小麦抗叶锈基因Lr38、Lr45分子标记及抗病相关分析
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摘要
由小麦叶锈菌(Puccinia triticina)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,选育并合理利用抗病品种是防治麦类锈病危害最为经济有效的途径之一。小麦抗叶锈病基因Lr38、Lr45为目前国内外有效抗叶锈病基因,本研究从Lr38、Lr45的分子标记、表达序列的分析以及蛋白质组分析等方面对小麦抗叶锈病基因Lr38、Lr45进行研究,为这两个基因的分子鉴定、辅助选择育种及抗病相关机制的研究奠定基础:
1.应用中间偃麦草E染色体组的SCAR标记成功标记了来源于中间偃麦草的小麦抗叶锈基因Lr38,命名为Y38SCAR982,连锁分析表明为与Lr38基因共分离的SCAR标记, 120个小麦品种检测的结果表明,可方便快捷的应用于分子标记辅助选择;
2.建立了Lr45基因的一个RAPD标记,并转化为稳定的SCAR标记,命名为YpSc20H1272;利用黑麦的RAPD片段pSc20H.2设计特异引物pSc20H23/24,建立了Lr45基因的SCAR标记,命名为YpSc20H750;利用黑麦特异SCAR标记引物H11R/F在TcLr45中扩增出特异片段,但连锁距离较远,该标记命名为YpScH650。以上所建立的TcLr45的3个SCAR标记,除YpSc20H750在黑麦5R染色体无检测位点外,都在黑麦的1R-7R染色体扩增出检测位点序列,本实验所用的100个小麦品种检测结果表明,可方便快捷的用于分子标记辅助选择。
3.首次对小麦抗叶锈基因Lr38与Lr45进行了SRAP与SSR (eSSR)组合的TRAP分析,获得了在小麦抗叶锈近等基因系TcLr38中特异的长度为277bp的片段序列,GenBank比对分析表明为一新的小麦序列,但在本实验所用的100个小麦品种中普遍扩增出该序列。同时采用SRAP与RGA组合的TRAP对小麦抗叶锈基因Lr38与Lr45进行了分析,获得TcLr45特异的片段,GenBank比对分析与基因组DNA序列无同源性,但与EST序列具有较高的同源性,表明所获得的序列为表达基因的序列,其中一条序列与Lr10相似性达90%,推测可能与抗病相关。
4.首次利用染色体步行NASS-PCR技术将Lr38基因Y38SCAR982片段的3′端序列延伸了1213bp,获得了一个完整的编码反转录转座子rve蛋白的ORF,编码蛋白序列为313个氨基酸的多肽。经BLASTP分析,与大麦的反转录转座子的rve整合酶核心区域蛋白序列(gb|AAV80394.1|)相似性达67%。
5.首次利用2-DE技术分析了小麦抗叶锈基因系材料TcLr38与背景材料Thatcher间的蛋白表达差异,获得了在Thatcher中差异表达的蛋白点,PMF分析与放氧蛋白复合前体OEC、蛋白激酶、β-葡聚糖激酶序列具有较高的同源性。
6.根据差异蛋白点OEC比对序列设计引物,获得了在TcLr38与Thatcher DNA中相同的1kb的条带,并在TcLr38与Thatcher cDNA以及TcLr38 DNA中相同的750 bp的条带。表明TcLr38基因组中除含编码放氧复合蛋白前体包含内含子的基因外,还具有直接转录放氧复合蛋白的序列。
Leaf rust, caused by Puccinia triticina, is one of the most severe diseases of common wheat (Triticum aestivum L.) all over the world. Breeding and application of resistant cultivars are the most economical and environment-friendly ways to reduce the damage caused by wheat leaf rust. Lr38 and Lr45 are the effective resistance genes to wheat leaf rust currently in the world. Related studies were carried out on both genes for molecular markers, and analysis of expression sequence as well as proteome, laying a solid foundation for the marker assisted selection (MAS) and identification of resistance mechanism for Lr genes.
1. A new specific molecular marker disgnated as Y38SCAR982 for wheat leaf rust resistance gene Lr38 was developed by using a SCAR marker derived from E-chromosome of Thinopyrum, and it was found that Y38SCAR982 was co-segregated with gene Lr38 in the tested F2 progeny and can be used in MAS feasibly.
