【作者】 秦跟基；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2001， 博士

【摘要】 小麦白粉病和赤霉病是世界小麦生产中的严重病害。南京农业大学细胞遗传所培育出高抗白粉病的小麦-簇毛麦6VS/6AL易位系，其携有高抗白粉病基因Pm21。研究并克隆小麦抗病基因，对控制小麦病害及研究小麦抗病的分子机理有非常重要的意义。本研究利用植物抗病基因具有一些共同的保守区如核苷酸结合位点（NBS），富含亮氨酸重复（LRR）和丝氨酸/苏氨酸蛋白质激酶域的特点，设计简并性引物通过PCR方法获得抗病基因片段，继而筛选6VS/6AL易位系的可转化人工染色体（TAC）基因组文库，分离完整的基因。 用根据NBS，LRR和丝氨酸/苏氨酸蛋白质激酶域设计的3对简并性引物，以6VS/6AL易位系的基因组DNA或cDNA及簇毛麦的基因组DNA为模板进行PCR扩增。扩增产物克隆到pGEM-T载体中，经测序，共获得具有NBS结构域片段的克隆18个，具有LRR结构域片段的克隆3个，具有丝氨酸/苏氨酸蛋白质激酶域片段的克隆1个。将克隆之间核苷酸序列同源性高于90％的克隆归为一类，共分为8类。这8类抗病基因类似序列（RGAs）均具有开放阅读框，并与已克隆的小麦抗条锈病基因Yr10，大麦抗白粉病基因Mlal和Mla6，拟南芥的抗病基因RPS2，番茄的抗病基因Cf-2、Cf-4、Cf-5和Cf-9以及其他一些抗病基因在保守区内具有高度的同源性。用小麦中国春缺体-四体初步将它们分别定位于小麦第一、第二和第五部分同源群上。 根据从小麦-簇毛麦6VS/6AL易位系的基因组DNA获得的一条296bp的LRR序列设计引物，以6VS/6AL易位系的基因组DNA为模板，用TAIL-PCR从两端延伸该片断达800bp左右。根据该序列设计了一对特异引物，分别以感病对照扬麦5号，抗病6VS/6AL易位系，6V代换系，硬粒小麦-簇毛麦双二倍体及簇毛麦的基因组DNA为模板进行特异性扩增。除扬麦5号未扩出带，其他均扩出一条730bp的带。再用不同品系小麦-簇毛麦6VS/6AL易位系以及其他品种的基因组DNA为模板进行PCR，都只能在含有Pm21的易位系中扩出730bp的目的带，而不含Pm21的品种则无扩增产物。 用克隆池PCR法用这对LRRt的特异引物筛选小麦-簇毛麦6VS/6AL易位系的基因组TAC文库。该文库由22块96孔板组成，每孔中含有约1000个混合的TAC克隆。为了用PCR筛选文库，首先提取这22×96个混合质粒，然后用特异引物分级筛选，最后获得一个可能的阳性TAC单克隆。将该TAC克隆用不同6碱基酶酶切，转膜后，以上述LRR片段作探针进行Southern杂交，每种酶切均有杂交带，表明该克隆为真正的阳性单克隆。其中用SeaⅠ酶酶切的该TAC克隆，仅在约4．okb处有一条杂交带。将这条带进行亚克隆后，用特异引物分步测序，共获得 273 hp序列。 该序列编码一个含有753个氨基酸的蛋白，暂命名为7iiLKKIo用出 刀软件在GeneBank中比较该基因的核昔酸序列，未发现有明显同源的序列，表明该基因是一个新基因。当用BbotZ、0软件在GeneB毗中比较该基因的氨基酸序列时，发现TaLRRI与番茄抗病垦因CfJ、o人q5和o9以及其他一些抗病基因在保守区内具有高度的同源性。再用 ClustaIXI．巴软件将 TaLRRI的氨基酸序列与番茄抗叶霉病基囚 CfZ和 Cf4的氨基酸序列进行多序列比较，结果表明 TaLRRI与 CfZ和 Cf4有相似的结构。这是首次从小麦中克隆到的与双于叶植物番茄的 CfJ和 Cf4有类似结构的细胞外受体类抗病候选基因。根据比较结果推断TaLRRI为一个含有LRR的膜结合糖蛋白，可划分为7个区域（AAI）。A区是可能的信号肽，B区是成熟蛋白的N－端，C区为25个LRR重复，D区是没有明显特点的区域，E区富含带负电荷的酸性氨基酸的区域，F区由29个不带电荷的氨基酸组成跨膜疏水区，G区富含带正电荷的碱性氨基酸的区域。C区共有13个可能的糖基化位点N以S汀卜 用电激转化法将该TAC克隆转人农杆菌LBA4404中。从农杆菌中提取质粒，再转入大肠杆菌，再从人肠杆菌提取质粒，然后用Hedll酶切，酶切带型与原未的该TAC质粒完全一样，哀明该TAC质粒可以在农杆菌中稳定存在。用该克隆转化小麦的实验正在进行中。 本研究获得的RGA序列之一N7与小麦抗条锈病基因bIO和大麦抗口粉病基因M7dj及ABiz6分别有65o和61o的核芍酸相似性，并具有R基囚NBS保守区。用N7的特异引物筛选6VS／6AL易位系的基因组TAC库，获得一个抗病候选基因的TAC克隆。 小麦抗赤霉病性状山数量基因控制，遗传机制复杂。利用抗赤霉病品种苏麦39和望水IJ，分别与感病品种Alondra’s杂交，取n代花药培养获得单倍体绿苗。绿茁转接到炼茵培养基上在4℃冰箱中越夏。初冬移栽大田。分孽期挖出小苗，用0．04％的秋水仙碱浴液浸根加倍。并用玉米和小麦杂交通过染色体捎除法获得小麦单倍体。共获得能稳定遗传的双单倍体群体（DH 2个。其中来自苏麦 3号 X Alondra’s的群体共有 102个株系，来自望水白 x Alondra’s的群体共有153个株系。在构建DH群体时对一些影响小麦单倍体获得效率的囚素进行了初步研究。更多还原

【Abstract】 Powdery mildew and scab are serious wheat diseases occurring worldwide. The translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa, developed by Cytogenetics Institute of Nanjing Agricultural University, has been proved to be highly resistant to powdery mildew. A resistant gene locus for powdery mildew, Pm21, has been identified in the chromosome arm 6VS in this translocation line. Efforts in studying and cloning of resistance(R) genes of wheat are of great importance both in control of wheat diseases and understanding of the