‘鸭梨’及其自交亲和性突变体‘金坠梨’花粉蛋白质组比较分析
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摘要
【目的】分析‘鸭梨’和‘金坠梨’花粉蛋白差异,探讨‘金坠梨’花粉侧自交亲和突变的机制。【方法】以‘鸭梨’及其自交亲和性芽变‘金坠梨’花粉为材料,利用双向电泳技术(two-dimensional electrophoresis,2-DE)对二者花粉总蛋白进行分离,并对差异蛋白进行功能和质谱分析,通过qRT-PCR进行相关差异基因的表达验证。【结果】分离得到23个差异蛋白,其中14个蛋白在‘金坠梨’中上调表达,9个蛋白在‘金坠梨’下调表达;对其中差异显著的10个蛋白点质谱检测结果表明,1个为半胱氨酸甲基转移酶(cysteine S-methyltransferase,4 803),1个为存储蛋白(Storage protein,5413),4个为蛋白酶类:果糖激酶(fructokinase,5 316)、顺乌头酸水合酶(aconitate hydratase,5 811)、烯酰-ACP-还原酶(Enoyl-ACP reductase,7 303)和JPR ORF1蛋白(JPR ORF1 protein,7 506),4个为HSP家族蛋白:HSP70伴侣26(HSP70-type chaperone 26,8 633)、HSP 90.5(8 822)和2个ER结合蛋白(ER-binding protein,4 209和8 805)。这些蛋白涉及到遗传信息处理、碳水化合物代谢、能量代谢、酶和氨基酸代谢等生理过程;qRT-PCR对8个基因进行验证,发现部分基因在蛋白水平与基因表达水平有明显差异。这些差异的蛋白在不同的信号途径中参与了‘金坠梨’花粉侧自交亲和突变。【结论】明确了‘鸭梨’和‘金坠梨’花粉侧自交亲和性突变中差异表达蛋白,为进一步研究梨亚科自交不亲和性的调控机理提供理论依据。
【Objective】The differentially expressed proteins of pollens from'Yali'pear(self-incompatibility) and'Jinzhui'pear(self-compatible mutant of'Yali') were analyzed, and the mechanism of selfcompatible mutation in'Jinzhui'pear was discussed.【Methods】The pollens of'Yali'pear and selfcompatible pollen-part mutant'Jinzhui'pear were used as the materials, and the pollen proteins were separated. Thereafter, the proteins were tested by dyeing technology and BIO-RAD GS-900 Calibrated Densitometer scanning. PDQuest 8.0.1 software was used to analyze the gel images, and the differential protein spots were obtained. The differentially expressed proteins were finally identified through enzymatic hydrolysis of differentially expressed proteins and LC-MS/MS analysis in the UniProt database.The raw file of mass spectrometry data was processed by the Mascot software, and meanwhile the quality of data was controlled. The results of identified proteins were obtained. Using the online website NCBI and Gene Ontology Database(GO), the identified proteins were compared and analyzed according to cell components, biological processes and molecular functions. Protein sequences were submitted to Kyoto Encyclopedia of Gene and Genomes(KEGG) and bi-directional best hit(BBH) method was used to compare similarities to find the most similar proteins and determine the KO(KEGGORTHOLOG)classification of retrieval proteins. According to the gene sequences of differential proteins, gene-specific primers were designed by NCBI on-line primer design tool Primer-BLAST, and the transcription level of related genes was verified by qRT-PCR.【Results】The results of two-dimensional electrophoresis(2-DE) of total pollen proteins showed that the protein spots were clearly visible and the separation quality was high. It was found that the protein spots were mainly distributed in the region of pH 3-10, the relative molecular weights were about 10-200 kDa, and about 550 protein spots could be detected. Using PDQuest 8.0.1 software, 23 differential proteins were isolated. All the differential proteins were mainly concentrated in isoelectric points(pH): 5-8, relative molecular weights(Mr): 40-200 kDa. Fourteen of the proteins were up-regulated in'Jinzhui'pear, and their numbers were 3 504, 4 209, 4 307, 4616, 5 316, 5 411, 5 413, 5 704, 5 717, 6 808, 7 103, 7 303, 7 419 and 8 805, respectively. Nine proteins were down-regulated in'Jinzhui pear, and their numbers were 4 401, 4 803, 5 802, 5 811, 6 611, 7 506,8 633, 8 822 and 9 301, respectively. According to the expression levels of different proteins, the differences in 14 up-regulated proteins and 9 down-regulated proteins in'Jinzhui'pear were analyzed. It was found that the expression levels of six proteins, 4 209, 4 616, 5 316, 5 413, 7 303 and 8 805, were extremely significantly higher(p < 0.01) than those of'Yali'pear, while the expression levels of eight other differential proteins, 3 504, 4 307, 5 411, 5 704, 5 717, 6 808, 7 103 and 7 419, were significantly higher(p < 0.05) than those of'Yali'pear. Among the 9 down-regulated differentially expressed proteins in'Jinzhui'pear pollen, 4 differentially expressed proteins(5 802, 5 811, 8 633 and 8 822) were extremely significantly lower(p < 0.01) than those in'Yali'pear, and 5(4 401, 4 803, 6 611, 7 506 and9 301) were significantly lower(p < 0.05) than those in'Yali'pear. Twenty-three differentially expressed proteins were isolated by PDQuest 8.0.1 software, and fourteen of which were up-regulated and nine were down-regulated. Mass spectrometry was used to detect 10 protein spots of 23 differentially