【作者】 张谦；

【导师】 张志毅；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 毛白杨(Populus tomentosa Carr.)是我国特有的白杨派乡土树种,具有分布广、速生、抗逆性强、材质优良等特性,深受生产者欢迎。然而,毛白杨在生长发育过程中常常受到病原物的危害,尤其是毛白杨锈病,使得其生长与生产受到严重影响,已引起人们的广泛关注。目前有关毛白杨抗病研究十分薄弱,在抗病遗传资源筛选、抗病品种选育、抗病基因定位与克隆等方面仍属空白,这远远落后于黑杨派与青杨派树种的抗病研究,也与毛白杨生产中迫切期待解决病害的需求形成了鲜明的反差。为此,本研究通过毛白杨锈病活体接种试验,筛选到具有强抗病能力的无性系;运用PCR方法,从中克隆到NBS型抗病基因同源序列,对这些基因的进化关系与表达模式进行分析,并从中筛选到1个毛白杨锈病抗性相关基因(DQ324288);通过RACE-PCR分析,克隆到DQ324288基因家族的2个成员,并对其组织表达特性与诱导表达规律进行研究。在构建抗病基因原核表达载体、RNAi干扰载体和正义表达载体基础上,开展原核表达以及烟草和杨树的遗传转化研究,获得一批转基因植株,并对转基因烟草进行抗病试验。此外,以毛白杨RGA为探针,运用生物信息学方法从毛果杨基因组中克隆到74个NBS型抗病基因,研究这些基因的结构特点和进化关系,并对这些毛果杨基因在三倍体毛白杨各组织器官中的表达特性以及在多种生物和非生物诱导胁迫下的表达规律进行分析。通过上述研究可得到以下主要结果:1.本研究以先前筛选得到的抗逆性强的28个毛白杨杂种无性系为试材,以致病性强的马格栅锈菌(Melampsora magnusiana Wanger)为病原物,进行毛白杨锈病活体接种试验,结果发现,在供试材料中,有13个具有不同发病程度的感病无性系和15个无发病症状的抗病无性系,通过分析进一步筛选得到2个具有过敏性反应的强抗病三倍体毛白杨[(P.tomentosa×P bolleana)×P tomentosa]无性系。利用抗病R基因NBS结构域保守区域设计简并引物,运用PCR方法从抗病无性系中克隆得到59个NBS型抗病基因同源序列(RGA)。依据进化关系,59个RGA可被划分为10个亚家族,其中具有完整开放阅读框(ORF)的54个RGA可被进一步划分为TIR型与non-TIR型。序列比对分析发现,59个RGA在毛果杨基因组内共有96个高度同源区域,它们分布在基因组的37个位点,表明三倍体毛白杨基因组内具有丰富的NBS型抗病基因。另外,在亚家族1至3中,异义突变与同义突变的平均比值(ω)显著小于1,说明毛白杨NBS序列承受着极强的纯化选择压力,但NBS区域的一些氨基酸位点的ω值显著大于1,这些位点承受着正向选择压力。同时,在这3个亚家族的RGA中还检测到大量的基因交换,这说明基因交换在NBS序列的纯化过程中发挥着重要作用。荧光定量PCR分析发现,序列差异显著的21个RGA中有18个在三倍体毛白杨的成熟叶片、茎和根中组成型表达,但不同基因间的表达水平存在显著差异,其中2个基因的表达具有成熟树皮特异性,4个基因具有成熟叶片特异性,14个基因具有地上部分特异性,这些基因可能具有抵抗组织器官特异性病害的功能。2.通过序列比对分析,从59个毛白杨RGA中筛选到基因DQ324288,它与美洲黑杨叶锈病抗性位点MER的基因组序列以及其中的6012G11基因同源性高达93%;而且在28个毛白杨无性系内均发现有此同源序列,并可在叶片中呈组成型特异表达,其表达水平与毛白杨无性系的抗病能力呈正相关,表明DQ324288基因可能为毛白杨锈病抗性相关基因。在此基础上,采用RACE-PCR方法克隆到该基因家族2个成员的全长cDNA序列,分别命名为PtDRG01和PtDRG02基因。序列分析与实验结果表明,它们分别为TIR-NBS-LRR和TIR-NBS基因,在基因组内具有多拷贝,与DQ324288基因具有相同的组织表达特性,而且受伤诱导、甲基茉莉酸和水杨酸处理后表达上调,但对暗培养和农杆菌侵染无响应。生物信息学分析结果表明,PtDRG01与PtDRG02基因所编码蛋白的等电点pHi分别为8.165和10.325,且蛋白内存在大量亲水性二级结构,表明它们为碱性、亲水性蛋白。原核表达研究均可检测到目标长度的特异表达蛋白,说明所获得的PtDRG01和PtDRG02基因均具有完整的编码框,而且蛋白表达量随温度上升和诱导时间延长而增加。为了研究PtDRG01基因的功能,本研究在PtDRG01基因的正义表达载体构建的基础上,开展农杆菌介导的烟草遗传转化研究,获得了一批转基因植株。分子检测表明,外源基因已成功导入烟草基因组并稳定存在,而且外源基因在基因组内的拷贝数是内源ACTIN基因的0.1或0.2倍,但在转基因烟草的不同无性系内具有不同的表达水平。烟草花叶病毒(TMV)接种试验结果发现,接种1周后的转基因烟草的发病程度和顶叶中的病毒数量显著低于未转基因对照植株,而且叶片中的病毒数量与外源基因的表达水平呈负相关;接种6周后,未转基因烟草新萌发顶端叶片出现畸形,而外源基因高效表达的转基因无性系Pt-11顶端叶片保持正常叶型,而且转基因顶端叶片的病毒数量也显著低于对照叶片,这些结果说明PtDRG01基因具有较强的抗TMV能力。此外,本研究还构建了PtDRG基因家族的RNA干扰表达载体,并采用基因枪法进行抗病三倍体毛白杨无性系的遗传转化研究,获得了一批卡那霉素抗性植株,PCR检测证实外源基因已整合到三倍体毛白杨基因组内。3.应用基因预测软件,从与毛白杨RGA高度同源的毛果杨基因组区域中克隆得到74个NBS型抗病基因。它们的外显子和内含子的长度、数量各不相同,而且所编码的氨基酸所含结构域种类、数量、长度也存在显著差异。依据基因结构组成,74个毛果杨抗病基因可分为9种类型,依据进化关系则可分为11个亚家族。从中筛选出结构与序列高度相似的8个Group抗病基因进行进化分析,结果发现,基因的平均ω值大于、小于或接近1,表明这些Group基因的进化整体上分别承受着正向选择、纯化选择或中性选择压力。除Group 6基因外,其余基因包含不同数量的正向选择氨基酸位点,其ω值均显著大于1,而且多数氨基酸位点不均匀地集中在基因中下游区域,表明抗病基因的中下游可能是决定基因作用对象特异性的主要区域。另外,在进化树的7个亚家族和8个基因组位点的基因中检测到许多基因转换,说明基因转换在基因进化中具有重要作用。为了进一步了解抗病基因与毛白杨的关系,本研究定量分析了74个毛果杨抗病基因在三倍体毛白杨6种组织器官(组培苗叶片、成熟叶片、幼嫩叶片、成熟树皮、幼嫩树皮与根)中的表达水平,结果表明,共有27个毛果杨抗病基因在三倍体毛白杨中呈现组成型表达,但不同基因间的表达水平存在显著差异。进一步比较分析发现,在树干顶端叶片、顶端幼嫩树皮和下部成熟树皮具有特异高效表达特性的基因分别有6个、2个和3个,表明这些基因可能参与组织器官特异的抗病反应。另外,24个基因在幼嫩树叶中的表达水平显著高于成熟树叶中的表达水平,22个基因在成熟树皮中的表达水平也显著高于幼嫩树皮中的表达水平,5个基因在各组织器官的表达水平均显著高于其它抗病基因的表达水平。逆境胁迫实验结果发现,表达水平受伤诱导下调的基因有2个,受伤诱导、暗培养、甲基茉莉酸(MeJA)处理、水杨酸(SA)处理和野生根癌农杆菌侵染而上调的分别有22个、20个、14个、6个和11个基因,表明三倍体毛白杨体内可能存在复杂的信号传递途径,它们相互交织在一起,协同调控着抗病基因的表达。上述研究初步揭示了毛白杨抗病资源、杨树抗病基因的进化与表达,获得了具有抗病功能的毛白杨锈病抗性相关基因,填补了毛白杨抗病研究在此领域的空白,并可为将来开展抗病基因分离、功能鉴定以及杨树抗病基因工程研究奠定坚实基础,并提供了科学借鉴。更多还原