molecular mechanism of disease resistance in wheat. The objects of this study are cloning resistant genes by~ the R gene homology strategy, based on the finding that plant resistant genes contain conserved sequences such as nucleotide binding site (NBS), leucine-rich repeats (LRR), and serine/threonine kinase domain. Three degenerate primers were designed based on NI3S, LRR or serine/threonine kinase domain and used to perform PCR with genomic DNA or cDNA from 6VS/6AL or genomic DNA from Haynaldiu villosa. Amplified products were cloned in pGEM-T vector and then sequenced. Eighteen clones characterized with NBS, three with LRR and one with serine/threonine kinase domain were obtained by sequencing analysis. These clones were classified to 8 groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORFs), and show high homology to Yr]O in wheat, Mlal and M1a6 in barley, RPS2 in Arabiadopsis. (f 2, Cf.-4, Cf 5 and Cf-9 in tomato and other cloned R genes with conserved motifs. They were preliminarily mapped to homeologous groups 1, 2 or 5 of the chromosomes of common wheat by nulli- tetrasomic analysis. TAIL-PCR was performed to amplify sequences flanking a 296-bp LRR fragment obtained from the 6VS/6AL line, and a total of 800-bp sequence containing this LRR was determined. A pair of specific primers was designed from the sequence and was used to amplify the genomic DNAs of Yangmai 5 (susceptible check), 6VS/6AL line, substitution line of 6V and amphidiploid of Triticum durum-Haynaldia 3 villosa. The results showed that a 730-bp PCR product was amplified in all lines with Pm21 but Yangrnai5. The same results were observed with other 6VS/6AL translocation lines and other susceptible cultivars. The LRR sequence specific primers were used to screen a genomic TAC library of 6VSI6AL consisting of 2x I 0~ clones. The library was stored in twenty-two 96-well plates, each well containing approximately 1000 TAC colonies. By using a pooled PCR screening procedure, a positive TAC clone was obtained. The clone plasmid DNA was digested with various 6-base restriction enzymes, then transferred to membrane, and conducted southern hybridization with the LRR fragment as a probe. A 4-kb Sea I fragment was identified and subcloned. Part of the sequence (2731 bp) was determined by specific primer walking. The sequence encodes a predicted protein with 753 amino acids, designated as TaLRR1. The nucleotide sequence was compared with the GeneBank database with software Blast2.0. No high scored homologous sequence was found, indicating TaLRRI is a novel gene. When the amino acid sequence of TaLRRI was compared with the GeneBank database with software Blast2.0, the result show that it has strong alignments with the amino acid sequence of Cf 2, Cf-4, Qf-5 and Zf 9 in tomato and othe更多还原