【作者】 王闻哲；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2008， 博士

【摘要】 Ced-9是从动物线虫（C.elegance）中克隆出来的抗凋亡基因,属于Bcl-2家族,与哺乳动物中的抗凋亡基因Bcl-2同源。前人的研究结果已经证实凋亡抑制基因Bcl-X_L和Ced-9能够促进植物细胞对生物和非生物胁迫的抗性,但是其作用机制仍知之甚少。本研究以烟草为模式材料,开展铝（Al）、盐诱导其细胞程序性死亡（programmed cell death,PCD）的研究,并通过转细胞凋亡抑制基因Ced-9/Bcl-2提高烟草和水稻的耐铝抗盐性及其机理分析,获得如下主要结果:1、用DNA ladder检测证明了Al诱导的植物细胞死亡是类似于凋亡的PCD过程。2、通过测定转基因烟草的相对根长、苏木精染色等指标,首次证明凋亡抑制基因Ced-9在烟草细胞内具有一定的促进耐Al功能。通过苏木精染色比较转基因和野生型烟草在不同Al浓度下根部Al的积累量,证实Ced-9可缓解Al胁迫下烟草根部Al的积累量。研究表明过量表达Ced-9的转基因烟草植株也表现出比野生型更强的抗盐能力。3、用DNA ladder检测比较转基因和野生型烟草在Al或盐胁迫条件下的植物细胞PCD,发现Ced-9能阻断Al或盐诱导PCD的发生,从而提高烟草的耐胁迫能力。4、研究发现Ced-9能通过下调植物细胞内Al和盐诱导的类天冬氨酸依赖性半胱氨酸蛋白酶（Caspases）的液泡酶（vacuolar processing enzyme,VPE）的表达水平,抑制Al和盐诱导的PCD发生。揭示PCD的负调控可能是潜在的耐Al机制之一,并且PCD的负调控作用对植物细胞耐受非生物胁迫具有广谱性。5、用荧光探针DCFH和Fluo-3结合共聚焦荧光显微技术,发现Al能显著诱导细胞内的活性氧自由基（reactive oxygen species,ROS）增加。研究还发现凋亡抑制基因Ced-9不能抑制Al诱导的ROS增加,但能直接调节胞内钙离子(Ca²⁺)水平。暗示Ced-9作用于Al胁迫引起的ROS信号的下游并延迟了Al激活的Ca²⁺信号水平的升高。推测Ced-9在植物细胞中的耐Al机制可能是通过延迟细胞内的Ca²⁺信号水平的升高来抑制Al引起的PCD。6、研究表明Ced-9不直接上调受Al诱导的基因NtGDI1、parB、NtPox的表达,这些基因的表达能抑制Al胁迫激发的ROS所引起的脂质过氧化作用。说明Ced-9不直接抑制Al胁迫引起的脂质过氧化作用。7、此外,还将Ced-9和Bcl-2基因通过根癌农杆菌（Agrobacteriumtumefaciens）EHA105介导转化水稻,获得以下结果:（1）将Bcl-2小片段连接到中间载体pGEM上以获得合适的酶切位点,构建中间载体pGEM-Bcl2,将酶切下来的Bcl-2小片段连接到载体pCAMBIA13011上,成功构建了表达载体pCAMBIA13011-Bcl2。（2）将pCAMBIA13011-Bcl2以及实验室已有的pCAMBIAl3011-Ced9通过农杆菌介导法转化水稻,用潮霉素对转基因种子筛选获得抗性植株,并进行PeR和Gus活性检测。（3）转基因植株后代遗传分析,获得转基因纯系,并用RT-PCR检测到转基因后代植株的基因表达。（4）利用Tail-PCR技术对其中一株转基因水稻进行了T-DNA插入位点检测。发现水稻基因组上对应位置在水稻8号染色体上。更多还原

【Abstract】 Ced-9 is a Bcl-2 analog from Caenorhabdities elegans that promotes cell survival.It has been shown that apoptotic suppressors such as Bcl-X_L and Ced-9 enhance the resistance of plant cells to biotic or abiotic stresses,but detailed mechanisms still are largely unknown.Here,we choose tobacco（Nicotiana tabacum L.）as an excellent model for studying various aspects of Al or salt induced programmed cell death（PCD）.And we want to uncover the mechanism and enhance the resistant to aluminum （Al）and salt stress by overexpressing Ced-9/Bcl-2 in tobacco and rice. Results obtaind in the present study are summarized as follows:1.We proved a process of PCD induced by Al in tobacco by DNA ladder analysis.2.Data of root growth length,hematoxylin staining in treatment solution containing different concentrations of Al indicate that tobacco cells overexpression Ced-9 gene are more resistance to Al.Detailed analyses show that Ced-9 reduces Al uptake amounts in plant cells.3.Afterwards,total DNA were extracted from plant cells treated with Al or salt,and DNA ladder analyses suggest that Ced-9 increases tolerance via blocking Al-or salt-induced PCD.4.Caspases as a character of apoptosis,plays an indispensible role in animal cells.Here,we show Ced-9blocks Al-or salt-induced programmed cell death（PCD）by downregulation of Al-or salt-induced transcriptional levels of Caspase-like vacuolar processing enzyme（VPE）.Our research suggest that negative regulation of PCD probably is one of the potential Al-resistance mechanisms and has a broad-spectrum resistance to abiotic stresses in plant cells.5.Up to now,little is clearly understood about Al-resistance mechanism involving intracellular signal transduction and regulation mechanisms.Staining of Al-treated roots with 2’,7’ -dichloro fluorescein diacetate（H₂DCFDA）had suggested that Al treatment causes ROS production.Ced-9 may indirectly advance the repression of the lipid peroxidation caused by Al stress in the early process of Al-elicited ROS. Our data suggest that Ced-g acts downstream of oxidative burst to Al stress and delays the elevation of[Ca²⁺]_cytcaused by Al stress and Ced-9 enhances Al tolerance probably via delayed elevation of intracellular Ca²⁺levels and inhibition of Al-induced PCD in plant cells.6.NtGDI1、parB、NtPox are Al-induced early response genes. Overexpression of these genes could diminish the oxidative damage caused by Al stress.In our study,Ced-9 may indirectly advance the repression of the lipid peroxidation caused by Al stress in the early process of Al-elicited reactive oxygen species（ROS）.7.In this paper,we also transferred Ced-9 and Bcl-2 genes into rice and by Agrobacterium tumefaciens EHA105.The results are showed as followes:（1）Using recombination techniques,the gene fragment Bcl-2 was incorporated into pGEM-7zf（+）and formed a new vector pGEM-Bcl2,in order to get the right endonuclease sites.And then the Bcl-2 gene was inserted into plant expression vector pCAMBIA13011 under the control of CaMV 35S promoter.（2）The Ced-9 and Bcl-2 genes were transformed into rice calli by Agrobacterium tumefaciens EHA105.PCR analysis,hygromycin selection and Gus assay of transformants confirmed that the Ced-9 and Bcl-2 genes have been integrated into the chromosomal DNA of these transgenic plants.（3）T1 seeds were harvested and obtained homozygotic lines of transgenic rice after hygromycin selection.Then we confirmed transgenic lines expressing Ced-9 and Bcl-2 by RT-PCR. （4）Tail-PCR（thermal asymmetric interlaced PCR）was used to analyze the locus of T-DNA insertion.In the transgenic rice,we determined the loci of T-DNA insertions in one line.T-DNA was predicated to insert the chromosomeⅧ.更多还原