【Abstract】 Populus tomentosa Carr. is an indigenous tree species of white poplar (in section Leuce) in China and widely employed for forestation and landscape in northern China. However, this poplar species is susceptible to a wide variety of diseases, particularly the leaf rust caused by Melampsora magnusiana Wagner, which often severely affect the poplar growth and yield. In contrast to tremendous progress in the identification and characterization of resistance loci in poplars (in section Tacamahaca and Aigeiros) as well as of putative disease resistance genes from genome sequences of P. trichocarpa, little is known about disease resistance in P. tomentosa so far.In this study, inoculation bioassays were performed on hybrids of P. tomentosa with leaf rust fungi and screened out 2 highly resistant clones of white poplars. Many resistance gene analogs (RGAs) were isolated from one resistant clone using PCR-based approach for the analyses of evolution and expression profile of disease resistance genes in white poplar and one RGA (DQ324288) was identified that showed putative involvement in resistance against leaf rust in P. tomentosa. Two members of DQ324288 gene family were cloned using RACE-PCR analysis and the expression profile of these 2 genes was analyzed using real time PCR analyses. Based on the construction of prokaryotic expression vector, RNA interference vector and sense expression vector, prokaryotic expression analysis and genetic transformation on resistant poplar and tobaccos were conducted to reveal the potential function of the above two genes. In addition, a number of resistance genes were identified from the genome of P. trichocarpa based on the RGAs of white poplar, and organization, evolution as well as expression profile of these genes in a triploid white poplar clone were studied. The main results from the above mentioned studies are described as below:1. The inoculation assays with leaf rust fungi were performed on 28 clones of hybrids of P. tomentosa and identified 13 susceptible clones with various degrees of rusting sypmton and 15 resistant clones without sympton, of which 2 clones of triploid white poplars [(P. tomentosa × P. bolleana) × p tomentosa] were considered highly resistant to leaf rust since hypersensitive response was observed. Based on the highly resistant poplars and presence of a nucleotide binding site (NBS) domain in majority of cloned plant disease resistance genes (R genes), 59 resistance gene analogs (RGAs) were identified by using PCR analysis with degenerate primers. The 59 RGAs were phylogenetically classified into 10 subfamilies and 54 RGAs with open reading frames (ORFs) were further grouped into two classes, toll and interleukin-1 receptor (TIR) and non-TIR. BLAST searches with reference to the genomic sequence of P. trichocarpa found 96 highly homologous