【作者】 彭辉；

【导师】 麻浩；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 生物和非生物胁迫严重影响农作物的产量和品质。NAC基因在植物发育和生长调节以及逆境应答分子网络中扮演着极其重要的角色,是作物抗逆遗传工程的优良候选资源。目前关于NAC基因的研究主要集中于拟南芥和水稻等少数模式作物。鉴于同源基因在不同物种间可能存在功能差异,广泛地从不同植物中分离和研究NAC基因是有必要的。鹰嘴豆是世界第三大豆类作物,它生育期短,抗逆性强,基因组小,用于挖掘抗逆关键基因和解析植物生长发育及耐逆分子机制拥有巨大的潜力。然而,目前该作物在分子生物学方面的研究较为缺乏,国内尚未见报道。为了构筑鹰嘴豆分子生物学研究基础并挖掘逆境胁迫相关基因,本实验室于2007年利用新疆抗逆种质构建了干旱胁迫相关的cDNA文库。在该cDNA文库中,我们发现6条独立的NAC样基因片段、1个GPRP基因ORF以及1条ACTIN基因片段。鉴于NAC及GPRP基因可能具有重要的生理功能且在鹰嘴豆中还没有被克隆,我们分离和鉴定了这6个NAC和1个GPRP基因,并广泛地进行发育及胁迫相关的表达分析,还通过转基因研究初步分析了其中3个NAC基因的功能。此外,考虑到在鹰嘴豆中至今没有可用于表达分析的内标基因,我们还克隆了1个ACTIN基因,并初步调查了其作为内标基因使用的可行性。本研究获得的主要结果如下：借助cDNA末端快速扩增技术(RACE),CARNAC1～6(Cicer arietinum NAC gene)基因被克隆,相应cDNA长度分别为753bp、706bp、927bp、1108bp、987bp和921bp,依次编码6个长度为239、191、285、339、291和307aa的蛋白质。CARNAC1-6基因的基因组序列均含有两个长度不一但位置保守的内含子。多重序列比对分析揭示,CARNAC1-6蛋白N端形成保守的NAC结构域,但C端序列的变异性较大。DNA gelb1ot实验结果显示,CARNAC1-6在基因组中均以单或低拷贝的形式存在。亚细胞定位实验证实6个CarNAC:GFP融合蛋白均位于洋葱表皮细胞核中。酵母单杂交实验结果显示CARNAC1～6蛋白的转录激活功能位于C端。以上发现说明,CARNAC1～6属于典型的NAC基因,其编码产物在鹰嘴豆体内可能也具有转录激活活性。利用半定量RT-PCR技术,CARNAC1～6基因在多个发育过程中、多种胁迫及激素处理下的表达模式被调查分析。除了CarNAC3和CarNAC4基因,其他基因均表现出不同的组织特异性表达规律。(CarNAC1,3基因的表达受到衰老的强烈诱导,而CarNAC6基因的表达受到衰老的显著抑制。在不同发育阶段的籽粒以及逐渐萌发的种子胚中,6个CarNAC基因都展现出特定的表达规律,具有协同表达的特征,表明CARNAC1～6基因在生理功能上既存在差异又相互补充。在叶片中,CARNAC1～6基因都受到干旱的诱导表达,只是响应的时间和强度存在一定的差异。CarNAC1,4,5,6基因的表达受到盐胁迫的显著增强。高温(37℃)促使CarNAC4,5基因的转录水平快速增高。CarNAC1,4,6基因的表达在不同时间点受到低温(4℃)的诱导。叶片的机械损伤引起CarNACl,5基因表达水平的显著上升。外源激素处理的结果显示,受到脱落酸(ABA)上调表达的NAC基因有4个(CarNAC2,3,4,6),茉莉酸甲酯(MeJA)有1个(CarNAC4),乙烯利(ET)有4个(CarNAC1,3,4,6),水杨酸(SA)有4个(CarNAC1,3,4,5),生长素(IAA)有5个(CarNAC1,3-6),赤霉素(GA3)有3个(CarNAC1,4,6), H2O2有4个(CarNAC1,3,4,6)。此外还发现,CarNAC3,6基因的转录受到6-苄基腺嘌呤(6-BA)的显著抑制。这些实验结果说明CarNAC转录因子既涉及植物生长发育的调节又参与多种环境胁迫的响应。CarNAC2基因的过量表达导致拟南芥种子萌芽延迟、发芽势低,幼苗茎短小、主根显著增长,成年植株地上部体型矮小、抽苔开花早,结荚量小。CarNAC2的高同源基因拟南芥XND1由于特异性地抑制木质部纤维细胞次生壁的合成,也导致转基因植株株型矮小,表明二者的功能可能存在共性。半定量RT-PCR实验结果显示,CarNAC3与其他NAP亚家族成员(拟南芥NAP、野生小麦NAM-B1和大豆GmNAC1)一样,在叶片中受到衰老的诱导表达,然而与它们不同的是,过量表达CarNAC3基因的拟南芥植株生长发育正常,没有出现衰老加剧的现象,说明它与其他NAP亚家族成员在涉及衰老生理的功能上存在差异。CarNAC3的表达还受到干旱和ABA的显著上调,其转基因植株展现了较强的抗旱性并对ABA高度敏感,说明该基因是ABA依赖型干旱应答因子。CarNAC3基因的异位表达没有影响拟南芥植株的生长发育过程但提高了它的耐旱能力,在作物抗旱基因工程方面展现出较大的应用潜力。CarNAC6基因在拟南芥中的过量表达未影响植株的表型和发育进程,但在苗期显著增强其耐渗透胁迫的能力。此外,过量表达CarNAC6基因的转基因植株展现出较野生型植株更高的ABA敏感性,说明该基因为ABA依赖型胁迫应答因子。对于转基因植株,六个与逆境生理密切相关的拟南芥基因中,有4个(COR15A, RD22,RD29A,KIN1)的表达被显著上调,说明CarNAC6的过量表达促进了这些基因的转录,进而增强转基因植株的抗渗透胁迫能力。像CarNAC3基因一样,CarNAC6基因在作物抗逆基因工程上也具有潜在的应用价值。CarPRP1 (Cicer arietinum L. glycine- and proline-rich protein)基因包含两个内含子,编码一个长为186aa的XYPPX家族多肽,在基因组中存在3-4个拷贝。CarPRP1:GFP融合蛋白被定位于细胞膜和细胞核中。CarPRP1基因广泛地表达在幼苗的根、茎、叶以及花、发育中的种子和荚中,只是在种子和荚中的表达量相对较高。该基因的表达不受叶片衰老过程的明显影响,但在籽粒发育和种子萌芽过程中出现规律性变化。随着籽粒的逐渐膨大,CarPRPl基因的转录逐渐被增强。而随着种子胚芽的逐渐伸长,胚中CarPRPl基因的表达量逐渐降低。此外,CarPRP1基因的表达还受到干旱、低温、高盐以及机械损伤等胁迫和ABA、IAA、GA3以及H202等化学处理的诱导。CarPRPl基因在拟南芥中的过量表达能显著增强植株抗盐和冷冻胁迫的能力。在非处理情况下,6个抗逆相关基因(COR15A, COR47, ERD10, RD22, RB29A, KIN1)在转基因和野生型植株中的表达没有明显差异,但经干旱处理啊3小时后,所有这些基因在转基因植株中的转录水平显著高于野生型植株,这至少是CarPRPl基因能增强植株抗逆性的部分分子机理。CarPRPl基因在作物抗逆基因工程上具有潜在的应用价值。借助RACE技术,第一个鹰嘴豆ACTIN基因(CarACT1:Cicer arietinum L. actingene)被克隆。CarACT1基因的cDNA全长1418bp,编码一个长377aa的蛋白,其基因组序列含有4个长度不一的内含子。系统进化分析显示ACTIN基因核苷酸序列在整个生物界都是高度保守的。半定量RT-PCR实验结果显示,CarACT1基因广泛地表达在各个器官、组织以及不同的发育时期中。以CarACT1基因为假定内参照,CAP2(Cicer arietinum L. APETALA2)基因表现出与前人报道一致的表达模式,因而可初步确定CarACT1能够作为鹰嘴豆基因表达分析中的内参照使用。第一个鹰嘴豆内标基因的确立为RT-PCR以及核酸杂交技术在基因表达分析研究中的应用奠定了基础。更多还原

