【作者】 马金彪；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 小麦条锈菌（Puccinia striiformis f.sp.tritici）是世界范围内危害小麦正常生长的病原物。由其引发的小麦条锈病具有流行性强、危害范围广、危害严重等特点,成为世界粮食安全生产的制约因素。过去,人们对该病害的流行学、组织学、细胞学等方面进行了详尽的研究,获得了基础学研究的许多理论和方法。随着分子生物学研究技术的日新月异,人们逐渐把工作重点转移到抗病基因分子标记以及筛选利用方面,而对条锈病亲和性互作发病分子机理和病原菌致病功能基因领域研究较少。由于小麦条锈菌存在种内毒性小种差异,而且能够通过变异和突变的方式产生新的毒性小种,这是造成小麦抗条锈性品种在短期内丧失抗性的主要因素。因此,从基因水平去全面阐述小麦与条锈菌亲和互作分子机理和基因表达特征,揭示小麦与条锈菌亲和互作分子机理,将为研究该病害的发病机理奠定物质基础。本研究重点是从转录基因水平总括调控小麦与条锈菌亲和互作基因表达谱。挖掘小麦与条锈菌亲和互作中表达的寄主感病基因和条锈菌在这一生理过程中表达的调控基因。本论文第一部分内容首次对构建的小麦与条锈菌亲和性cDNA文库进行大规模测序,通过对所获得的EST进行生物信息学分析,分离获得了大量调控小麦与条锈菌亲和互作过程中特异表达基因;通过SqRT-PCR和qRT-PCR技术分析亲和互作中来源于小麦和条锈菌特异表达基因的转录表达谱;通过VIGS技术对一些感兴趣候选基因进行初步功能验证分析。本论文第二部分则通过生物信息学研究技术,以现有的小麦秆锈菌基因组序列为参照,通过比较基因组学的方法将条锈菌的EST与秆锈菌的基因组序列进行了同源性比对分析。首次绘制获得了小麦条锈菌转录基因物理图谱。主要研究成果如下:1.对已经构建好的小麦与小麦条锈菌亲和性cDNA文库进行大规模测序,获得5796条高质量的EST序列。经CAP3聚类后拼接成583条contigs和2163条singletons,共获得2743条非重复序列。在所有contig序列中,有300条序列包含了2条EST,169条包含了3-5条EST,57条包含了6-10条EST,43条包含了11-35条EST,11条包含了36-70条EST,有三条contigs分别包含了89、93、150条EST。这说明,这些基因在小麦与条锈菌亲和互作过程中高丰度转录表达。将获得的非重复EST序列与NCBI中的非冗余蛋白质数据库进行本地Blast分析,通过EST蛋白功能注释发现,结果发现有1922条可以找到最佳匹配序列,占70%左右。824条在数据库中无同源性序列,为no hits found,占30%。具有已知蛋白功能的Unigene中,1450条同源于水稻、拟南芥、玉米、苜蓿、大麦、小麦等植物基因编码的蛋白,占52.8%;472条同源于酵母、玉米瘤黑粉、稻瘟菌、曲霉菌、脉孢菌、杆锈菌、叶锈菌、蚕豆锈菌等病原真菌蛋白,且大多数为假象蛋白和未知蛋白,占16.3%。这些EST数据为今后挖掘调控病原菌与寄主亲和互作基因提供了物质基础。2.本研究用半定量和定量PCR两种技术,对候选基因进行了转录表达分析。半定量结果显示,所选择的条锈菌基因在小麦接种条锈菌（亲和性反应）后不同阶段转录表达情况有所差异。一种为随着条锈菌侵染时间的变化,条锈菌基因呈现上调表达趋势,推测这类基因为条锈菌组成性表达的基因。如actin、核糖体基因等持家基因,随着侵染时间的延续,条锈菌组织在小麦组织内所占的比例逐步增大,因此这些基因被检测到的可能性也越大。另一种只在侵染后期表达,前期未检测到任何表达信号,转录表达开始在接种120h之后,判断此类基因的表达可能与小麦与条锈菌建立了更广泛的寄生关系相关,因为此时有大量的吸器形成,这些基因可能与条锈菌吸器的寄生功能有关。同时在未接种小麦叶片对照CK中我们没有检测到条锈菌来源的基因表达。qRT-PCR分析了10个来自于寄主小麦的基因,从表达模式上来看WRIS₆71和WRIS₁264在亲和与非亲和互作中均无太大的变化。而WRIC₁16、WRIS₂33、WRIS₂972、WRIS₈67、WRIS₁790在非亲和组合中的表达量要高于在亲和组合中的表达量。此外,我们所选的候选基因中WRIC₄02,WRIS₃105,WRIS₃974在亲和互作中的表达量略高于非亲和组合。从VIGS的结果来看,四个基因WRIC₄02-γ-23（C2）,WRIC₄23-γ-23（B4）,WRIS₃105-γ-23（C4）和WRIS₂972-γ-CY23（A8）在BSMV-VIGS体系下被有效沉默后,使本来对CY23具有抗性反应的小麦suwon11叶片上产生了孢子,反应从抗病转为感病。但是,在亲和互作体系下抑制这四个基因的表达并未出现与对照表型上的差异。说明这四个基因在小麦与条锈菌互作过程中参与了寄主抗病反应。3.通过利用4219条来源于条锈菌夏孢子、萌发芽管和吸器的EST对小麦秆锈菌基因组序列进行Blast同源性比对,发现1432条条锈菌EST与杆锈菌基因组序列有高度同源性（e-value<1.00E-5）。结果显示,不同生育期来源的EST与杆锈菌基因组同源性比对结果有差异。其中,来源于夏孢子的EST与杆锈菌的同源性最高,达到54.51%。来源于萌发芽管的EST与杆锈菌基因组的同源性为51.21%。而来源于吸器的EST只有13.64%能够与杆锈菌基因组比对上。在比对的所有条锈菌EST中,平均有33.94%与秆锈菌有同源性。用Blast程序,对条锈菌EST进行本地同源性比对,参照数据库为在http://www.broadinstitute.org/annotation/genome/puccinia group/MultiHome.html网站上公布的秆锈菌392条基因组序列,获得条锈菌237张转录基因物理图谱,大小范围在2878-3081398bp之间,大部分在5000bp-20000bp左右。大部分EST在图谱上存在多点分布的特点。超过70%的图谱上只分布了不超过20个基因,但是在1、2、3和17号图谱上分布了超过100个基因。通过比较三个不同文库EST比对结果发现来源不同发育阶段的EST在图谱上分布区域不同。通过与秆锈菌基因组序列比对发现1432个条锈菌基因中911个是有外显子的基因,占所比对基因总数的63.62%。而521个基因不具有外显子特征。用基因特异引物对所绘制的基因物理图谱进行试验验证。为验证条锈菌转录基因物理图谱的正确性,我们选择在图谱上50kb范围内的两个基因来设计基因特异引物,用来在条锈菌BAC文库中筛选阳性克隆,如果这两个基因同时出现在一个BAC克隆里,说明这两个基因在物理图谱上具有很好的共线性。通过选用42个基因组成21个组进行试验,结果有12组能够在同一个BAC克隆中扩增出条带,结果显示,大部分条锈菌转录序列在图谱上具有共线性。本研究为克隆小麦条锈菌功能基因和开展小麦条锈菌基因组测序提供了物质基础,并具有指导意义。更多还原

