小麦抗叶锈近等基因系TcLr45分子标记开发与凝集素类受体蛋白激酶基因的克隆
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摘要
由小麦叶锈菌(Puccinia triticina Eriks.)引起的小麦叶锈病是影响世界小麦稳产高产的重要病害之一,也是影响我国小麦生产的重要因素。抗叶锈病基因的应用成为防治该病害最经济、有效的方法。小麦抗叶锈病基因Lr45来源于日本小黑麦,目前我国尚无关于克服该基因的叶锈菌株的报道,因此,Lr45为我国小麦叶锈菌有效的抗病基因,具有一定的应用潜力。本研究通过分子标记以及抗病相关基因克隆的方法对小麦抗叶锈病基因Lr45进行研究,为该基因的分子鉴定以及抗病相关机制的研究奠定基础。本论文主要结果包括以下几个方面:
1、利用定位在黑麦2R染色体的3个SSR标记、6个RAPD标记、1个STS标记以及小麦2A染色体上的4个EST-SSR标记、31个SSR标记筛选与Lr45连锁的分子标记,发现黑麦2R染色体上的SSR标记SCM43、SCM75和小麦2A染色体上的SSR标记WMC63在亲本TcLr45与Thatcher之间表现多态性。
2、利用小麦抗叶锈病基因Lr25的SCAR标记Lr25F20/Lr25R19对同样来源于黑麦的小麦抗叶锈近等基因系TcLr26、TcLr45和TcLr25以及感病对照Thatcher进行检测,仅在TcLr45中扩增出一条1859bp的条带。经52个小麦抗叶锈近等基因系以及6个普通小麦、5个黑麦和3个小黑麦品种的验证,更正该标记为小麦抗叶锈近等基因系TcLr45特异的分子标记,命名为LR45R/F,遗传距离为14cM。避免了该标记今后在分子辅助选择中的错误使用。
3、首次利用EST-CAPS技术对小麦抗叶锈近等基因系TcLr45及感病亲本Thatcher进行多态性分析。2A染色体短臂C-2AS5-0.78区域的25对EST引物中有2对引物在亲本之间扩增出差异,100个引物/酶切组合中有4组在TcLr45和Thatcher之间表现多态性。
4、根据已克隆的小麦抗叶锈病基因Lr34序列中的凝集素类受体蛋白激酶基因设计引物,在小麦抗叶锈近等基因系TcLr45的cDNA中扩增出单一条带,经克隆、测序,该序列为凝集素类受体蛋白激酶基因的部分片段。以该序列为靶序列进行RCAE-PCR,获得基因的cDNA全长片段为2347bp,共编码730个氨基酸。经SMART软件分析,该基因具有典型的凝集素类受体蛋白激酶结构,为L类型,将该基因命名为LecRK-LR45。
Leaf rust, caused by Puccinia triticina Eriks., is one of the most severe diseases of common wheat (Triticum aestivum L.) throughout the world, and also the important factor affecting the wheat yield in China. The application of leaf rust resistance genes became the most economical and effective method to control the disease. Wheat leaf rust resistance gene Lr45 derived from the Japnases triticale, until now there is no report has been found about the leaf rust strain overcoming this gene in China, therefore Lr45 is an effective gene and with potential application in China. Related studies were carried out on wheat leaf rust resistance gene Lr45 for developing molecular markers and cloning resistance related gene, which laid a solid foundation for the molecular identification and annotation the resistance mechanism of Lr gene. The major results are as follows:
1. 3 SSR markers, 6 RAPD markers and 1 STS marker located on rye 2R chromosome and 4 EST-SSR markers, 31 SSR markers located on wheat 2A chromosome were screened the linkage molecular markers for Lr45. The SSR markers SCM43, SCM75 located on rye chromosome 2R and WMC63 from wheat chromosome 2A amplified the polymorphism between TcLr45 and Thatcher, but with a long genetic distance.
2. Wheat leaf rust near-isogenic line TcLr26 and TcLr45 which derived the same heredity background as TcLr25, and susceptible background genotype Thatcher were detected with the SCAR marker Lr25F20/R19 of wheat leaf rust resistance gene Lr25. A unique 1859bp band amplified in TcLr45. The amplification results of 52 wheat leaf rust near-isogenic lines, 6 wheat varieties, 5 rye varieties and 3 triticale varieties validated Lr25F20/R19 is a specific molecular maker with 14cM genetic distance for wheat leaf rust resistance gene Lr45, and named LR45R/F. The results corrected the location of the SCAR marker Lr25F20/R19, and will avoid the unreliable of the marker-assisted selection.
3. EST-CAPS was the first time carried on wheat leaf rust near-isogenic line TcLr45 research. 2 of 25 EST markers and 4 of 100 primers/restriction enzyme combinations which located on wheat chromosome 2A short arm C-2AS5-0.78 domain amplified the polymorphism between TcLr45 and Thatcher.