2. A RAPD marker for Lr45 was acquired and converted into a stable SCAR marker named as YpSc20H1272. A SCAR marker, named YpSc20H750, for Lr45 was obtained based on specific RAPD fragment pSc20H.2 of Secale cereale. A new specific molecular marker for Lr45 was developed based on the fragment amplified with a SCAR marker primers (H11R/F) derived from S. cereale, and named as YpScH650, with a long genetic distance to Lr45. The three SCAR markers can be used to detect 1R to 7R chromosomes loci of S. cereale, except for YpSc20H750 in which no alleles loci were detected on 5R. These markers can be used in MAS feasibly based on the detection of 100 wheat cultivars used in this paper.
3. Resistant genes of wheat leaf rust, Lr38 and Lr45, were investigated first time by TRAP, combined with SRAP and SSR or EST-SSR. A 277 bp specific fragment was acquired in TcLr38. The specific fragment has no homologous sequence in GenBank, but the same size fragment was amplified in most of the 100 wheat cultivars. The genes Lr38 and Lr45 were studied by TRAP as well, with a combination of SRAP with RGA. A specific fragment was acquired in TcLr45, which showed highly homology with EST sequence, but no homology with DNA sequences in GenBank database. One sequence of the specific fragment had 90% homology with Lr10, indicating that it may closely related to disease resistance.
4. An elongated fragment of 1213 bp from the 3′end of Y38SCAR982 in Lr38 gene was obtained first time by chromosome walking NASS-PCR. A full length of 2195 bp sequence with a complete ORF, encoding 313 amino acid of retro-transposons rve, has 67% homology with rve integrase core domain sequence of Hordeum.
5. The 2-D maps of TcLr38 and Thatcher were analyzed first time. One different expression protein spot was identified in Tathcher, and was analyzed and searched its homologous proteins in the database based on NCBI by using MALDI-TOF-MS. Oxygen-bursting complex precursor (OEC), a kinase-like and beta galactosidase-like protein showed highly homology with a part of the differential protein polypeptide.
6. A pair of specific primers were designed based on the OEC, and a fragment of 750 bp expected product was acquired when amplification was carried out on cDNA of TcLr38, Thatcher and DNA of TcLr38. 1000 bp products was acquired just in DNA of TcLr38 and Thatcher. So, a conclusion can be drawn that there were two kinds of OEC genes in TcLr38 genome, one with introns, and the other with protein sequence of oxygen-bursting complex.
1.应用中间偃麦草E染色体组的SCAR标记成功标记了来源于中间偃麦草的小麦抗叶锈基因Lr38,命名为Y38SCAR982,连锁分析表明为与Lr38基因共分离的SCAR标记, 120个小麦品种检测的结果表明,可方便快捷的应用于分子标记辅助选择;
2.建立了Lr45基因的一个RAPD标记,并转化为稳定的SCAR标记,命名为YpSc20H1272;利用黑麦的RAPD片段pSc20H.2设计特异引物pSc20H23/24,建立了Lr45基因的SCAR标记,命名为YpSc20H750;利用黑麦特异SCAR标记引物H11R/F在TcLr45中扩增出特异片段,但连锁距离较远,该标记命名为YpScH650。以上所建立的TcLr45的3个SCAR标记,除YpSc20H750在黑麦5R染色体无检测位点外,都在黑麦的1R-7R染色体扩增出检测位点序列,本实验所用的100个小麦品种检测结果表明,可方便快捷的用于分子标记辅助选择。
3.首次对小麦抗叶锈基因Lr38与Lr45进行了SRAP与SSR (eSSR)组合的TRAP分析,获得了在小麦抗叶锈近等基因系TcLr38中特异的长度为277bp的片段序列,GenBank比对分析表明为一新的小麦序列,但在本实验所用的100个小麦品种中普遍扩增出该序列。同时采用SRAP与RGA组合的TRAP对小麦抗叶锈基因Lr38与Lr45进行了分析,获得TcLr45特异的片段,GenBank比对分析与基因组DNA序列无同源性,但与EST序列具有较高的同源性,表明所获得的序列为表达基因的序列,其中一条序列与Lr10相似性达90%,推测可能与抗病相关。
4.首次利用染色体步行NASS-PCR技术将Lr38基因Y38SCAR982片段的3′端序列延伸了1213bp,获得了一个完整的编码反转录转座子rve蛋白的ORF,编码蛋白序列为313个氨基酸的多肽。经BLASTP分析,与大麦的反转录转座子的rve整合酶核心区域蛋白序列(gb|AAV80394.1|)相似性达67%。
5.首次利用2-DE技术分析了小麦抗叶锈基因系材料TcLr38与背景材料Thatcher间的蛋白表达差异,获得了在Thatcher中差异表达的蛋白点,PMF分析与放氧蛋白复合前体OEC、蛋白激酶、β-葡聚糖激酶序列具有较高的同源性。
6.根据差异蛋白点OEC比对序列设计引物,获得了在TcLr38与Thatcher DNA中相同的1kb的条带,并在TcLr38与Thatcher cDNA以及TcLr38 DNA中相同的750 bp的条带。表明TcLr38基因组中除含编码放氧复合蛋白前体包含内含子的基因外,还具有直接转录放氧复合蛋白的序列。
