谷子抗锈基因RUS分子标记和抗病相关基因的克隆与分析
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摘要
由单胞锈菌(Uromyces setariae-italicae)引起的谷子锈病是威胁谷子生产安全的重要病害之一,常造成严重经济损失。选育并合理利用抗病品种是减轻锈病最经济、有效、安全的方法。同时,抗病基因的研究也是抗病育种的基础,克隆谷子抗锈或抗锈相关基因不仅可以加快抗病育种工作,而且有助于揭示谷子抗锈机理,为开发快速、高效的防治技术提供理论依据。本研究以谷子抗锈/感锈病材料及其杂交的F2代群体为材料,对谷子抗锈基因连锁的分子标记及抗病相关基因的克隆等方面进行了研究,为谷子抗锈基因的分子鉴定、辅助选择及抗病机制相关研究奠定基础。本论文内容包括以下几方面:
1.应用AFLP标记技术筛选获得了10对能够揭示抗感材料之间多态性的AFLP引物组合;应用所获得的AFLP引物组合对谷子抗锈/感锈病材料及其F2代群体中的抗感基因池进行扩增,获得了18个抗锈材料中特有的差异条带;生物信息学分析结果表明,2个差异条带与玉米的W22 pol基因和水稻的丝氨酸-苏氨酸蛋白激酶基因的同源性分别为86%和92%,其他差异条带未发现同源的基因,推测可能为新的基因。
2.根据差异条带的测序结果设计PCR特异引物,成功地将AFLP标记“E+GC/M+TC”转化为结果稳定、操作简单的SCAR标记“SCEM-187”。以转化成功的SCAR标记对抗锈基因进行定位,结果表明标记“SCEM-187”与谷子抗锈基因间的遗传距离为27.4 cM。
3.应用RGA技术对谷子基因组DNA和cDNA进行扩增,获得了7个抗病材料十里香中特异的NBS类型抗病基因同源序列。NBS类型抗病基因同源序列均含有P-loop和Kinase 2α结构域,与水稻含有的NB-ARC保守结构域的蛋白、水稻和玉米的NBS-LRR类型抗病蛋白、小麦的抗病蛋白等的同源性分别在47%～98%之间。聚类分析结果表明,7个RGA片段分为三类:第一类包括RUS1-1、RUS1-2、RUS1-3和RUS1-6,与水稻Pib基因聚在一起,属于NBS-LRR类型抗病基因;第二类包括RUS1-5和RUS1,与LZ-NBS-LRR类型的拟南芥RPP8和HRT基因聚在一起;第三类包括RUS1-4,与番茄NBS-LRR类型I2基因聚在一起。
4.根据番茄Fen和水稻Xa21等蛋白激酶基因保守结构域设计引物,从抗病材料十里香中获得了1个STK类型抗病基因同源序列。BLASTX分析结果表明,STK类型抗病基因同源序列与水稻中推测的丝氨酸/苏氨酸受体激酶PR5K、小麦LRK33和抗锈激酶Lr10同源性在77%～78%之间。
5.根据已知的RUS1序列,利用Genome Walking和Reverse PCR技术获得了RUS1的全长DNA(GenBank登录号:FJ467296)和cDNA序列。DNA全长2673 bp,cDNA为2193 bp,编码731个氨基酸,含有3个外显子和2个内含子。RUS1基因编码产物分子量为82.588 kD,等电点(pI)为8.20。半定量RT-PCR分析结果表明,谷子NBS类型抗病基因同源序列RUS1表达方式为组成型表达。随着接菌时间的延长,RUS1表达量增加,参与谷子抗锈病反应。
6.生物信息学分析确定了RUS1基因具有NBS、LRR、HD等一系列植物抗病基因的保守结构域,且与NBS-LRR类抗病Pib、HRT和RPP8的亲缘关系较近。确定了RUS1基因属于NBS-LRR类型的抗病基因。RUS1蛋白的二级结构以α-螺旋为主,其次为无规线团;RUS1基因与大麦NBS-LRR类型抗病蛋白同源性为58%,与水稻含有NB-ARC结构域的蛋白同源性为63%,与小麦的抗病蛋白同源性为59%。
7. Southern杂交结果表明,RUS1以多拷贝形式存在于谷子十里香基因组DNA中。
8.根据已知的RUS1基因序列,利用Genome Walking技术获得了RUS1基因的启动子序列,长度为675 bp。成功构建了RUS1∷GUS双元载体。GUS染色结果表明,含有双元载体RUS1∷GUS的农杆菌经染色后溶液变蓝,表明该启动子具有活性。
9.通过PCR扩增获得含有启动子序列的RUS1基因。成功构建了植物表达载体p1300∶RUS1。
10.利用SSH技术对抗锈病材料十里香接种锈菌后诱导基因的表达情况进行了研究,共获得了11个诱导表达的序列。BLASTX分析结果表明,1个序列与小麦、大麦和水稻的SGT1和水稻、玉米的skp1等位基因g2抑制因子的同源性在91%～97%之间;4个序列未发现同源的序列,可能为新的基因;其他序列与水稻保守的假定蛋白、葡萄未知蛋白、水稻和玉米光合系统I亚基IX、玉米未命名蛋白和核糖体蛋白S14的同源性在58%～96%之间。
Uromyces setariae-italicae is a destructive pathogen of Setariae-italicae Beauv. all over the world and causes significant yield losses. Resistance cultivars are the most economical and environmentally-friendly way to reduce the damage caused by millet leaf rust. Researches on resistant genes are important and provide the base for the resistance breeding. Not only can our results accelerate the process of plant breeding, but also lay a foundation for the elucidate resistance mechanism and development of rapid and efficient control strategy. Taking Setaria italica