【目的】对辣椒差减文库中挑选出的4个育性相关候选基因进行克隆与表达分析,探讨其与辣椒细胞核雄性不育的关系。【方法】通过半定量RT-PCR技术分析候选EST在辣椒可育株和不育株中的表达情况。利用RACE技术获得4个育性相关基因的cDNA全...【目的】对辣椒差减文库中挑选出的4个育性相关候选基因进行克隆与表达分析,探讨其与辣椒细胞核雄性不育的关系。【方法】通过半定量RT-PCR技术分析候选EST在辣椒可育株和不育株中的表达情况。利用RACE技术获得4个育性相关基因的cDNA全长,结合生物信息学方法进行序列比对和蛋白理化性质分析。采用半定量RT-PCR检测基因在不同器官(花药、子房、花瓣、萼片和叶片)和花药小孢子不同发育时期(四分体时期、单核早中期、单核靠边期和双核期)的表达量。【结果】根据比对注释结果,将4个基因分别命名为CaSEP1、CaPROF、CaOle e 6和CaPCP。CaSEP1全长1 108 bp,编码221个氨基酸,含有一个MADS结构域和一个K结构域;CaPROF全长767 bp,编码131个氨基酸,含有一个PROF结构域;CaOle e 6全长523 bp,编码85个氨基酸,含有一个Ole e 6结构域;CaPCP全长563 bp,编码66个氨基酸。氨基酸序列比对及系统发育树分析表明,CaSEP1与番茄SEP1的氨基酸序列相似性最高,并且亲缘关系最近;CaPROF与茄科作物烟草和番茄的前纤维蛋白相似性较高,且与番茄的前纤维蛋白亲缘关系最近;CaOle e 6与番茄中的对应蛋白存在一定的相似性,亲缘关系也最近;CaPCP则与茶树中的对应蛋白相似性最高,亲缘关系最近。表达分析显示,CaSEP1在可育株花药、子房、花瓣等花器官中表达量一致,且高于萼片和叶片;CaPROF在可育株花药中的表达明显高于叶片,在其他器官中不表达;CaOle e6在可育株花药中的表达明显高于其他器官;CaPCP只在可育株的花药中表达。CaSEP1在可育株小孢子不同发育时期表现为先逐渐升高,至单核靠边期达到最高,而后在双核期表达量明显下降,在不育株中则表现为伴随着小孢子发育表达量逐渐升高;该基因在四分体时期两材料的表达量相当,在单核早中期和单核靠边期可育株中的表达量明显高于不育株,而双核期可�展开更多
: For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages,...: For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only full-length cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and large-sized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.展开更多
文摘【目的】对辣椒差减文库中挑选出的4个育性相关候选基因进行克隆与表达分析,探讨其与辣椒细胞核雄性不育的关系。【方法】通过半定量RT-PCR技术分析候选EST在辣椒可育株和不育株中的表达情况。利用RACE技术获得4个育性相关基因的cDNA全长,结合生物信息学方法进行序列比对和蛋白理化性质分析。采用半定量RT-PCR检测基因在不同器官(花药、子房、花瓣、萼片和叶片)和花药小孢子不同发育时期(四分体时期、单核早中期、单核靠边期和双核期)的表达量。【结果】根据比对注释结果,将4个基因分别命名为CaSEP1、CaPROF、CaOle e 6和CaPCP。CaSEP1全长1 108 bp,编码221个氨基酸,含有一个MADS结构域和一个K结构域;CaPROF全长767 bp,编码131个氨基酸,含有一个PROF结构域;CaOle e 6全长523 bp,编码85个氨基酸,含有一个Ole e 6结构域;CaPCP全长563 bp,编码66个氨基酸。氨基酸序列比对及系统发育树分析表明,CaSEP1与番茄SEP1的氨基酸序列相似性最高,并且亲缘关系最近;CaPROF与茄科作物烟草和番茄的前纤维蛋白相似性较高,且与番茄的前纤维蛋白亲缘关系最近;CaOle e 6与番茄中的对应蛋白存在一定的相似性,亲缘关系也最近;CaPCP则与茶树中的对应蛋白相似性最高,亲缘关系最近。表达分析显示,CaSEP1在可育株花药、子房、花瓣等花器官中表达量一致,且高于萼片和叶片;CaPROF在可育株花药中的表达明显高于叶片,在其他器官中不表达;CaOle e6在可育株花药中的表达明显高于其他器官;CaPCP只在可育株的花药中表达。CaSEP1在可育株小孢子不同发育时期表现为先逐渐升高,至单核靠边期达到最高,而后在双核期表达量明显下降,在不育株中则表现为伴随着小孢子发育表达量逐渐升高;该基因在四分体时期两材料的表达量相当,在单核早中期和单核靠边期可育株中的表达量明显高于不育株,而双核期可�
基金国家自然科学基金，the Key Sci-Technology Project of Zhejiang Province,China
文摘: For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only full-length cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and large-sized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.
