【作者】 刘金戈；

【导师】 姚泉洪； 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2007， 博士

【摘要】 干旱、高盐和极端温度非生物胁迫,严重影响了植物的生长和作物的产量。因此,系统研究植物对环境胁迫应答的机制就显得尤为重要。植物在感受环境胁迫信号后,会激活相应的信号传导途径,诱导大量胁迫应答基因表达,产生相应的胁迫应答反应,在这一系列的应答反应过程中,转录因子起着十分关键的作用。根据转录因子结构以及DNA结合活性等特点,将其分成若干个家族,如AP2/EREBP、Myb、HSF、bZIP等,这些转录因子在胁迫响应过程中发挥着重要的作用。本研究从八棱海棠和水稻中共克隆了5个转录因子的基因,它们分别属于AP2/EREBP和HSF类转录因子基因家族,并对它们进行了生物信息学分析和功能鉴定。利用RACE（rapid amplification of cDNA ends）技术从八棱海棠cDNA文库中克隆到1个新的编码AP2/EREBP类转录因子的全长cDNA序列,命名为MrDBP1。MrDBP1基因的开放阅读框（ORF）由483bp组成,编码1个由160个氨基酸组成的DREB类转录因子。构建的同源进化树表明,MrDBP1在进化关系上与DREB亚族转录因子较ERF类转录因子更近,属于DREB家族的A5亚群。Southern杂交的结果表明MrDBP1基因在八棱海棠基因组中很可能以多拷贝的形式存在,或者存在两个与MrDBP1基因同源性很高的同源基因。对MrDBP1蛋白的亚细胞定位分析表明,转录因子MrDBP1具有明显的核定位能力。使用实时荧光定量PCR对MrDBP1基因在不同组织中以及不同逆境胁迫下相对的转录水平进行了定量分析。结果表明,MrDBP1基因的表达在种子中最高,并且受干旱的诱导,而与低温和高盐的关系不大。利用酵母单杂交方法从水稻cDNA文库中分离了1个AP2/EREBP类和3个HSF类转录因子。这些基因的序列分析表明,这个AP2/EREBP类转录因子属于DREB家族的A1亚群,命名为RCBF2。而这3个HSF类转录因子中,OsHSF6和OsHSF7属于热休克转录因子的A类亚群,而OsHSF12属于C类亚群。对这些基因进行表达谱分析,结果表明低温,干旱和高盐都能够诱导RCBF2基因的表达,并且Ca²⁺和MeJA也能够轻微诱导RCBF2基因的表达。此外,OsHSF6和OsHSF7对高温应答模式十分相似,即高温处理十分钟内其mRNA的水平均达到了最高,随后逐渐降低。为了进一步分析OsHSF7的功能,我们通过蘸花法转化拟南芥,获得了过量表达OsHSF7基因的拟南芥植株。对转基因植株的分析表明,OsHSF7基因的表达导致了拟南芥植株抗高温能力的增强,这主要是由于在转基因拟南芥植株内OsHSF7促进了下游HSP,GolS等基因的过量表达。最后,我们分析了八棱海棠,水稻和拟南芥中DREB1B/CBF1同源基因,即八棱海棠的MrCBF1,水稻的OsDREB1A和拟南芥的AtCBF1功能上的差异。结果表明,这3个基因功能上存在着很大的相似性,但是,也存在着很大的差异。即表型上的差异和诱导下游抗性基因表达强度的差异。三类转基因植株分别均表现出不同程度的生长滞后,D35S∷MrCBF1植株在长日照条件下,播种后六个月仍未抽薹,D35S∷OsDREB1A植株播种后4个月开始抽薹,而D35S∷AtCBF1植株和野生型拟南芥分别四个月和三个月已经完成了一个生命周期。对于诱导下游基因的表达,D35S∷MrCBF1植株中,COR15A,RD29A和KIN1基因的表达明显高于D35S∷AtCBF1和D35S∷OsDREB1A植株中这些基因的表达。更多还原

【Abstract】 Plants are constantly challenged by various biotic and abiotic stresses such as drought,high salinity,low temperature and disease in their natural environment,which cause adverse effects on the growth of plants and serious reductions in the quantity and quality of agriculturally important crops.Encountering environmental stress,many signal transduction pathways are activated and plenty of genes involved in abiotic stress response are induced to express in plants.In the process of environmental stress response,transcription factors play important role.Till now,a number of transcription factor families have been found involving in plant stress responses,and each contain a different type of DNA-binding domain,including AP2/EREBP,Myb,WRKY,and bZIP and so on.In this study,we isolated five novel transcription factors,MrDBP1 form Malus robusta Rehd.,RCBF2,OsHSF6,OsHSF7,OsHSF12 from Oryza Sativa L, which belonged to AP2/EREBP and HSF,respectively.In this study,a novel AP2/EREBP type transcription factor,MrDBP1,was isolated using rapid amplification of cDNA ends(RACE) method.The full-length cDNA of MrDBP1 has a 483bp open reading frame(ORF) and encode a DREB subgroup transcription factor consisting in 160 amino acids.The result of Southern blot showed MrDBP1gene might be has several copies or high level of homologous genes in Malus robusta Rehd.genome.Subcellular location of MrDBP1 showed that the transcription factor MrDBP1 had obvious ability of nucleus location.The result of RT-PCR indicated that the expression of MrDBP1gene was induced by drought stress,but not low-temperature and high salinity and the transcript level of MrDBP1gene was the highest in Malus robusta Rehd.seeds.In our study,a transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L by yeast one-hybrid method.Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved EREBP/AP2 domain of 59aa and a potential nuclear localization sequence.The result of rice semi-quantitative RT-PCR(s-Q RT-PCR) indicated the expression of RCBF2 gene was induced by cold,dehydration and high-salinity,but not by ABA,and the transcription of RCBF2 gene accumulated primarily in rice immature seeds,growing point and shoots. The expression of OsHSF6 and OsHSF7 gene was induced immediately and largely by high temperature,and the mRNA transcript of OsHSF7 was found to be most abundant in leaf.This indicated that OsHSF7 might involve in heat stress reception and response. Over-expression of OsHSF7 in transgenic Arabidopsis could not induced over-expression of most target heat stress-inducible genes of HSFs.However,after two hours heat stress treatment,the transcript of some HSF target genes containing HSE in their promoter regions were more abundant in transgenic plants than in wild type, meanwhile,those transgenic plants also revealed higher tolerance to heat stress. Collectively,our results indicated that OsHSF7 might play an important role in responding high temperature and be potentially useful for producing transgenic monocots,which were induced to over-express some protective genes,such as HSP, GolS,under high temperature stress,and resulted in the tolerance to heat.In addition,the function of homologous genes of DREB1B/CBF1 was analyzed and compared in Malus robusta Rehd.,rice and Arabidopsis.The results suggested the function of three genes was similar.However,there were some differences the function of three genes in phenotype and expression of downstream genes.Three types of transgenic plants all presented retardant phenomenon at different extent,respectively. Under long light condition,D35S::MrCBF1 plants had still not flowered after six months development,D35S::OsDREB1A plants started to flower after four months development,while D35S::AtCBF1 plants and wild type plants had finished a life cycle in four or three months,respectively.Furthermore,as the ability about induction of expression of down stream genes as concerned,the level of expression of COR15A, RD29A and KINI genes in D35S::MdCBF plants was higher than that in D35S::AtCBF1 and D35S::OsDREB1A plants.更多还原