小麦抗白粉病基因Pm6、Pm21、Pm23RGAP标记的鉴定
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摘要
本研究中，结合小麦抗性鉴定结果，选择河南省麦区具有良好抗病性，含不同抗白粉病基因的13个小麦品系，同时选感病品种Chancellor作为对照。根据抗病基因保守序列，合成7对RGA引物，进行RGAP分析。
通过RGAP引物的筛选，发现引物R11R／R11F在含有Pm6基因的Timgalen中扩增出1102bp的多态性片段C2，在感病品种Chancellor和含其它抗白粉病基因品系中则扩增不出该片段。对该片段进行克隆测序，DNA序列分析发现，C2与Genbank中的小麦抗叶锈病基因Lr10(序列登录号为AY270159.1)、小麦中的抗病基因类似序列RGA2(序列登录号为AF326781.1)、小麦NBS-LRR类蛋白mRNA序列(序列登录号为AY084050.1)，有较高的相似性。C2推定的蛋白质保守区域分析，结果表明，C2的氨基酸序列包含抗病基因的类似序列，有1个不完整的核苷酸结合位点(NB-ARC)和2个亮氨酸重复(LRR)保守结构域。
进一步对C2做序列延伸，结果向5'端成功延伸852bp，与C2拼接后为1954bp。对拼接序列进行Blast序列分析发现，该片段相对于C2，与相应序列相比DNA类似性升高。氨基酸序列保守区域分析结果表明，拼接序列的NB-ARC区域比C2延伸23个氨基酸，类似性由原来的27.9％提高到35.4％。并且与登录号分别为AAW78913.1、AAK84082.1、AAQ01786.1、XP_483560.1、BAA76281.2和ABA98920.1已知的抗病蛋白序列有较高的类似性。
利用软件GENSCAN 1.0进行基因预测分析，结果表明13-C2含有2个外显子，编码396个氨基酸，cDNA长1188bp。利用软件Tmpred对13-C2编码序列分析发现该蛋白含有2或3个跨膜螺旋。
在7对RGAP引物中只有R11R／R11F一对引物，能够在携带Pm21基因的扬麦5／sub6v中扩增出1317bp的多态性片段，在感病品种Chancellor和其它抗白粉病基因载体品系中则扩增不出该片段，对该多态性片段回收、克隆、测序结果进行Blast分析，发现C3第1-199核苷酸、第520-792核苷酸两个区域，与Genbank中的已知序列有较高的类似性，这些序列中均含有抗性蛋白的保守区域。例如：与小麦抗叶锈病基因Lr10的序列(AY270159.1)比较结果显示，有两区域的类似性达89％和86％。发现C3的氨基酸序列包含抗病基因的类似序列，有1个不完整的核苷酸结合位点(NB-ARC)。并与已知的抗性蛋白序列有一定的类似性。
C3列向3'延伸长度为387bp，向5'延伸长度为1028bp，与C3拼接后长达2562bp。对拼接序列13-C3进行Blast分析，发现其第20-699、824-1133、1451-1723、核苷酸三个区域，与Genbank中的已知序列Y14573.1、AF326781.1、AY270159.1、AY084050.1、AY663391.1、AY485643.1有较高的同源性。其中与小麦抗叶锈病基因Lr10序列(AY270159.1)，有两个区域类似性分别达87％和89％。拼接序列13-C3的氨基酸保守区域分析结果表明，NB-ARC区域的5'端比C3延伸23个氨基酸，类似性由原来的21.3％提高到29.2％。并且与Genbank中含已知的抗病蛋白序列AAW78913.1、AAK84082.1和AAQ01786.1有较高的类似性
进一步挑选26个纯合的F_2感抗单株初步验证其连锁性，发现有较高的连锁性。要想实现
Abstract:In this study, we selected thirteen wheat lines carring different powdery mildew resistance genes, which had good resistance in wheat planting zone of henna province, and took the susceptible cultivar chancellor as the control, considering the results of resistance identification. According to the conserved sequence of resistance gene, seven pairs of RGA primers were designed for RGAP analysis.
a special fragment C2 was amplified by the primer pair R11R/R11F in the Timgulan which carring Pm6 gene through screening the RGAP primers.It didn't appear in other lines tested. This fragment was cloned and sequenced,it was 1102bp. The homology analysis of DNA sequence showed, C2 had higher homology with known sequences in the genebank, such as the sequence of wheat leaf rust resistance gene Lrl0(AY270159.1), Triticum monococcum actin (ACT-1) gene RGA2 (A F326781.1) and Triticum aestivum NBS-LRR-like protein mRNA (AY084050.1). The analysis of inferred protein showed that its amino acid sequence include the analog sequence of resistance gene,an incomplete NB-ARC locus and two LRR sequences.The homology of conserved domain had 27.9% and 45.2% with known resistance protein sequences respectively.
The 5' end of C2 were successfully extended to 852bp by Tail-PCR technique. The extended sequence became 1954bp.after analying the homology of the fragment, we found its nucleotide domains had higher homology than C2's which compared with known sequences in genebank ;The homology the conserved domains of amino acid arrived at 35.4% from the former 27.9%., The NB-ARC domain of extended sequence had 23 amino acid more than C2, and The extended amino acid sequence have higher homology with known resistance protein sequences. Such as AAW78913.1、 AAK84082.1、 AAQ01786.1、 XP-4835601.1、 BAA76281.2 and ABA 98920.1.
Forecasting and analysis the jointed sequence 13-C2 by GENSCAN 1.0 TMpred, the results showed that 13-C2 had two exon one is initiation type, and the other is midst type, the forecasting peptide chain composed of 396 amino acid, and the cDNA length is 1188bp which have two or three helixes spanning velum.
