棉花线粒体基因cRT-PCR改良及其在寻找CMS相关基因中的应用
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摘要
植物线粒体是含有遗传物质的细胞器,为细胞生命活动提供必需的能量。农业生产中,线粒体基因的变异容易导致细胞质雄性不育,为作物杂交育种提供了高效的杂交母本。然而,植物线粒体基因组结构复杂,不同类型的细胞质雄性不育的发生往往由特异的基因突变所导致,为雄性不育基因的克隆带来了较大的困难。针对棉花线粒体基因的特性,改进了cRT-PCR技术,RNA连接酶将线粒体mRNA的5′和3′末端连接环化后,应用随机引物进行反转录,在保证与特异引物获得相同的完整线粒体基因转录本的基础上,实现了同时对所有线粒体编码基因的cRT-PCR,方便对线粒体编码基因的结构进行全局分析,并且通过改良方法对棉花CMS基因进行探索,从而快速找到异常因素,为线粒体功能基因组分析提供了新的方法。
Plant mitochondria is a type of organelles containing genetic material,which provides necessary energy for cells. In agricultural production, genovariation of mitochondrial gene may lead to cytoplasmic male sterility, providing a convenient female parent for hybird breeding. Despite successful utilization of CMS and restoration of fertility(Rf) in breeding programs, the underlying mechanisms of these processes remain elusive because the mitochondrial genome structure is complex, and occurrence of different types of CMS is often caused by specific mutations consequently limiting gene cloning. This study summarized the techniques to improve cRT-PCR technology and present detailed characteristics mitochondrial gene involved in CMS. After RNA ligase cyclizes the 5′ and 3′ ends of mitochondrial mRNA, random primers were used for reverse transcription to ensure the same integrity as specific primers. Based on the mitochondrial gene transcript, cRT-PCR was performed on all mitochondrial coding genes, which provided insight of structure of mitochondrial coding genes. Therefore, we used this system to explore cotton CMS genes, thus quickly figuring out abnormal factors and providing a new method for mitochondrial functional genomic analysis.
Plant mitochondria is a type of organelles containing genetic material,which provides necessary energy for cells. In agricultural production, genovariation of mitochondrial gene may lead to cytoplasmic male sterility, providing a convenient female parent for hybird breeding. Despite successful utilization of CMS and restoration of fertility(Rf) in breeding programs, the underlying mechanisms of these processes remain elusive because the mitochondrial genome structure is complex, and occurrence of different types of CMS is often caused by specific mutations consequently limiting gene cloning. This study summarized the techniques to improve cRT-PCR technology and present detailed characteristics mitochondrial gene involved in CMS. After RNA ligase cyclizes the 5′ and 3′ ends of mitochondrial mRNA, random primers were used for reverse transcription to ensure the same integrity as specific primers. Based on the mitochondrial gene transcript, cRT-PCR was performed on all mitochondrial coding genes, which provided insight of structure of mitochondrial coding genes. Therefore, we used this system to explore cotton CMS genes, thus quickly figuring out abnormal factors and providing a new method for mitochondrial functional genomic analysis.
引文
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[2] Yang P,Han J,Huang J,et al..Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton [J].PLoS ONE,2014,9(11):e112320.
[3] Kuhn J,Binder S.RT-PCR analysis of 5′to 3′end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria[J].Nucl.Acids Res.,2002,30(2):439.
[4] Zhang Q Y,Lin Y G.Rice mitochondrial genes are transcribed by multiple promoters that are highly diverged[J].J.Plants,2006,48(12):1473-1477.
[5] Hang R,Deng X,Liu C,et al..Circular RT-PCR assay using Arabidopsis samples[J].Bio.Protocol.,2015,doi:10.21769/BioProtoc.1533.
[6] Luo D,Xu H,Liu Z,et al..A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice[J].Nat.Genet.,2013,45(5):573.
[7] Wang Z,Zou Y,Li X,et al..Cytoplasmic male sterility of rice with Boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing[J].Plant Cell,2006,18(3):676-687.
[8] Gobron N,Waszczak C,Simon M,et al..A cryptic cytoplasmic male sterility unveils a possible gynodioecious past for Arabidopsis thaliana[J].PLoS ONE,2013,8(4):e62450.
[9] Zhu T,Liang C,Meng Z,et al..CottonFGD:An integrated functional genomics database for cotton[J].BMC Plant Biol.,2017,17(1):101.
[10] Slomovic S,Schuster G.Circularized RT-PCR (cRT-PCR):Analysis of the 5′ ends,3′ ends,and poly(A) tails of RNA[J].Methods Enzymol.,2013,530(4):227-251.
[11] Yu Y,Liu C,Wang B,et al..Characterization of 3,17 β-hydroxysteroid dehydrogenase in Comamonas testosteroni.[J].Chem.Biol.Inter.,2015,234:221-228.
[12] 张晓.棉花胞质雄性不育系与保持系线粒体基因组差异研究[D].北京:中国农业科学院,博士学位论文,2011.