Beauv. cultivar Shilixiang (resistant to leaf rust) and Yugu No.1(susceptible to leaf rust) and their F2 progenies as material, research on screening of AFLP marker which closely linked with Setaria italica Beauv. leaf rust resistant gene and cloning of resistant gene was done. Result will contribute to the marker assisted selection (MAS) and identification of resistance mechanism of Setaria italica Beauv. against leaf rust infection. The main results as follows:
1. AFLP technology was employed to isolate Setaria italica Beauv. leaf rust resistant gene. 10 primer pairs could reveal the polymorphism of resistance and susceptible parents. Bioinformatics analysis result indicated that two bands had 86% and 92% homology with pol gene of Zea mays cultivar W22 and serine/threonine protein kinase of Oryza sativa respectively. Other bands had no significant similarity with sequence in GenBank database and may be new genes.
2. Primers were designed according to known sequences. SCAR marker“SCEM-187”converted from AFLP marker“E+GC/M+TC”which closely linked with leaf rust resistant gene of Setaria italica Beauv. was obtained through the optimized PCR condition. Using SCAR marker“SCEM-187”, 118 F2 progenies were employed to analysis separate ratio of resistance and susceptible plants. Results showed that“SCEM-187”linked to resistant gene of Setaria italica Beauv. against leaf rust infection with a distance of 27.4 cM.
3. RGA(resistance gene analogs) analysis was employed on genomic DNA and cDNA of Setaria italica Beauv.. Seven NBS-like sequences contained conserved motifs P-loop and kinase 2αwhich were the characteristics of NBS type resistant gene of plant was obtained. These sequences had 47% to 98% homology with protein contained NB-ARC domain of Oryza sativa, NBS-LRR resistance protein of Oryza sativa and so on. Phylogenetic analysis indicated that there were three categories of RGAs. One group included RUS1-1, RUS1-2, RUS1-3 and RUS1-6 which were similar to NBS-LRR type disease resistant gene Pib of Oryza sativa. The second group included RUS1-5 and RUS1 which were similar to LZ-NBS-LRR type disease resistance gene RPP8 and HRT of Arabidopsis. The third group included RUS1-4 which was similar to NBS-LRR type disease resistant gene I2 of tomato.
4. Using the primers which designed according to the conserved domain of Fen and Xa21 protein kinase, one STK-like sequence was obtained. BLASTX result indicated that it had 77% to 78% homology with putative receptor serine/threonine kinase PR5K of Oryza sativa, LRK33 and resistance-related receptor-like kinase Lr10 of Triticum aestivum.