regions distributed in 37 loci, suggesting the abundance and divergence of NBS encoding genes in the triploid poplar genome. Within subfamilies 1 to 3, the average nonsynonymous/synonymous substitution (ω) rates were < 1, indicating purifying selection on these RGAs, but some sites were clearly under diversifying selection with ω > 1. Many intergenic exchanges were also detected among these RGAs, indicating the probable role in homogenizing the NBS domains. Quantitative real-time PCR analysis revealed dramatic variations in the transcript level of 18 RGAs in the mature leaves, bark and roots of the triploid poplar and identified 2 RGAs that had significantly higher level of transcripts in bark, 4 RGAs in mature leaves, and 14 in the above ground portion of poplars, suggesting their probable roles respectively involved in resistance against the pathogens attacking the organs.2. A RGA (DQ324288 gene) was identified out of triploid poplar RGAs that shared high similarity (93%) with the genomic sequence of P. deltoides cultivar S9-2 MER locus, conferring resistance to three races of rust fungi M. larici-populina Kleb, and with the 60I2G11 gene within the MER locus. DQ324288 had homologous genes in twenty-eight triploid poplar clones, and was expressed constitutively and specifically in leaf tissue. Its expression level was increasing along with the increase in the level of resistance of the hosts against leaf rust, indicating its close relationship with the resistance against this pathogen. RACE-PCR analysis revealed two members of this gene family: PtDRGOl and PtDRG02 gene, encoding TIR-NBS-LRR and TIR-NBS proteins, respectively. These two genes displayed similar expression profile with DQ324288 gene, and positive responses to wounding, MeJA and SA rather than darkness and A. tumefaciens. Bioinformatic analysis showed that the deduced proteins of two genes were soluble and hydrophilic with respective pHi 8.165 and 10.325. The high level expression of fusion proteins of two genes in Escherichia coli was induced the IPTG and the expression level was elevated with the increase in induction temperature and time. The PtDRGOl gene was cloned into an expression vector and transferred to tobaccos via A. tumefaciens-mediated transformation. The integration of foreign genes into the genome of transgenic tobaccos was confirmed by PCR analyses with two sets of primers and the number of foreign genes in the genome of tobaccos were identified to be0.1-fold or 0.2-fold of that of native ACTIN gene by quantitative real-time PCR analysis. The expression of PtDRG01 gene varied dramatically among different transgenic lines. One-week-period inoculation with tobacco mosaic virus (TMV) revealed that the transgenic tobaccos contained less number of viruses than non-transgenic ones and that the number of viruses was negatively correlated with the expression level of PtDRG01 gene. Moreover, six-week-period inoculation induced abnormal morphology of apical leaves on the non-transgenic tobaccos and normal morphology of apical leaves on the transgenic tobaccos with