Leaf rust, caused by Puccinia triticina, is one of the most severe diseases of common wheat (Triticum aestivum L.) all over the world. Breeding and application of resistant cultivars are the most economical and environment-friendly ways to reduce the damage caused by wheat leaf rust. Lr38 and Lr45 are the effective resistance genes to wheat leaf rust currently in the world. Related studies were carried out on both genes for molecular markers, and analysis of expression sequence as well as proteome, laying a solid foundation for the marker assisted selection (MAS) and identification of resistance mechanism for Lr genes.
1. A new specific molecular marker disgnated as Y38SCAR982 for wheat leaf rust resistance gene Lr38 was developed by using a SCAR marker derived from E-chromosome of Thinopyrum, and it was found that Y38SCAR982 was co-segregated with gene Lr38 in the tested F2 progeny and can be used in MAS feasibly.
2. A RAPD marker for Lr45 was acquired and converted into a stable SCAR marker named as YpSc20H1272. A SCAR marker, named YpSc20H750, for Lr45 was obtained based on specific RAPD fragment pSc20H.2 of Secale cereale. A new specific molecular marker for Lr45 was developed based on the fragment amplified with a SCAR marker primers (H11R/F) derived from S. cereale, and named as YpScH650, with a long genetic distance to Lr45. The three SCAR markers can be used to detect 1R to 7R chromosomes loci of S. cereale, except for YpSc20H750 in which no alleles loci were detected on 5R. These markers can be used in MAS feasibly based on the detection of 100 wheat cultivars used in this paper.
3. Resistant genes of wheat leaf rust, Lr38 and Lr45, were investigated first time by TRAP, combined with SRAP and SSR or EST-SSR. A 277 bp specific fragment was acquired in TcLr38. The specific fragment has no homologous sequence in GenBank, but the same size fragment was amplified in most of the 100 wheat cultivars. The genes Lr38 and Lr45 were studied by TRAP as well, with a combination of SRAP with RGA. A specific fragment was acquired in TcLr45, which showed highly homology with EST sequence, but no homology with DNA sequences in GenBank database. One sequence of the specific fragment had 90% homology with Lr10, indicating that it may closely related to disease resistance.
4. An elongated fragment of 1213 bp from the 3′end of Y38SCAR982 in Lr38 gene was obtained first time by chromosome walking NASS-PCR. A full length of 2195 bp sequence with a complete ORF, encoding 313 amino acid of retro-transposons rve, has 67% homology with rve integrase core domain sequence of Hordeum.
5. The 2-D maps of TcLr38 and Thatcher were analyzed first time. One different expression protein spot was identified in Tathcher, and was analyzed and searched its homologous proteins in the database based on NCBI by using MALDI-TOF-MS. Oxygen-bursting complex precursor (OEC), a kinase-like and beta galactosidase-like protein showed highly homology with a part of the differential protein polypeptide.
6. A pair of specific primers were designed based on the OEC, and a fragment of 750 bp expected product was acquired when amplification was carried out on cDNA of TcLr38, Thatcher and DNA of TcLr38. 1000 bp products was acquired just in DNA of TcLr38 and Thatcher. So, a conclusion can be drawn that there were two kinds of OEC genes in TcLr38 genome, one with introns, and the other with protein sequence of oxygen-bursting complex.
引文
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