辣椒GMS育性相关候选基因的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2014.16.012

摘要/Abstract

摘要： 【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 e 6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，而双核期可育株中的表达量比不育株低；CaPROF、CaOle e 6和CaPCP均在可育株小孢子发育后期（主要为单核靠边期和双核期）表达，在不育株花药发育的各个时期均不表达。【结论】4个候选基因的序列比对和表达分析结果表明它们与辣椒雄蕊育性关系密切，为揭示雄性不育机理和调控辣椒育性提供了重要信息。

Abstract: 【Objective】To investigate the correlativity between the four screened ESTs and the genic male sterility, full length cDNA of the four genes were obtained, and the expression patterns of different materials and different organs were analyzed.【Method】The expression patterns of the four genes were detected by RT-PCR. Full length cDNA were cloned using the rapid-amplification of cDNA ends technique (RACE). Physical and chemical properties of proteins were analyzed with the bioinformatics softwares. RT-PCR was employed to detect expression patterns of the genes in different organs (anther, ovary, petal, sepal and leaf) and anthers from different developmental stages (tetrad stage, early- or mid-uninucleate stage, late-uninucleate stage and binucleate stage).【Result】According to the results of Blastx, the four genes were named as CaSEP1, CaPROF, CaOle e 6 and CaPCP, separately. Full length of CaSEP1 is 1 108bp, which codes 221 amino acids and contains a MADS domain and a K domain. The full length CaPROF includes 767 base pairs and 131 amino acids, which has a PROF domain. CaOle e 6 is 523 bp in full length, codes 85 amino acids with an Ole e 6 domain. Full length of CaPCP includes 563 base pairs and 66 amino acids. Amino acid sequence alignments and phylogeny reconstructions indicate that CaSEP1 is most similar to the tomato SEP1 protein and they have the closest relationship. CaPROF is more similar to profilins from tobacco and tomato than other species and shows the closest genetic distance with tomato profiling, CaOle e 6 is similar to the tomato Ole e 6 protein and they are classified into the same cluster, CaPCP shows the most similarity with tea pollen like protein and they are closest in genetic distance. RT-PCR showed that CaSEP1 expressed only in the reproductive organs of the fertile line with higher expression in anther, ovary and petal than sepal and leaf. CaPROF expressed significantly higher in anther than leaf of the fertile line, while no expression in other organs. CaOle e 6 expressed significantly higher in anther than other organs of the fertile line. CaPCP showed specific expression in anther of the fertile line. The expression of CaSEP1 increased gradually before decreasing in the binucleate stage of fertile anther, while it increased gradually in sterile anther during microspore development. The gene shows a similar expression between fertile line and sterile line at the tetrad stage of microspore development. At early- or mid-uninucleate stage and late-uninucleate stage, the gene expresses higher in fertile line. While the expression in fertile line is lower than sterile line during binucleate stage of microspore development. CaPROF, CaOle e 6 and CaPCP only express at the late stages (late-uninucleate stage and binucleate stage).【Conclusion】Analysis of sequences and expression profilings of the four genes indicates close relationships to male fertility. This study provides important information for revealing the mechanism of male sterility and regulating male fertility in pepper.

Key words: pepper , genic male sterility , expression , gene clone , sequence analysis

刘辰, 马宁, 付楠, 李欣, 郭爽, 沈火林. 辣椒GMS育性相关候选基因的克隆及表达分析[J]. 中国农业科学, 2014, 47(16): 3264-3276.

LIU Chen, MA Ning, FU Nan, LI Xin, GUO Shuang, SHEN Huo-Lin. Cloning and Expression Analysis of Fertility-Related Genes for the Genic Male Sterile Line in Pepper (Capsicum annuum L.)[J]. Scientia Agricultura Sinica, 2014, 47(16): 3264-3276.

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