【作者】 曹张军；

【导师】 商鸿生； 井金学； 张相岐；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2004， 博士

【摘要】 小麦条锈病是世界范围内小麦上最重要的病害之一。国内外研究和生产实践证明，种植抗锈良种是控制该病害最经济、有效、易行和对环境安全的核心措施，但一个品种种植几年后抗病性往往“丧失”。造成该现象出现的原因是育种学家只注意利用个别优良基因，而忽视了抗病基因多样性，且不注意选择高品位抗病基因，从而导致小麦品种的遗传基础狭窄，形成以小麦简单的群体抗病遗传结构应对小麦条锈菌复杂的毒性变异压力的不稳定互作系统。许多科学工作者提出了增加小麦抗源多样性，丰富小麦抗病基因，寻找高品位抗病基因保持小麦品种抗病持久性等以使小麦病害系统稳定性，延长抗病品种使用寿命的各种方法。本实验鉴定出了一个新的优秀抗条锈基因并对其进行了分子遗传图谱的构建，另外对另一优良的抗病类型— 高温抗条锈性的分子机制进行了初步研究。　１．　一个新的抗条锈基因— ＹｒＨｕａ 的鉴定及分子作图　华山新麦草（Ｐｓａｔｈｙｎｒｏｓｔａｃｈｙｓ　ｈｕａｓｈａｎｉｃａ　Ｋｅｎｇ，２Ｎ＝１４）是分布于中国秦岭山脉华山段的一个我国的特有物种，西北植物所利用染色体工程技术等已将其染色体导入普通小麦，形成了系列易位系、代换系和附加系，成为开发利用这一珍稀种质资源基础试材。我们前期研究华山新麦草抗病机制中发现其抗我国已发现的所有小麦条锈菌生理小种，是可资利用的宝贵抗病资源，同时得到一些染色体工程材料表现良好的抗性反应，易位系 Ｈ９０２０－１７－５ 是其中之一。关于来自华山新麦草抗病基因的研究尚属空白。因此本文对来自华山新麦草抗小麦条锈病基因在小麦中的经典遗传及分子遗传进行了研究，现将研究结果简要介绍如下：　(1). 本研究应用小麦条锈菌生理小种 ＣＹ３０、ＣＹ３１、ＣＹ３２ 分别对易位系 Ｈ９０２０－１７－５的供体亲本华山新麦草、受体 ７１８２ 及易位系自身进行接种鉴定。结果表明，该易位系的抗条锈基因来自华山新麦草。　(2). 以该易位系与感病品种铭贤 １６９ 正反交、回交获得的 Ｆ２代、ＢＣ１代，对其抗性遗传分析表明该易位系的抗条锈性由显性单基因控制，　暂定名为 ＹｒＨｕａ。　<WP=7>2 来自华山新麦草抗小麦条锈病基因的遗传分析和小偃 6 号高温抗条锈性差异表达基因的鉴定 (3). 对 Ｈ９０２０－１７－５ 与感病品种铭贤 １６９ 杂交 Ｆ２代分离群体利用微卫星（ＳＳＲ）技术进行抗条锈基因定位。采用了 １６６ 对 ＳＳＲ 引物，其中 ８６ 对在两新本间表现多态性，进一步在 Ｆ２ 代群体中分析，得出 ＹｒＨｕａ 与位于 ６Ａ 染色体长臂上的 Ｘｇｗｍ１６９ 标记连锁，遗传距离为 ２８．７ｃＭ，故该基因位于 ６Ａ 染色体长臂上。　 (4). 应用 ＡＦＬＰ 技术研究以找到更近的标记，共应用了 ８１ 对引物，结果获得了两个ＡＦＬＰ 标记 ＰＭ１４（３０１）和 ＰＭ４２（２４７）与 ＹｒＨｕａ 紧密连锁，距离分别是 ５．４ｃＭ 和 ２．７ｃＭ，且位于该基因两侧。　 (5). 为了方便育种实践中该基因的利用，应用酶切位点多态性结合 ＰＣＲ 将 ＰＭ１４ 标记转化为长度是 ２９６ｂｐ 的可用的单条带标记。　 本研究首次在普通小麦中对来自我国特有的珍稀物种— 华山新麦草的抗条锈基因进行了经典遗传及分子作图研究，同时将 ＡＦＬＰ 标记转化为分子辅助育种中容易利用的单条带标记。该实验结果为易位系 Ｈ９０２０－１７－５ 做为抗源应用提供依据，同时得到的标记可以作为进一步该基因精细作图的基础，并可用于分子标记辅助育种，以利于该抗病基因利用，加快育种进程。　 ２．　高温条件下小偃 ６ 号－小麦条锈菌非亲和互作中差异表达基因的鉴定与分析　 高温抗病性（Ｈｉｇｈ－ｔｅｍｐｅｒａｔｕｒｅ　Ｒｅｓｉｓｔａｎｃｅ，　ＨＴＲ）是在较高温度下所诱导表达的一种低反应型抗病性，具有非小种专化型抗病性特点，在小麦解决抗病性“丧失”问题有广阔的应用前途。普通小麦品种小偃 ６ 号是利用染色体工程技术将长穗偃麦草基因导入普通小麦中选育而成的品种，自上一世纪 ８０ 年代推广以来，至今在田间仍保持较好的抗病性。经本实验室研究其抗性表现为全生育期高温抗性，并对其抗病性机理在生理生化、组织病理学等方面进行了研究。本论文是在以前工作的基础上，进一步对其抗病分子机理进行了研究，取得了新的结果：　（１）　建立了小偃 ６ 号与条锈菌相互作用分子研究体系，确定了适宜的取样时间在花斑出 现之后至坏死出现。　（　２）使用了 ４５ 个 引 物 组 合 对 样 品 进 行 分 析 ， 检 测 了 大 约 有 ２７００　个 ＴＤＦｓ　（Ｔｒａｎｓｃｒｉｐｔ－ｄｅｒｉｖｅｄ　Ｆｒａｇｍｅｎｔｓ）的表达情况。观察到 ２２ 个 ＴＤＦｓ 在病菌及高温双重作用下表现为上调或诱导表达。　（３）　利用小麦微管蛋白 ｔｕｂｌｉｎ 作为内参，对得到的差异表达条带 ＴＤＦ６－１ 及 ＴＤＦ１７－２ 进行了半定量 ＰＣＲ 验证，这两个基因片段表现为高温条件下上调表达，与 ｃＤＮＡ－ＡＦＬＰ 结果一致。　（４）更多还原

