抗逆调节转录因子CBF1基因提高多年生黑麦草的抗旱能力

通过逆境诱导型启动子rd29B为驱动,分别构建出含有抗逆调节转录因子CBF1基因的表达载体pBAC122,pBAC127,其中pBAC127以CaMV35S启动子驱动的bar基因作为选择标记。用高压氦气基因枪PDS1000/He分别将表达载体导入多年生黑麦草(Lolium perenne)品种Topgun的幼胚、成熟胚和愈伤组织。经除草剂Bialaphos抗性筛选和植株再生,获得了36棵转基因植株。经PCR,Dot-blotting的分子检测,CBF1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到135~200 mg/L。叶片脯氨酸含量测定表明,经干旱处理或使用15%PEG处理,转基因植株叶片脯氨酸含量比未处理时显著提高,部分转基因植株提高幅度明显高于非转基因植株。经过25 d人工温室干旱处理,有3棵植株显示出存活迹象,复水后,有1棵植株(C122-7)恢复正常生长。从而表明,利用逆境诱导型启动子(rd29B)来调控外源CBF1基因的表达,能显著改良黑麦草的抗旱能力。

Improvement of Perennial Ryegrass (Lolium perenne ) Tolerance to Drought by Gene Transferring of Stress-inducible Transcription Factor CBF1 Gene

In this research,two expression vectors pBAC122 and pBAC127 containing CBF[STBX]1 gene were constructed,CBF1 gene is driven by rd29B promoter,and bar gene driven by CaMV35S promoter as selection marker.By biolistic bombardment of PDS1000/He system,both expression vectors(pBAC122 was co-transferred with p35SIH3) were transformed into callus,immature embryos and mature embryos of perennerial ryegrass(Lolium perenne),respectively.After screening by Bialaphos and plant regeneration culture,36 transgenic plants were produced.The results of PCR and Dot-blotting analysis showed that foreign CBF1 gene were well integrated into some transgenic plant's genome of ryegrass,respectively.Treatment of transgenic plants under water stress or 15%PEG,the free proline content of transgenic plants increase markedly,the increased extent was obviously higher than non-transgenic wild type.After 25 days of drought stress,3 transgenic lines behave green and living,and 1 transgenic lines restored living after watering.Our research suggested that using drought-inducible rd29B gene promoter to enhance the expression of foreign CBF1 gene in ryegrass can greatly improve its tolerance to drought.