4. A pair of pimers was disigned based on the lectin-like receptor kinases gene sequence of wheat leaf rust resistance gene Lr34. A single band was amplified in the cDNA of TcLr45. The fragment was cloned and sequenced, and it is a part of lectin-like receptor kinases gene. Based on the target sequence, the complete sequence of cDNA, 2347bp and encoding 730aa, was obtained through RACE-PCR. SMART software was used to analysis this gene found that it has classic LecRKs structures, and belongs to L type, named as LecRK-LR45.
1、利用定位在黑麦2R染色体的3个SSR标记、6个RAPD标记、1个STS标记以及小麦2A染色体上的4个EST-SSR标记、31个SSR标记筛选与Lr45连锁的分子标记,发现黑麦2R染色体上的SSR标记SCM43、SCM75和小麦2A染色体上的SSR标记WMC63在亲本TcLr45与Thatcher之间表现多态性。
2、利用小麦抗叶锈病基因Lr25的SCAR标记Lr25F20/Lr25R19对同样来源于黑麦的小麦抗叶锈近等基因系TcLr26、TcLr45和TcLr25以及感病对照Thatcher进行检测,仅在TcLr45中扩增出一条1859bp的条带。经52个小麦抗叶锈近等基因系以及6个普通小麦、5个黑麦和3个小黑麦品种的验证,更正该标记为小麦抗叶锈近等基因系TcLr45特异的分子标记,命名为LR45R/F,遗传距离为14cM。避免了该标记今后在分子辅助选择中的错误使用。
3、首次利用EST-CAPS技术对小麦抗叶锈近等基因系TcLr45及感病亲本Thatcher进行多态性分析。2A染色体短臂C-2AS5-0.78区域的25对EST引物中有2对引物在亲本之间扩增出差异,100个引物/酶切组合中有4组在TcLr45和Thatcher之间表现多态性。
4、根据已克隆的小麦抗叶锈病基因Lr34序列中的凝集素类受体蛋白激酶基因设计引物,在小麦抗叶锈近等基因系TcLr45的cDNA中扩增出单一条带,经克隆、测序,该序列为凝集素类受体蛋白激酶基因的部分片段。以该序列为靶序列进行RCAE-PCR,获得基因的cDNA全长片段为2347bp,共编码730个氨基酸。经SMART软件分析,该基因具有典型的凝集素类受体蛋白激酶结构,为L类型,将该基因命名为LecRK-LR45。
Leaf rust, caused by Puccinia triticina Eriks., is one of the most severe diseases of common wheat (Triticum aestivum L.) throughout the world, and also the important factor affecting the wheat yield in China. The application of leaf rust resistance genes became the most economical and effective method to control the disease. Wheat leaf rust resistance gene Lr45 derived from the Japnases triticale, until now there is no report has been found about the leaf rust strain overcoming this gene in China, therefore Lr45 is an effective gene and with potential application in China. Related studies were carried out on wheat leaf rust resistance gene Lr45 for developing molecular markers and cloning resistance related gene, which laid a solid foundation for the molecular identification and annotation the resistance mechanism of Lr gene. The major results are as follows:
1. 3 SSR markers, 6 RAPD markers and 1 STS marker located on rye 2R chromosome and 4 EST-SSR markers, 31 SSR markers located on wheat 2A chromosome were screened the linkage molecular markers for Lr45. The SSR markers SCM43, SCM75 located on rye chromosome 2R and WMC63 from wheat chromosome 2A amplified the polymorphism between TcLr45 and Thatcher, but with a long genetic distance.
2. Wheat leaf rust near-isogenic line TcLr26 and TcLr45 which derived the same heredity background as TcLr25, and susceptible background genotype Thatcher were detected with the SCAR marker Lr25F20/R19 of wheat leaf rust resistance gene Lr25. A unique 1859bp band amplified in TcLr45. The amplification results of 52 wheat leaf rust near-isogenic lines, 6 wheat varieties, 5 rye varieties and 3 triticale varieties validated Lr25F20/R19 is a specific molecular maker with 14cM genetic distance for wheat leaf rust resistance gene Lr45, and named LR45R/F. The results corrected the location of the SCAR marker Lr25F20/R19, and will avoid the unreliable of the marker-assisted selection.
3. EST-CAPS was the first time carried on wheat leaf rust near-isogenic line TcLr45 research. 2 of 25 EST markers and 4 of 100 primers/restriction enzyme combinations which located on wheat chromosome 2A short arm C-2AS5-0.78 domain amplified the polymorphism between TcLr45 and Thatcher.
4. A pair of pimers was disigned based on the lectin-like receptor kinases gene sequence of wheat leaf rust resistance gene Lr34. A single band was amplified in the cDNA of TcLr45. The fragment was cloned and sequenced, and it is a part of lectin-like receptor kinases gene. Based on the target sequence, the complete sequence of cDNA, 2347bp and encoding 730aa, was obtained through RACE-PCR. SMART software was used to analysis this gene found that it has classic LecRKs structures, and belongs to L type, named as LecRK-LR45.
引文
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