抗逆调节转录因子DREB1B基因转化多年生黑麦草的研究

Perennial Ryegrass Transformed with the Adversity-Resistant Transcription Factor DREB1B Gene

以逆境诱导型启动子rd29B为驱动，分别构建了含有抗逆调节转录因子DREB1B基因的表达载体pBAC123、pBAC128，选择标记为bar基因。用高压氦气基因枪PDS1000／He分别将表达载体和p35SIH3导人多年生黑麦草（Loliumperenne）品种Topgun成熟胚的愈伤组织。经除草剂Bialaphos抗性筛选和植株再生，获得了62株转基因植株。经PCR、Dot—blotting分子检测，DREB1B基因已整合到多年生黑麦草部分转基因株系的基因组中。用5种不同浓度的除草剂涂抹黑麦草叶片，非转基因植株表现为不抗，而转基因植株最高可以抗135～200mg／L。脯氨酸含量测定表明，使用15％PEG8000处理后，转基因植株的叶片脯氨酸含量比非转基因植株提高1倍左右。经25d人工温室干旱处理，有5棵转基因植株存活；复水后，有3棵植株恢复正常生长。结果表明，利用逆境诱导型启动子（rd29B）来增强外源DREB1B基因的表达，能显著改良黑麦草的抗旱能力。

Driven with the adversity-inducible promoter rd29B,two expression vectors carrying the adversity-resistant transcription factor DREB1B gene,pBAC123 and pBAC128, and one selection marker,bar gene were constructed. By means of PDS1000/He,the expression vectors were separately transformed into the calluses of mature embryos of Topgun,a perennial ryegrass variety（Lolium perenne）. Sixty two transgenic plantlets were obtained from the callused by bialaphos resistance selection and regeneration culture. PCR and Dot-blotting tests showed that DREB1B gene has been transformed into the genomes in some of the transgenic plants, after the leaves of the ryegrass plants were smeared with bialaphos at five different concentrations,the non-transgenic plants appeared not resistant and the transgenic plants appeared resistant to bialaphos at the concentration as high as 135-200 mg/L. Proline determination showed that treated with 15% PEG8000,the transgenic plants had a proline content in their leaves that was about one times higher than that in the leaves of the non-transgenic plants. Under drought in growth chamber for 25 days, there were five transgenic plants left alive of which, three returned to their normal growth after they were watered. It was shown that the enhanced expression of exogenous DREBIB gene was capable of improving the drought resistance of ryegrass.