【Abstract】 Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici （Pst）, is one of the most destructive diseases of wheat worldwide. Yield losses caused by stripe rust over a large area can be up to 50%, while individual fields can be destroyed when the disease is badly prevalent. Traditionally, research about wheat stripe rust focused on incompatible interactions, explaining the host resistant mechanism and resistance genes exploitation. More recently, increasing attention has been focused on pathogen factors that promote compatible interactions and disease development in plant tissue.So this research gets a lots of EST sequence by sequencing a compatible interaction cDNA library between wheat and stripe rust. We analyses these sequences by bioinformatics methods and well known the gene transcription mode. The RT-PCR and the qRT-PCR were used to analysis the transcription expression of several genes which homologue to plant and fungi in different stage and tissue. At the same time, VIGS technique was used in verify the gene function in the system of interaction of wheat and stripe rust. The last, we successful constructed a gene transcription physical map of stripe rust. The main results of our work are as follow.1. From 6,002 randomly picked clones that were sequenced from 5’ end. 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,163 singletons to give a set of 2,746 unique sequences （unisequences）. EST similarity search using the BLASTx program was carried out by comparing sequences in the non-redundant protein database of GenBank. About 50% of the ESTs showed significant matches to gene coding proteins with known functions; 20% of the ESTs were similar to gene coding proteins with unknown functions; 30% of the ESTs did not show significant similarities to any sequences in public databases. 52.8% ESTs, which was the largest category, showed the highest homology with plants; 16.3% ESTs showed higher homology with fungi and the category of no hits found accounted for 30%.2. To validate the expression patterns of genes generated in cDNA library of wheat-Pst compatible interaction. We tested some gene generating from the compatible interaction using the SqRT-PCR and real-time PCR. Several stripe rust gene have differential expression pattern in the stage of compatible interaction between wheat and stripe rust. Some genes are up-regulated after the rust infection the wheat. We speculated those gene are house keeper genes. For example, actin and ribosome genes. Those genes are expressed by rust with high level. Other genes only expressed at the end infection stage. No signals were detected at the early stage of rust infection. Those genes maybe express by hausteria of rust controlling the rust to acquire the nutrient from the wheat. At the some time, we didn’t detected any expression signal of those genes in the healthy wheat. Both WRIS₆71 and WRIS₁264 genes have no more expression change in compatible and incompabtible interaction of wheat and stripe rust. WRIC₁16, WRIS₂33, WRIS₂972, WRIS₈67, WRIS₁790 genes have high level expression in incompabtible interaction of wheat and stripe rust. Three of genes WRIC₄02,WRIS₃105,WRIS₃974 are expressed with