采用农杆菌介导法,以下胚轴为转化受体,将溶血性磷脂酸酰基转移酶(LPAAT)和酮脂酰辅酶A合成酶(KCS)基因导入2种不同基因型(“Drakkar”和“Maplus”)的甘蓝型油菜,共获得247株再生植株,其再生频率分别为15.1%和2.0%。PCR检测和Southern杂交分析证实,外源基因已整合到油菜基因组中,转化频率分别为3.6%和0.7%。种子中芥酸含量因外源基因的导入使零芥酸油菜“Drakkar”提高到10.5%,高芥酸品种“Maplus”提高了5.0%,最高达62.8%。
A protocol for Agrobacterium tumefaciens-mediated transformation of Brassica napus hypocotyls was designed. A strain with a LPAAT (lysophosphatidic acid acyltransferase) gene from Limnanthes douglasii and a KCS (beta-ketoacyl-CoA synthase) gene under control of a napin promoter was co-cultivated with hypocotyls from two genotypes of B. napus, erucic-acid-free cv. ‘Drakkar’ and high-erucic-acid cv. ‘Maplus’. In total, 247 plants were regenerated, with the regeneration percentage of 15.1% for ‘Drakkar’ and 2.0% for ‘Maplus’, respectively. Transformants were confirmed by PCR of the LPAAT gene and NAP/KCS expression cassette, and Southern blotting analysis, indicating that the foreign genes were integrated into the genome of rapeseed. The transformation frequencies of B. napus cv. ‘Drakkar’ and ‘Maplus’ were 3.6% and 0.7%, respectively. Seeds of the transformants showed a changed fatty-acid profile. The content of erucic acid was up to 10.5% in the seeds of transformants of erucic-acid-free cv. ‘Drakkar’ and increased by 5% in the seeds of transformants of cv. ‘Maplus’, compared with the control . The highest content of erucic acid in some individuals of transgenic ‘Maplus’ was 62.8%.
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