3拷贝,epsp102拷贝数为1～2拷贝。萌发种子、三叶期及分蘖期对草甘膦抗性检测显示:转基因萌发种子对草甘膦的抗性提高15倍;三叶期对草甘膦的抗性提高3～4倍。分蘖期剂量效应检测结果显示:非转基因对照在施0.1倍以上的致死剂量农达时,植株显著失水,而转基因株系水分生理正常。两地两季的农艺性状考察显示:与对照相比转基因株系的穗数明显增多,转基因株系ep-3和ep102-20结实率和单株重与对照没有显著差异;以ep-3和ep102-20作父本,与4个恢复系杂交的F2代对草甘膦的抗性没有降低,两个组合的结实率显著增加,SK/ep-3组合结实率显著下降,其余组合的结实率与对照持平。
The rice （Oryza satire L.） epsp gene encodes an enzyme called 5-enol-pyruvylshikimate-3-phosphate （EPSP） synthase, which has been. shown as the primary target of the herbicide glyphosate. Both wild type and epsp mutant, namely epsp 102 mutagenized by Error-prone polymerase chain reaction, were introduced into rice restorer line Minghui 86 via Agrobacterium tumefaciens-mediated, 84 and 109 independent clones were obtained respectively. Glufosinate resistance in T1 and T2 progeny were confirmed under field condition by screened with roundup twice at commercially recommended dose in each generation. T3 was used for molecular detection, glyphosateresistant test and agronomic trait evaluation. Southern blot analysis of glyphosate-resistant transgenic progeny indicated that lines with epsp exhibited 2-3 bands, while those lines with epsp102 showed 1-2 bands, that means epsp102 confer higher glyphosate-resistant to rice. Transgenic T3 seedlings grew normally on the solution with 0.05 mmol/L and above glyphosate and formed well-developed roots and rootlets. While wild type seedlings grew abnormally and exhibited poor root development. At three-leaf period spraying with glyphosate both transgenic rice could do tolerance 20 mmol/L glyphosate compared to the wild type of 5 mmol/L. Water physiology experiment shows the transgenic plant with normal water ratios at roundup concentration range from 0.01 to 1 time commercially recom- mended dose, while wild type water lose significantly at 0.1 time dose. Base on above results, three lines include ep-3 content epsp, ep102-15 and ep102-20 content epsp102 were selected out for agronomic traits evaluation. The results of field .testing show that the spike number of transgenic rice were more than that of wild type, ep-3 and ep102-20 lines show no significant difference in percentages of ripen grain and grain yield per plant. The transgenic hybrid lines were produced from hybridization of ep-3 and ep102-20 with 4 elites restores lines. F2 were investigated. The results indicated that of the total 8 combinations there were 2 combinations with higher yield, 1 combination with lower yield and others showed no significant difference compared to wild type.
出处
《分子植物育种》
CAS
CSCD
2008年第5期830-836,共7页
Molecular Plant Breeding