【Abstract】 Biotic and abiotic stresses severely affect quantity and quality of crop product. The plant-specific NAC (for NAM, ATAF1,2 and CUC2) transcription factors, which have been found playing important roles in plant growth, development and stress responses, are taken as good genetic resource for the improvement of crop tolerance to environmental stresses. Previous researches about NAC proteins mainly focused on a few model plants such as Arabidopsis and rice. In view of functional difference of orthologues from different species, further investigation on the functions of NAC genes from other plant species will be helpful to understand the common and special molecular mechanisms of plant development and stress responses. Chickpea(Cicer arietinum L.) is the third important legume crop in terms of cultivation area. It is an annual plant with a short life, a small genome size and strong resistance to biotic and abiotic stresses. And chickpea has been suggested as a model plant for investigation of physiological mechanisms of plant development and responses to stresses. However, chickpea is scarcely studied in the molecular biology, especially in china. To build foundation for molecular biology research and to identify the stress responsive genes, we have constructed a drought-related cDNA library using the stress resistance germplasm from Sinkiang. Six expressed sequence tags (EST) of NAC genes, an EST of actin gene and an contig of GPRP (glycine-and proline-rich protein) gene were found in chickpea cDNA library. Since NAC proteins have important physiology function and no NAC gene is cloned in chickpea, we isolated and identified six NAC genes based on these ESTs. Furthermore, expression patterns of these NAC genes were globally investigated in various developmental processes and under diverse stresses. And function of three NAC genes were analyzed using transgenic Arabidopsis. In view of no reference gene for expression analysis in chickpea, we also isolated an actin gene and estimated usability of this gene for normalization purposes. In addition, a GPRP gene also was cloned and characterized functionally. Main results of this study are as follows.CARNAC1～6 (Cicer arietinum NAC gene) genes were obtained using rapid amplification of cDNA end (RACE) technique and their cDNA length were 753,706,927,1108,987 and 921bp, respectively. The putative corresponding proteins were composed of 239,191,285, 339,291 and 307 amino acids, respectively. Genomic DNA sequences of all CarNAC genes contained two location-conserved introns with different length. Multiple sequences alignment revealed that the N-terminus of CARNAC1～6 proteins had conserved NAC domain and the C-terminus represented a more variable region. Based on the DNA gel blot analysis, all CarNAC genes were found to be either single or low copy number in the chickpea genome. All CarNAC:GFP fusion proteins were localized in the nucleus of onion epidemical cells by subcellular localization assay. Moreover, the trans-activation activity CARNAC1～6 proteins were proved to be located in the C-terminal region by trans-activation activity analysis in yeast. These data suggested that CARNAC1～6 were typical NAC genes and the corresponding proteins probably were transcriptional activators in chickpea.Expression