【作者】 朱波；

【导师】 侯喜林； 姚泉洪；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2008， 博士

【摘要】 干旱、盐害、极端温度、化学毒害和氧化胁迫等非生物胁迫严重影响农业生产,并导致环境退化。植物在生长发育过程中,需要对各种逆境和发育信号作出反应,这就要求对各种功能基因的表达进行精确调控。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。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NAC、Mvb、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。本研究利用RACE(Rapid amplification of cDNA ends)技术从不结球白菜cDNA文库中克隆到1个新的编码NAC类转录因子的全长cDNA序列,命名为BrNAC。BrNAC基因的开放阅读框(ORF)由837bp组成,编码1个由278个氨基酸组成的NAC类转录因子。推导的氨基酸序列与油菜NAC5-8有97%的同源性,与油菜NAC5-7、NAC3同源性分别为92%和86%,与拟南芥ATAF2的同源性达89%。二级结构分析表明BrNAC具有许多其它NAC家族的共同特性。构建的同源进化树表明,BrNAc在进化关系上与AFAF2亚族转录因子最近。使用实时荧光定量PCR对BrNAC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,BrNAC基因的表达在茎、叶中最高,并且受冷和机械损伤和高盐的诱导,而与NAA和ABA的关系不大。利用基于PCR的两步PTDS方法(PCR-based two-step DNA synthesis,PTDS)技术成功的合成了来源于白杨SP1基因和昆虫的抗冻肽ThpI基因。PTDS基因合成方法,是一个较为简单,高效,高保真度,低成本的基于两步PCR方法合成长DNA序列方法。同以前的合成方法比较,该PTDS还具有时间短,通常是6到7天,该方法还比较容易获得不同种类,不同长度,甚至大于6kb的DNA片段,适合高GC比,高度重复序列,复杂二级结构的DNA合成。因而,PTDS方法提供了一个能够合成不同种类、具有复杂结构的长片段基因的可行的技术。为了进一步分析SP1基因和ThpI基因的功能,本研究构建了农杆菌.植物双元表达载体,通过蘸花法转化拟南芥,获得转SP1基因和ThpI基因的拟南芥植株。结果表明,过量表达SP1基因的拟南芥获得了很明显高温抗性。通过检测野生型和转SP1基因的拟南芥的叶绿素含量、叶绿素荧光、含水量、脯氨酸及MDA含量等多种生理指标,验证了SP1基因过量表达提高拟南芥高温抗性的生理机制;过量表达ThpI基因的拟南芥提高了植物的低温抗性,通过检测野生型和转ThpI基因的拟南芥的电导率、脯氨酸、MDA含量、SOD活性等多种生理指标,验证了ThpI基因过量表达提高植物耐寒性的生理基础。更多还原

【Abstract】 Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity, and oxidative stress, are serious threats to agriculture and result in the deterioration of the environment. Plants need to response to multiple stresses and developmental signaling during the growth and development. Thus the expression of functional genes should be accurately adjusted and controlled. Many signal transduction pathways are activated and plenty of genes involved in abiotic stress response are induced to express in plants. At present, 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 NAC, Myb, WRKY, and bZIP and so on.In this paper, a novel NAC type transcription factor, nambed BrNAC, was isolated using rapid amplification of cDNA ends (RACE) method from Brassica campestris ssp.chinensis. The full-length cDNA of BrNAC has an 837bp open reading frame (ORF) and encode a NAC subgroup transcription factor consisting in 278 amino acids. At the nucleotide sequence level, BrNAC has 97% sequence similarity to Brassica napus NAC5-8, 92% to NAC5-7, 86% to NAC3 genes and 89% to Arabidopsis thaliana AFAF2. The secondary structure analysis revealed that BrNAC strongly resembled other NAC genes. BrNAC was more close to AFAF2 in its homologous evolutional tree. The result of RT-PCR indicated that the expression of BrNAC gene was induced by cold stress, mechanical damage and high salinity, but not NAA and ABA, and the transcript level of BrNAC gene was the highest in Brassica campestris ssp. chinensis stem and leave.SP1 gene from Populus tremula and ThpI gene from Choristoneura fumiferana were successively synthesized by PCR-based two-step DNA synthesis (PTDS). It is a simple, high fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. Compared to the previously published methods, the PTDS method is rapid (6 to 7 days) and suitable for synthesizing different species and different segments, long segments of DNA (5 to 6-kb) with high G+C contents, repetitive sequences, or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures.We analyzed the function of expression of SP1 and ThpI gene in Arabidopsis thaliana plants, through constructing the Agrobacterium tumefaciens GV3101 containing plant binary expression vector. The floral dip method was used to transfer them into A. thaliana. The results showed that the SP1 transgenic plants exhibited improved growth than wild type control plants after 45℃high temperature stress for 16 h. The chlorophyll and water contents and chlorophyll fluorescence were decreased much more in wild type than in transgenic plants; Moreover, the transgenic plants had higher proline contents and lower malondialdehyde contents after high temperature stress. After low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild plants. The transgenic plants had higher proline contents and higher SOD activity. Moreover, they had lower electrolyte leakage percentage and lower malondialdehyde contents after cold stress.更多还原