expressed proteins, including 1 amino acid: DL-methionine(4 803), 1 hexamerin(5 413), 4 enzymes:fructokinase,(5 316), aconitate hydratase,(5 811), Enoyl-ACP reductase(7 303) and JPR ORF1 protein(7 506), and four HSP family proteins: HSP70-type chaperone 26(8 633), HSP90.5(8 822) and two ERbinding proteins(4 209, 8 805). Through GO annotation, 10 differential proteins were successfully annotated. According to molecular functional MF classification, the five proteins numbered 4 803, 5 316,5 811, 8 633 and 8 822 belonged to the specific biological process. The 10 differential proteins could be divided into five functional groups by KEGG annotation, including genetic information processing(4 209,8 633, 8 805 and 8 822), carbohydrate metabolism(5 316 and 5 811), energy metabolism(5 413), enzyme(7 303 and 7 506) and amino acid metabolism(4 803). The mRNA expression of fructokinase(5 316), unidentified protein(4 803) and HSP90.5(8 822) in'Jinzhui'pear were higher than those in'Yali'pear by qRT-PCR. The expression levels of ER binding protein(4 209), enoyl-ACP-reductase(7 303), ER binding protein(8 805), HSP70 chaperone 26 and JPR ORF1 protein in'Jinzhui'pear were lower than those in'Yali'pear. Only HSP70 chaperon 26(8 633), JPR ORF1 protein(7 506) and fructokinase(5 316) expressed consistently at protein level and transcriptional level. However, there were some differences in the expression of some proteins at both transcriptional level and protein level such as enoyl-ACP-reductase(7 303) and two ER-binding proteins(4 209 and 8 805) were up-regulated at the protein level, while down-regulated at the transcriptional level. Unidentified proteins(4 803) and HSP 90.5(8 822) were down-regulated at the protein level, but up-regulated at the transcriptional level.These results indicated that the transcription and translation of these protein genes were different.【Conclusion】The possible functions of differentially expressed proteins in'Yali'pear(self-incompatibility)and'Jinzhui'pear(self-compatible mutant of'Yali'pear), identified by two-dimensional electrophoresis(2-DE), were analyzed, and the correlation with the change of self-compatible mutation was also discussed. These results would provide important reference for further research on the mechanism of selfincompatibility in Pomnideae.
【Objective】The differentially expressed proteins of pollens from'Yali'pear(self-incompatibility) and'Jinzhui'pear(self-compatible mutant of'Yali') were analyzed, and the mechanism of selfcompatible mutation in'Jinzhui'pear was discussed.【Methods】The pollens of'Yali'pear and selfcompatible pollen-part mutant'Jinzhui'pear were used as the materials, and the pollen proteins were separated. Thereafter, the proteins were tested by dyeing technology and BIO-RAD GS-900 Calibrated Densitometer scanning. PDQuest 8.0.1 software was used to analyze the gel images, and the differential protein spots were obtained. The differentially expressed proteins were finally identified through enzymatic hydrolysis of differentially expressed proteins and LC-MS/MS analysis in the UniProt database.The raw file of mass spectrometry data was processed by the Mascot software, and meanwhile the quality of data was controlled. The results of identified proteins were obtained. Using the online website NCBI and Gene Ontology Database(GO), the identified proteins were compared and analyzed according to cell components, biological processes and molecular functions. Protein sequences were submitted to Kyoto Encyclopedia of Gene and Genomes(KEGG) and bi-directional best hit(BBH) method was used to compare similarities to find the most similar proteins and determine the KO(KEGGORTHOLOG)classification of retrieval proteins. According to the gene sequences of differential proteins, gene-specific primers were designed by NCBI on-line primer design tool Primer-BLAST, and the transcription level of related genes was verified by qRT-PCR.