patterns of CARNAC～6 genes were globally investigated in various developmental processes and under diverse stress and chemical treatments. All CarNAC genes had different tissue-specific expression profiles except for CarNAC3 and CarNAC4 genes. Expression of CarNAC1,3 genes was strongly induced by leaf age, whereas expression of CarNAC6 gene was significantly inhibited. Meanwhile, CARNAC1～6 showed differential expression patterns with cooperativity during seed development and germination. CARNAC1～6 showed differential expression patterns with cooperativity during seed development and germination, indicating that the physiological functions of CARNAC1～6 genes are different as well as mutually complementary. Transcription of CARNAC1～6 genes was enhanced by drought treatment in different degree at different time points. Moreover, four (CarNAC1, 4-6), two (CarNAC4,5), three (CarNAC1,4,6) and two (CarNAC1,5) NAC genes were significantly induced by high salinity, high temperature (37℃), low temperature (4℃) and mechanical wound, respectively. Additionally, four (CARNAC2～4,6), one (CarNAC4), four (CarNAC1,3,4,6), four (CarNAC1,3-5), five (CarNAC1,3-6), three (CarNAC1,4,6) and four (CarNAC1,3,4,6) NAC genes were significantly up-regulated by abscisic acid (ABA), methyl jasmonate (MeJA), ethephon (Et), salicylic acid (SA), indole-3-acetic acid (IAA), gibberellin (GA3) and H2O2, respectively, whereas two genes (CarNAC3,6) were down-regulated by N-6-benzyl-adenine (6-BA) treatment. These findings suggested that functions of all six CarNAC transcription factors were involved in plant growth and development regulation as well as environmental stress responses. Transgenic Arabidopsis plants expressing CarNAC2 gene showed some abnormalities such as germination delay, shorter stem of seedling, longer root of seedling, dwarfish aerial part, early blossoming and low propagation coefficient. XND1 (xylem NAC domain), an orthologue of CarNAC2 gene in Arabidopsis, which dramatically suppresses secondary wall deposition in the xylary fiber, stunts plant development in transgenic Arabidopsis, suggesting that CarNAC2 gene has physiology function similar to that of XND1 gene.Expression of CarNAC3 gene, like that of other NAP (NAC-like, activated by APETALA3/PISTILLATA) subgroup members including Arabidopsis NAP, NAM-B1 from ancestral wild wheat and soybean GmNAC1, was found to be induced by leaf age using semi-quantitative RT-PCR assay. However, overexpression of NAP, NAM-B1 or GmNAC1 accelerate senescence process of transgenic plants, whereas CarNAC3 gene did not, suggesting there are functional differences between Car NAC3 and other three orthologues in senescence physiology. Transcription of CarNAC3 gene was significantly up-regulated by drought stress and ABA treatment. Furthermore, transgenic Arabidopsis plants expressing Car NAC3 gene showed enhanced tolerance to drought stress and high sensitivity to ABA, indicating that this gene is an ABA-dependent gene response to drought stress. Since overexpression of CarNAC3 gene did not affect plant development but enhanced tolerance to dehydration, it has the potential value for crop drought-resistant genetic engineering.Overexpression of CarNAC6 gene did not alter the phenotype and developmental process of transgenic