【Abstract】 Stripe rust, caused by Puccinia striiformis f.sp. tritici, is one of the most serious diseasesof wheat in the worldwide, and planting resistant cultivars is the most effective andeconomical methods to control this disease. But one cultivar “loses”its resistance in severalyears because of resistant gene single and overcomed easy by pathogen. HTR(High-temprature Resistance) is a sort of excellent resistance to contrilloling stripe rust and itis durable resistance. So, for controlling stripe rust, identifying new resistance genes andstudying the molecular basis of HTR is of importance. Consequently, the research describedin this thesis focused on the above two aspects. 1. Identification and molecular mapping of YrHua, a new stripe rust resistance gene Ptathyrostachys huashanica Keng is a special species and distributed in Huashanbranches of the Qinling moutain chain, which is resistance to all stripe rust races in China.Crossing triticum aessivum 7182 with P. huashanica, a translocation to be attained and namedit H9020-17-5. H9020-17-5 is resistance to stripe rust, too. It suggests that the resistance genein H9020-17-5 comes from Ptathyrostachys huashanica Keng through inoculatingPtathyrostachys huashanica Keng, 7182 and H9020-17-5 with stripe rust races CY30, CY31,CY32. To mapping the resistance gene, H9020-17-5, Mingxian169, BC1 and F2 progeny(which were developed by set dialed crossing between H9020-17-5 and Mingxian169), wereevaluated for resistance in the seedling stage to Chinese P.s. tritici races under controlledtemperature in the green house, and it suggests that H9020-17-5 control stripe rust resistanceby single gene whatever it is male or female. Named it YrHua. Genomic DNA was extractedfrom 119 F2 plants and used for cosegregation analysis. 166 SSR markers and 81 AFLPmarkers were used to identify molecular markers for this resistance gene. Analysis of variance,as well as simple and composite interval mapping, were applied. The map were constructed<WP=10>Abstract 5detecting this gene 28.7cM to the wheat microsatellite (WMS or SSR) marker Xgwm169,which on the long arm of chromosome 6A, and 5.4cM and 2.7cM to the AFLP markersPM14(301)and PM42(247), respectively. For the convenience of marker-assisted selection inwheat breeding, one of the two AFLP markers was converted to PCR marker using a pair ofspecial primers designed based on the DNA sequence of PM14(301) and the polymorphism ofrestriction site. Our research results provided a useful tool for marker-assisted selection andyet laid the foundation of the fine mapping and map based cloning YrHua gene.2. Identification and analyse different expression genes involved in Xiaoyan6-Puccinia striiformis f.sp. tritici incompatible interaction under high-temprature In order to understand the molecular basis of resistance responses to stripe rust in wheatHTR, cDNA-AFLP (cDNA amplified fragment length polymorphism) analysis was used toidentify differentially expressed transcripts during the Xiaoyan6-Puccinia striiformis f.sp.tritici incompatible interaction under high-temprature. In the research, threerestriction-enzymes including MseI and two isocaudarnes (EcoRI and MunI) were used, andthe highly virulent race CY30 was selected as the experimental system. According to result of our laboratory, finding very few different fragments betweeninoculated and non-inoculated seedings before nesrotic lesions were observed. Therefore, thedifferently expressed genes after nesrotic lesions were observed in this research.Approximately 2700 TDFs (transtript-derived fragments) were screened, and found 22up-regulated or induced expression TDFs. Of these fragments, five were found to behomologous to previously characterized genes based on Blast analysis, and some of theirdifferential expression patterns were further verified using semi-quantitative RT-PCR. TDF6-2 encoded a gene which is homologous with a gene from Vibrio vulnificus,更多还原