甘蓝型油菜抗草甘膦基因遗传转化及抗性鉴定

为自主研发抗除草剂的甘蓝型油菜，从细菌（Isotericola variabilis）中克隆I.variabilis-EPSPS*基因,使油菜产生草甘膦抗性。通过农杆菌介导法将I.variabilis-EPSPS*转入甘蓝型油菜自交系育127，获得转I.variabilis-EPSPS*基因单株E₁。草甘膦耐受性结果表明，转I.variabilis-EPSPS*基因T₁代在不同浓度草甘膦的培养基中能够正常生长，而非转基因对照生长严重受阻。I.variabilis-EPSPS*成功整合于油菜基因组并可稳定遗传，草甘膦处理后表达量增加，而且在草甘膦浓度改变时仍保持稳定表达。在温室用41%草甘膦异丙胺盐商品制剂（农达）600倍稀释液（1/3田间生产推荐中剂量）处理转I.variabilis-EPSPS*基因E₁的T₁植株，体内莽草酸累计量远低于非转基因对照。通过对角果长、每角果粒数和千粒重等农艺性状考察发现，转基因植株和对照相比无显著性差异。田间喷施农达400倍液（1/2田间生产推荐中剂量），发现转基因E₁T₁植株正常生长而对照全部死亡。鉴于I.variabilis-EPSPS*基因在转基因油菜中能够稳定遗传并赋予了油菜草甘膦除草剂抗性，认为该抗草甘膦转基因油菜是一份新种质。

Genetic transformation and resistance evaluation of glyphosate resistance gene in Brassica napus

A novel glyphosate-resistant gene （designated as I. variabilis-EPSPS?） was cloned from bacterium of Isoptericola variabilis. In this research, I.variabilis-EPSPS* was overexpressed in Brassica napus L. cv Yu 127, under the control of constitutive CaMV35S promoter. Seeds from I.variabilis-EPSPS* transgenic plants could germinate and grow well in the presence of increasing concentrations of glyphosate（40-80 mg L^-1）. In contrast, wild type line had significantly reduced shoot and root length. qRT-PCR indicated that expression level of I.variabilis-EPSPS* increased after isopropylamine salt （Roundup） treatment, and while expression remained unchanged. After spraying 600× dilution of 41% glyphosate Roundup （1/3 recommended dose in field）, the cumulative amount of shikimic acid in I.variabilis-EPSPS* transgenic T₁ plants was lower than wild type. Additionally, agronomic performance demonstrated no significant difference in silique length, seeds per silique, and 1000-seed weight （etc.） between I.variabilis-EPSPS* E₁T₁ and wild type in greenhouse. Foliar glyphosate application in field showed that glyphosate at concentration of 759.8 mg L^-1 had no detrimental effect on the growth of I.variabilis-EPSPS* plants. Furthermore, the glyphosate-sprayed transgenic plants were preriodically monitored until maturity, and it was observed that they exhibited normal growth and morphological characteristics similar to those of WT which was not spayed with glyphosate in the greenhouse. In summary, the I.variabilis-EPSPS* gene and its transgenic glyphosate-resistant B. napus L. line E₁ could be the new materials for genetical modified rapeseed breeding resistant to glyphosate herbicide.