5. Using the method of Genome Walking and reverse PCR, DNA and cDNA sequence of RUS1 was obtianed. RUS1 included a 2673 bp DNA sequence with a 2193 bp coding region, 3 exons and 2 introns and it encode 731 aa. Molecular mass of RUS1 protein was 82.588 kDa and the pI of it was 8.20. The RUS1 gene could be induced by Uromyces setariae-italicae and was a constitutive gene with low abundance in the genome by semi-quantitative RT-PCR. The expression was increasing with the time of inoculation.
6. Bioinformatic result showed that RUS1 contained conserved NBS, LRR and HD domains which were the characteristics of NBS type resistant gene of plant. RUS1 belonged to NBS-LRR class resistant gene and had the most similarity to Pib, HRT and RPP8. Swissplot software analysis result showed that alpha helix was the main type in the predicted secondary structure of RUS1 protein. RUS1 had 58% to 63% homology with NB-ARC domain containing protein of Oryza sativa, NBS-LRR disease resistant protein of Hordeum vulgare and disease resistance protein of Triticum aestivum.
7. Southern blotting result displayed that there were multi-copies of RUS1 in the Setaria italica Beauv. genome DNA.
8. Promoter sequence of RUS1 gene was obtained. The length of it was 675 bp. RUS1 gene promoter and pCAMBIA1300 vector were fused to construct RUS1∷GUS vector. GUS histochemistry staining result showed that promoter could activate gene expression.
9. RUS1 gene (include promoter sequence) was obtained by the method of PCR.RUS1 gene and pCAMBIA1300 vector were fused to construct p1300∶RUS1 vector.
10. Technology of SSH was employed to analyze gene express of Setaria italica Beauv. after inoculated with Uromyces setariae-italicae. Eleven sequences related to resistant gene of Setaria italica Beauv. against leaf rust infection were obtained. One sequence had 91% to 97% homology with SGT1 of Oryza sativa, Triticum aestivum, Hordeum vulgare and suppressor of g2 allele of skp1 of Oryza sativa and Zea may. Four sequences had no significant similarity with sequence in GenBank database and may be new genes. Others sequences had 58% to 96% homology with hypothetical protein of Oryza sativa, unnamed protein of Vitis vinifera, photosystem I subunit IX of Oryza sativa and Zea mays, unnamed protein and ribosomal protein S14 of Zea mays.
1.应用AFLP标记技术筛选获得了10对能够揭示抗感材料之间多态性的AFLP引物组合;应用所获得的AFLP引物组合对谷子抗锈/感锈病材料及其F2代群体中的抗感基因池进行扩增,获得了18个抗锈材料中特有的差异条带;生物信息学分析结果表明,2个差异条带与玉米的W22 pol基因和水稻的丝氨酸-苏氨酸蛋白激酶基因的同源性分别为86%和92%,其他差异条带未发现同源的基因,推测可能为新的基因。
2.根据差异条带的测序结果设计PCR特异引物,成功地将AFLP标记“E+GC/M+TC”转化为结果稳定、操作简单的SCAR标记“SCEM-187”。以转化成功的SCAR标记对抗锈基因进行定位,结果表明标记“SCEM-187”与谷子抗锈基因间的遗传距离为27.4 cM。