【Results】The results of two-dimensional electrophoresis(2-DE) of total pollen proteins showed that the protein spots were clearly visible and the separation quality was high. It was found that the protein spots were mainly distributed in the region of pH 3-10, the relative molecular weights were about 10-200 kDa, and about 550 protein spots could be detected. Using PDQuest 8.0.1 software, 23 differential proteins were isolated. All the differential proteins were mainly concentrated in isoelectric points(pH): 5-8, relative molecular weights(Mr): 40-200 kDa. Fourteen of the proteins were up-regulated in'Jinzhui'pear, and their numbers were 3 504, 4 209, 4 307, 4616, 5 316, 5 411, 5 413, 5 704, 5 717, 6 808, 7 103, 7 303, 7 419 and 8 805, respectively. Nine proteins were down-regulated in'Jinzhui pear, and their numbers were 4 401, 4 803, 5 802, 5 811, 6 611, 7 506,8 633, 8 822 and 9 301, respectively. According to the expression levels of different proteins, the differences in 14 up-regulated proteins and 9 down-regulated proteins in'Jinzhui'pear were analyzed. It was found that the expression levels of six proteins, 4 209, 4 616, 5 316, 5 413, 7 303 and 8 805, were extremely significantly higher(p < 0.01) than those of'Yali'pear, while the expression levels of eight other differential proteins, 3 504, 4 307, 5 411, 5 704, 5 717, 6 808, 7 103 and 7 419, were significantly higher(p < 0.05) than those of'Yali'pear. Among the 9 down-regulated differentially expressed proteins in'Jinzhui'pear pollen, 4 differentially expressed proteins(5 802, 5 811, 8 633 and 8 822) were extremely significantly lower(p < 0.01) than those in'Yali'pear, and 5(4 401, 4 803, 6 611, 7 506 and9 301) were significantly lower(p < 0.05) than those in'Yali'pear. Twenty-three differentially expressed proteins were isolated by PDQuest 8.0.1 software, and fourteen of which were up-regulated and nine were down-regulated. Mass spectrometry was used to detect 10 protein spots of 23 differentially expressed proteins, including 1 amino acid: DL-methionine(4 803), 1 hexamerin(5 413), 4 enzymes:fructokinase,(5 316), aconitate hydratase,(5 811), Enoyl-ACP reductase(7 303) and JPR ORF1 protein(7 506), and four HSP family proteins: HSP70-type chaperone 26(8 633), HSP90.5(8 822) and two ERbinding proteins(4 209, 8 805). Through GO annotation, 10 differential proteins were successfully annotated. According to molecular functional MF classification, the five proteins numbered 4 803, 5 316,5 811, 8 633 and 8 822 belonged to the specific biological process. The 10 differential proteins could be divided into five functional groups by KEGG annotation, including genetic information processing(4 209,8 633, 8 805 and 8 822), carbohydrate metabolism(5 316 and 5 811), energy metabolism(5 413), enzyme(7 303 and 7 506) and amino acid metabolism(4 803). The mRNA expression of fructokinase(5 316), unidentified protein(4 803) and HSP90.5(8 822) in'Jinzhui'pear were higher than those in'Yali'pear by qRT-PCR. The expression levels of ER binding protein(4 209), enoyl-ACP-reductase(7 303), ER binding protein(8 805), HSP70 chaperone 26 and JPR ORF1 protein in'Jinzhui'pear were lower than those in'Yali'pear. Only HSP70 chaperon 26(8 633), JPR ORF1 protein(7 506) and fructokinase(5 316) expressed consistently at protein level and transcriptional level. However, there were some differences in the expression of some proteins at both transcriptional level and protein level such as enoyl-ACP-reductase(7 303) and two ER-binding proteins(4 209 and 8 805) were up-regulated at the protein level, while down-regulated at the transcriptional level. Unidentified proteins(4 803) and HSP 90.5(8 822) were down-regulated at the protein level, but up-regulated at the transcriptional level.These results indicated that the transcription and translation of these protein genes were different.【Conclusion】The possible functions of differentially expressed proteins in'Yali'pear(self-incompatibility)and'Jinzhui'pear(self-compatible mutant of'Yali'pear), identified by two-dimensional electrophoresis(2-DE), were analyzed, and the correlation with the change of self-compatible mutation was also discussed. These results would provide important reference for further research on the mechanism of selfincompatibility in Pomnideae.
引文
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[2]滕元文.梨属植物系统发育及东方梨品种起源研究进展[J].果树学报,2017,34(3):370-378.TENG Yuanwen. Advances in the research on phylogeny of the genus Pyrus and the origin of pear cultivars native to East Asia[J]. Journal of Fruit Science,2017,34(3):370-378.
[3] DE NETTANCOURT D. Incompatibility in angiosperms[J]. Sex Plant Reproduction,1997,10:185-199.
[4]胡彬,蒋建雄,易自力.植物配子体自交不亲和机制研究进展[J].中国农学通报,2012,28(18):168-173.HU Bin,JIANG Jianxiong,YI Zili. Advances on mechanisms of gametophytic self-incompatibility in plant[J]. Chinese Agricultural Science Bulletin,2012,28(18):168-173.
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