【作者】 王军卫；

【导师】 张改生； 张晓东；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2005， 博士

【摘要】 干旱是严重影响作物生长和产量的主要环境限制因素之一。在干旱胁迫下,植物表达的基因分为两类:1.基因编码的产物为一些功能蛋白和酶类,如LEA,水通道蛋白,脯氨酸合成酶等;2.基因编码的产物为传递信号和调控基因表达的转录因子,如bZIP,MYC,DREB等。对于第二类基因,鉴于这些基因的表达调控其下游一系列功能基因,目前已成为抗旱基因工程的重点。DREB（Dehydration Responsive Element Binding）和CBF1（CRT/DRE binding factor 1）是拟南芥菜中发现的一个顺式调控元件,在逆境胁迫下,DREB和CBF1转录因子可激活其它多达12个依赖DRE顺式作用元件的抗逆功能基因,如RD29、COR15、RD17,从而使植株多方面的抗逆性（抗旱、抗冻及抗盐）得到提高,因此克隆DREB1B和CBF1基因,并转化到小麦品种中,以提高小麦的对干旱胁迫的抗性。 本实验以冬小麦8901,5-98,99—92和保丰104品种为受体品种,以基因枪微弹轰击转化携带有目的基因DREB1B和CBF1基因的逆境诱导表达类型质粒pBPC128F和pBPC127F载体获得的转基因再生植株79株为材料,经除草剂Basta（100mg/L）抗性鉴定T₁代,部分转基因株系表现出明显的抗性。 转外源转录因子DREB1B基因株系采用点杂交与PCR结合的方法对转基因小麦T₀和T₁代快速进行DNA水平的分子鉴定,同时作了RNA的分子检测,结果表明所选转基因植株中外源基因能够正常转录。 对经过检测的转DREB1B基因株系采用与抗旱性密切相关的相对含水量,叶绿素含量,脯氨酸含量,光合速率四个生理指标进行测定,有10个株系在四个生理指标上相比对照都有明显提高,其中相对含水量均在15%以上,明显高于CK（7.19～9.13%）;叶绿素含量中,叶绿素a含量均在0.07μg/ml以上,叶绿素b含量在0.14μg/ml以上,高于CK的叶绿素a（0.02～0.05μg/ml）和叶绿素b（0.5～0.9μg/ml）;脯氨酸含量在1000μg/g以上,是对照（222.1～604.44μg/g）的2倍多;光合速率均在8.0μmol CO₂/m²/s以上,高于CK（2.27～2.47μmol CO₂/m²/s）。 室内抗旱模拟试验表明,转基因株系停止浇水15天后,叶片仍然表现绿色,而对照叶片则失绿、枯干;复水10天后,转基因株系恢复活力,对照则死亡。 转外源转录因子CBF1基因株系选用地高辛标记的CBF1和bar基因作为探针,进行点杂交检测。结果表明,无论是用CBF1探针,还是bar基因探针,证明了外源基因已整和更多还原

【Abstract】 Drought is one of environmental stresses that effects on the growth of plants and the productivity of crops. The products of these genes induced by drought stress can be classified into two groups: those that directly protect against environmental stresses, and those that regulate gene expression and signal transduction in the stress response. The first group includes proteins and enzymes that likely function by protecting cells from dehydration, such as late-embryogenesis-abundant （LEA） proteins, enzymes required for biosynthesis of proline, and aquaporins（AQP）, et al. The second group of gene products includes transcription factors, protein kinases, and enzymes involved in phosphoinositide metabolism, such as bZIP, MYC, DREB. Recently scientist mostly concerns the second group of gene. DREB （Dehydration Responsive Element Binding） and CBF1（CRT/DRE binding factor 1） are a cis-acting element identified in Arabidopsis. Expression of the DREB and CBF1 under control of promoter in transgenic plants gave rise to strong constitutive expression of the 12 stress-inducible genes, of which the dehydration-responsive element （DRE） is essential in the promoter regions. , such as RD29、COR15、RD17 et al. and increased tolerance to drought, salt loading, and freezing stresses. In this study, DREB1B and CBF1 gene were isolated from Arabidopsis, transferred into wheat, and in order to improve wheat drought-tolerance.Plasmid pBAC128F（7.0kb） and pBAC127F（6.9kb）, which contain RD29B （induced by drought stress） promoter driven DREB1B gene and CBF1 gene, and CaMV 35S promoter plus maize （Zeamays L.） Adh1 first intron driven bar gene were injected immature calluses as explants of common wheat cultivars, 8901, 5-98, 99-92, Baofenl04 and Jinghua No.1 by biolistic microprojectile bombardment （PDS-1000/He）. And 79 plants were regenerated and the leaves of T₁ transgenic lines were applied Basta（100mg/L）. Some transgenic lines took on obvious resistance.PCR, DNA and RNA dot blotting analysis of transgenic lines proved that DREB1B gene has been integrated into genome of some lines, and was well expressed in offspring of different line.Relative water content （RWC）, contents of chlorophyll and proline, the rate of photosynthesis in leaves of T₂ lines were determined and analysised. Among these transgenic lines, 10 transgenic lines exhibited increaseing. RWC in leaves of transgenic lines is all above15%. It is higher than CK（7.19⁹. 13%）. Contents of chlorophyll a are all 0. 07 y g/ml, more than CK （0. 02—0. 05 y g/ml） . Contents of chlorophyll b are all 0. 14y g/ml, more than CK（0. 5—0. 9 y g/ml）. The content of proline in leaves of transgenic lines are more than 1000 y g/g, increased 2-3 folds. The rate of photosynthesis in leaves of transgenic lines are above 8. 0 y mol C02/m2/s, whereas CK are2. 27². 47 y mol C02/m2/s. Under drought condition, transgenic lines and wild type control stopped to water for 15 days. Leaves of transgenic lines kept green, while control showed fading. Then both transgenic lines and control were treated with water for 10 days, leaves of transgenic lines recovered green, while wild type control dead.Dot blotting analysis of transgenic lines with the probes of CBF1 gene and bar proved that CBFlgene has been integrated into genome of some transgenic lines. And transgenic plant lines identified by PCR with primers 5’-bar and 3-NOS showed that 2 transgenic plant lines had the 800bp DNA band. The result was the same as DNA dot blotting analysis.Our research suggests that enhancement expression of alien DREB1B and CBF1 transcriptional factors into wheat is an effective way to improve its drought tolerance ability.更多还原