3.应用RGA技术对谷子基因组DNA和cDNA进行扩增,获得了7个抗病材料十里香中特异的NBS类型抗病基因同源序列。NBS类型抗病基因同源序列均含有P-loop和Kinase 2α结构域,与水稻含有的NB-ARC保守结构域的蛋白、水稻和玉米的NBS-LRR类型抗病蛋白、小麦的抗病蛋白等的同源性分别在47%～98%之间。聚类分析结果表明,7个RGA片段分为三类:第一类包括RUS1-1、RUS1-2、RUS1-3和RUS1-6,与水稻Pib基因聚在一起,属于NBS-LRR类型抗病基因;第二类包括RUS1-5和RUS1,与LZ-NBS-LRR类型的拟南芥RPP8和HRT基因聚在一起;第三类包括RUS1-4,与番茄NBS-LRR类型I2基因聚在一起。
4.根据番茄Fen和水稻Xa21等蛋白激酶基因保守结构域设计引物,从抗病材料十里香中获得了1个STK类型抗病基因同源序列。BLASTX分析结果表明,STK类型抗病基因同源序列与水稻中推测的丝氨酸/苏氨酸受体激酶PR5K、小麦LRK33和抗锈激酶Lr10同源性在77%～78%之间。
5.根据已知的RUS1序列,利用Genome Walking和Reverse PCR技术获得了RUS1的全长DNA(GenBank登录号:FJ467296)和cDNA序列。DNA全长2673 bp,cDNA为2193 bp,编码731个氨基酸,含有3个外显子和2个内含子。RUS1基因编码产物分子量为82.588 kD,等电点(pI)为8.20。半定量RT-PCR分析结果表明,谷子NBS类型抗病基因同源序列RUS1表达方式为组成型表达。随着接菌时间的延长,RUS1表达量增加,参与谷子抗锈病反应。
6.生物信息学分析确定了RUS1基因具有NBS、LRR、HD等一系列植物抗病基因的保守结构域,且与NBS-LRR类抗病Pib、HRT和RPP8的亲缘关系较近。确定了RUS1基因属于NBS-LRR类型的抗病基因。RUS1蛋白的二级结构以α-螺旋为主,其次为无规线团;RUS1基因与大麦NBS-LRR类型抗病蛋白同源性为58%,与水稻含有NB-ARC结构域的蛋白同源性为63%,与小麦的抗病蛋白同源性为59%。
7. Southern杂交结果表明,RUS1以多拷贝形式存在于谷子十里香基因组DNA中。
8.根据已知的RUS1基因序列,利用Genome Walking技术获得了RUS1基因的启动子序列,长度为675 bp。成功构建了RUS1∷GUS双元载体。GUS染色结果表明,含有双元载体RUS1∷GUS的农杆菌经染色后溶液变蓝,表明该启动子具有活性。
9.通过PCR扩增获得含有启动子序列的RUS1基因。成功构建了植物表达载体p1300∶RUS1。
10.利用SSH技术对抗锈病材料十里香接种锈菌后诱导基因的表达情况进行了研究,共获得了11个诱导表达的序列。BLASTX分析结果表明,1个序列与小麦、大麦和水稻的SGT1和水稻、玉米的skp1等位基因g2抑制因子的同源性在91%～97%之间;4个序列未发现同源的序列,可能为新的基因;其他序列与水稻保守的假定蛋白、葡萄未知蛋白、水稻和玉米光合系统I亚基IX、玉米未命名蛋白和核糖体蛋白S14的同源性在58%～96%之间。
Uromyces setariae-italicae is a destructive pathogen of Setariae-italicae Beauv. all over the world and causes significant yield losses. Resistance cultivars are the most economical and environmentally-friendly way to reduce the damage caused by millet leaf rust. Researches on resistant genes are important and provide the base for the resistance breeding. Not only can our results accelerate the process of plant breeding, but also lay a foundation for the elucidate resistance mechanism and development of rapid and efficient control strategy. Taking Setaria italica Beauv. cultivar Shilixiang (resistant to leaf rust) and Yugu No.1(susceptible to leaf rust) and their F2 progenies as material, research on screening of AFLP marker which closely linked with Setaria italica Beauv. leaf rust resistant gene and cloning of resistant gene was done. Result will contribute to the marker assisted selection (MAS) and identification of resistance mechanism of Setaria italica Beauv. against leaf rust infection. The main results as follows:
1. AFLP technology was employed to isolate Setaria italica Beauv. leaf rust resistant gene. 10 primer pairs could reveal the polymorphism of resistance and susceptible parents. Bioinformatics analysis result indicated that two bands had 86% and 92% homology with pol gene of Zea mays cultivar W22 and serine/threonine protein kinase of Oryza sativa respectively. Other bands had no significant similarity with sequence in GenBank database and may be new genes.