high level in compatible than in incompabtible interaction. For test the function of those genes, VIGS was used and four genes, WRIC₄02-γ-23（C2）,WRIC₄23-γ-23（B4）,WRIS₃105-γ-23（C4）and WRIS₂972-γ-CY23（A8）have the resistance function and control the incompatible.3. A total of 4,219 Pst unisequences from the Ured, GermUred, and Haus libraries were searched for homologous sequences in the Pgt genome of 392 supercontigs. Of the Pst genes, 1,432 had significant homology （e value < 1.00E-5） to Pgt genomic sequences. The three libraries had different percentages of homologous genes with Pgt. The Ured library had the highest percentage, 54.51%, followed by the GermUred library （51.21%）, while the Haus library had the lowest percentage （13.64%）. In average, 33.94% of the 1,432 Pst genes had significant homology with the Pgt sequences. The 1,432 Pst genes were aligned to 237 linkage groups corresponding to 237 Pgt supercontigs. The 237 linkage groups ranged from 2,878 to 3,081,398 bp with the most of the linkages from 5.0 Kb to 2.0 Mb. Overall, the 1,432 genes matched 787,413 bp and spanned over 86.55 Mb of the Pgt genomic sequences. Because the majority of the 1,432 different genes were aligned to more than one sequence locus, a total of 4,604 gene loci were obtained. The fold of multiple loci per unique gene was unbalanced among the three libraries with 1.30 for the GermUred library, 1.53 for the Ured library and 10.58 for the Haus library. The number of genes varied from 1 to 153, excluding“Supercontig 392”that contained unassembled sequences, with an average of 19 genes per supercontig. Over 70% of the supercontigs have 20 genes or fewer while only 4 supercontigs （Supercontigs 1, 2, 3 and 17） had more than 100 genes. The genes from the three Pst libraries were unevenly aligned to the Pgt genome. Of the 237 supercontigs, the 712 unisequences were aligned to 134 supercontigs; the 441 unisequences were aligned to 121 supercontigs; and the 279 supercontigs were aligned to 213 supercontigs. The gene density （the number of base pairs per gene） ranged from 1,020 to 209,493 bp with an average of 20,500 bp. The majority of the supercontigs had a gene less than 30 Kb, which may be considered relatively gene-rich region. In contrast, a few supercontigs had a gene in over 60 Kb genome region, which may be considered as relatively gene-poor region. These results indicated that genes expressed in different Pst growth stages tended to be clustered on the genome. To validate the linkage relationships of Pst genes, a total of 84 forward and reverse primers were designed for 42 genes to form 21 pairs. The genes in each pair were selected based on their proximity within 50 Kb in the physic map. Clones that were positively amplified with the first pair of primers resulted from the three-dimensional pooling screening were amplified with the second pair of primers. Of the 21 pairs of genes tested, 12 pairs （57%） were successfully identified in same BAC clones. The results clearly showed that these genes in pairs are truly linked in the Pst genome.更多还原