high level transcripts of PtDRG01 gene, indicating that PtDRG01 gene has the potential to enhance resistance of tobaccos to TMV. In addition, a RNA interference expression vector of PtDRG gene family was constructed and transferred into the resistant triploid poplar through particle bombardment. Many transgenic poplars were obtained and the integration of RNAi sequences into the host genome was confirmed by the PCR analysis with two sets of gene specific primers.3. A total of 74 R genes with the NBS domains were identified from the genomic sequences of P. trichocarpa that were highly homologous to the RGAs from triploid white poplar. The extrons and introns in these genes varied significantly in both length and number, and the protein domains showed dramatic variations in organization, number and length. These 74 R genes were structurally classified into 9 classes with distinct protein domain organizations and phylogeneitcally divided into 11 subfamilies according to the degree of the nucleotide sequence similarity. Within 8 groups of genes with high similarity in nucleotide sequences and length, the average ω rates were > 1, < 1, or close to 1, respectively, indicating the positive selection, purifying selection or neutral selection on these resistance genes, but many sites within genes of 7 groups (except for group 6) were clearly under diversifying selection with co > 1. Many intergenic exchanges were also detected among these resistance genes, indicating the important roles that gene conversions play in the evolutionary process of resistance genes from P. trichocarpa. For a better understanding of resistance genes in white poplars, the expression patterns of these 74 genes in various organs of a triploid white poplar, under different growth conditions, were examined by using quantitative real-time PCR. Twenty-seven of 74 genes from P. trichocarpa were expressed in 6 examined organs at various levels. Six, two and three genes, respectively, displayed significantly higher expression levels in apical leaves, young bark and mature bark than in other organs, indicating that these genes may be involved in organ specific disease resistance. Twenty-four genes had dramatically greater expression in apical leaves than in mature leaves, 22 genes higher in the mature bark than in the young bark, and five genes systematically displayed dramatically stronger expression than other genes. Wounding induced an increase in the transcript level of 22 genes and a reduction for 2 genes. Twenty genes were up-regulated by darkness, 14 by methyl jasmonate acid (MeJA), 6 by salicylic acid (SA), and 11 by the compatible Agrobacterium tumefaciens, implying a complex interconnecting signal transduction pathways that regulates the expression of poplar R genes.Our results shed light on the genetic resources of poplar resistance, the evolution and expression profiling of poplar resistance genes, and the preliminary features of putative resistance gene against leaf rust in P. tomentosa, which will be helpful for the further characterization of their function.更多还原