2. Primers were designed according to known sequences. SCAR marker“SCEM-187”converted from AFLP marker“E+GC/M+TC”which closely linked with leaf rust resistant gene of Setaria italica Beauv. was obtained through the optimized PCR condition. Using SCAR marker“SCEM-187”, 118 F2 progenies were employed to analysis separate ratio of resistance and susceptible plants. Results showed that“SCEM-187”linked to resistant gene of Setaria italica Beauv. against leaf rust infection with a distance of 27.4 cM.
3. RGA(resistance gene analogs) analysis was employed on genomic DNA and cDNA of Setaria italica Beauv.. Seven NBS-like sequences contained conserved motifs P-loop and kinase 2αwhich were the characteristics of NBS type resistant gene of plant was obtained. These sequences had 47% to 98% homology with protein contained NB-ARC domain of Oryza sativa, NBS-LRR resistance protein of Oryza sativa and so on. Phylogenetic analysis indicated that there were three categories of RGAs. One group included RUS1-1, RUS1-2, RUS1-3 and RUS1-6 which were similar to NBS-LRR type disease resistant gene Pib of Oryza sativa. The second group included RUS1-5 and RUS1 which were similar to LZ-NBS-LRR type disease resistance gene RPP8 and HRT of Arabidopsis. The third group included RUS1-4 which was similar to NBS-LRR type disease resistant gene I2 of tomato.
4. Using the primers which designed according to the conserved domain of Fen and Xa21 protein kinase, one STK-like sequence was obtained. BLASTX result indicated that it had 77% to 78% homology with putative receptor serine/threonine kinase PR5K of Oryza sativa, LRK33 and resistance-related receptor-like kinase Lr10 of Triticum aestivum.
5. Using the method of Genome Walking and reverse PCR, DNA and cDNA sequence of RUS1 was obtianed. RUS1 included a 2673 bp DNA sequence with a 2193 bp coding region, 3 exons and 2 introns and it encode 731 aa. Molecular mass of RUS1 protein was 82.588 kDa and the pI of it was 8.20. The RUS1 gene could be induced by Uromyces setariae-italicae and was a constitutive gene with low abundance in the genome by semi-quantitative RT-PCR. The expression was increasing with the time of inoculation.
6. Bioinformatic result showed that RUS1 contained conserved NBS, LRR and HD domains which were the characteristics of NBS type resistant gene of plant. RUS1 belonged to NBS-LRR class resistant gene and had the most similarity to Pib, HRT and RPP8. Swissplot software analysis result showed that alpha helix was the main type in the predicted secondary structure of RUS1 protein. RUS1 had 58% to 63% homology with NB-ARC domain containing protein of Oryza sativa, NBS-LRR disease resistant protein of Hordeum vulgare and disease resistance protein of Triticum aestivum.
7. Southern blotting result displayed that there were multi-copies of RUS1 in the Setaria italica Beauv. genome DNA.
8. Promoter sequence of RUS1 gene was obtained. The length of it was 675 bp. RUS1 gene promoter and pCAMBIA1300 vector were fused to construct RUS1∷GUS vector. GUS histochemistry staining result showed that promoter could activate gene expression.
9. RUS1 gene (include promoter sequence) was obtained by the method of PCR.RUS1 gene and pCAMBIA1300 vector were fused to construct p1300∶RUS1 vector.
10. Technology of SSH was employed to analyze gene express of Setaria italica Beauv. after inoculated with Uromyces setariae-italicae. Eleven sequences related to resistant gene of Setaria italica Beauv. against leaf rust infection were obtained. One sequence had 91% to 97% homology with SGT1 of Oryza sativa, Triticum aestivum, Hordeum vulgare and suppressor of g2 allele of skp1 of Oryza sativa and Zea may. Four sequences had no significant similarity with sequence in GenBank database and may be new genes. Others sequences had 58% to 96% homology with hypothetical protein of Oryza sativa, unnamed protein of Vitis vinifera, photosystem I subunit IX of Oryza sativa and Zea mays, unnamed protein and ribosomal protein S14 of Zea mays.
引文
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