The primer pair R11R/R11F can amplify a special fragment C_3 in the Yangmai
通过RGAP引物的筛选，发现引物R11R／R11F在含有Pm6基因的Timgalen中扩增出1102bp的多态性片段C2，在感病品种Chancellor和含其它抗白粉病基因品系中则扩增不出该片段。对该片段进行克隆测序，DNA序列分析发现，C2与Genbank中的小麦抗叶锈病基因Lr10(序列登录号为AY270159.1)、小麦中的抗病基因类似序列RGA2(序列登录号为AF326781.1)、小麦NBS-LRR类蛋白mRNA序列(序列登录号为AY084050.1)，有较高的相似性。C2推定的蛋白质保守区域分析，结果表明，C2的氨基酸序列包含抗病基因的类似序列，有1个不完整的核苷酸结合位点(NB-ARC)和2个亮氨酸重复(LRR)保守结构域。
进一步对C2做序列延伸，结果向5'端成功延伸852bp，与C2拼接后为1954bp。对拼接序列进行Blast序列分析发现，该片段相对于C2，与相应序列相比DNA类似性升高。氨基酸序列保守区域分析结果表明，拼接序列的NB-ARC区域比C2延伸23个氨基酸，类似性由原来的27.9％提高到35.4％。并且与登录号分别为AAW78913.1、AAK84082.1、AAQ01786.1、XP_483560.1、BAA76281.2和ABA98920.1已知的抗病蛋白序列有较高的类似性。
利用软件GENSCAN 1.0进行基因预测分析，结果表明13-C2含有2个外显子，编码396个氨基酸，cDNA长1188bp。利用软件Tmpred对13-C2编码序列分析发现该蛋白含有2或3个跨膜螺旋。
在7对RGAP引物中只有R11R／R11F一对引物，能够在携带Pm21基因的扬麦5／sub6v中扩增出1317bp的多态性片段，在感病品种Chancellor和其它抗白粉病基因载体品系中则扩增不出该片段，对该多态性片段回收、克隆、测序结果进行Blast分析，发现C3第1-199核苷酸、第520-792核苷酸两个区域，与Genbank中的已知序列有较高的类似性，这些序列中均含有抗性蛋白的保守区域。例如：与小麦抗叶锈病基因Lr10的序列(AY270159.1)比较结果显示，有两区域的类似性达89％和86％。发现C3的氨基酸序列包含抗病基因的类似序列，有1个不完整的核苷酸结合位点(NB-ARC)。并与已知的抗性蛋白序列有一定的类似性。
C3列向3'延伸长度为387bp，向5'延伸长度为1028bp，与C3拼接后长达2562bp。对拼接序列13-C3进行Blast分析，发现其第20-699、824-1133、1451-1723、核苷酸三个区域，与Genbank中的已知序列Y14573.1、AF326781.1、AY270159.1、AY084050.1、AY663391.1、AY485643.1有较高的同源性。其中与小麦抗叶锈病基因Lr10序列(AY270159.1)，有两个区域类似性分别达87％和89％。拼接序列13-C3的氨基酸保守区域分析结果表明，NB-ARC区域的5'端比C3延伸23个氨基酸，类似性由原来的21.3％提高到29.2％。并且与Genbank中含已知的抗病蛋白序列AAW78913.1、AAK84082.1和AAQ01786.1有较高的类似性
进一步挑选26个纯合的F_2感抗单株初步验证其连锁性，发现有较高的连锁性。要想实现
Abstract:In this study, we selected thirteen wheat lines carring different powdery mildew resistance genes, which had good resistance in wheat planting zone of henna province, and took the susceptible cultivar chancellor as the control, considering the results of resistance identification. According to the conserved sequence of resistance gene, seven pairs of RGA primers were designed for RGAP analysis.
a special fragment C2 was amplified by the primer pair R11R/R11F in the Timgulan which carring Pm6 gene through screening the RGAP primers.It didn't appear in other lines tested. This fragment was cloned and sequenced,it was 1102bp. The homology analysis of DNA sequence showed, C2 had higher homology with known sequences in the genebank, such as the sequence of wheat leaf rust resistance gene Lrl0(AY270159.1), Triticum monococcum actin (ACT-1) gene RGA2 (A F326781.1) and Triticum aestivum NBS-LRR-like protein mRNA (AY084050.1). The analysis of inferred protein showed that its amino acid sequence include the analog sequence of resistance gene,an incomplete NB-ARC locus and two LRR sequences.The homology of conserved domain had 27.9% and 45.2% with known resistance protein sequences respectively.
The 5' end of C2 were successfully extended to 852bp by Tail-PCR technique. The extended sequence became 1954bp.after analying the homology of the fragment, we found its nucleotide domains had higher homology than C2's which compared with known sequences in genebank ;The homology the conserved domains of amino acid arrived at 35.4% from the former 27.9%., The NB-ARC domain of extended sequence had 23 amino acid more than C2, and The extended amino acid sequence have higher homology with known resistance protein sequences. Such as AAW78913.1、 AAK84082.1、 AAQ01786.1、 XP-4835601.1、 BAA76281.2 and ABA 98920.1.
Forecasting and analysis the jointed sequence 13-C2 by GENSCAN 1.0 TMpred, the results showed that 13-C2 had two exon one is initiation type, and the other is midst type, the forecasting peptide chain composed of 396 amino acid, and the cDNA length is 1188bp which have two or three helixes spanning velum.
The primer pair R11R/R11F can amplify a special fragment C_3 in the Yangmai
引文
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