Arabidopsis plants but enhanced tolerance to osmotic stress in seedlings. Additionally, the transgenic plants overexpressing CarNAC6 gene exhibited the higher ABA sensitivity than wild type plants, indicating that CarNAC6 is an ABA-dependent gene involved in sress response. Among six known stress-related genes, four genes(COR15A, RD22, RD29A, KIN1) were induced by overexpression of CarNAC6 gene in transgenic plants, suggesting that CarNAC6 protein can promote the transcription of these stress-related genes and then enhance tolerance to osmotic stress in transgenic plants. Like, CarNAC3 gene, CarNAC6 gene also has the potential value for crop drought-resistant genetic engineering.CarPRP1 (Cicer arietinum L. glycine- and proline-rich protein) gene containing two introns in genomic sequence encoded a XYPPX-repeat protein of 186 aa and had 3 or 4 copys in chickpea genome. The CarPRP1:GFP fusion protein was localized in cell nulclear and membrane. The transcripts of CarPRP1 appeared in many chickpea organisms including seedling leaves, stems, roots, flowers, developing seeds, and pods, but mostly accumulated in developing seeds and pods. The expression of CarPRP1 gene was not affected by leaf age but obviously fluctuated during seed development and germination. With expanding of seeds, the transcription of CarPRP1 gene was significantly enhanced. With elongation of germs, the expression of CarPRP1 gene gradually decreased in the embryo of germinating seeds. Furthermore, the expression of CarPRP1 gene was significantly induced by various stresses (drought, cold, high salility and mechanical wound) and several chemical treatments (ABA, IAA, GA3 and H2O2). Overexpression of CarPRP1 gene enhanced resistance to high salility and freezing stresses in transgenic plants. Although, the expression difference of six stresss-related genes between transgenic and wild type plants was not observed in normol conditions, their expression levels of transgenic plants were obviously higher than that of wild type plants, which can partially explain why CarPRP1 can enhance tolerance to environmental stresses in transgenic Arabidopsis. Our findings suggest CarPRP1 gene is a potential candidate for crop drought-resistant genetic engineering.CarACT1(Cicer arietinum L. actin) gene, the first actin gene from chickpea, was obtained using RACE technique and its cDNA length was 1418bp. The putative corresponding protein was composed of 377 amino acids. Genomic DNA sequence of CarACT1 gene contained four location-conserved introns with different length. Phylogenetic analysis revealed that all actin genes were conserved in the nucleotide level throughout the whole organic world. Transcript of CarACT1 was extensively detected by semi-quantitative RT-PCR in many organs of chickpea and during diverse developmental processes. Furthermore, expression pattern of CAP2 (Cicer arietinum L. APETALA2) was the same as previous findings using CarACT1 as a presumptive reference gene under drought and salt treatments. Therefore, we presumed that CarACT1 can serve as an internal reference gene for normalization of genes expression in the vegetative tissues of chickpea. The generation of the first reference gene in chickpea is useful to expression analysis through northern blot assay and RT-PCR method.更多还原