Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display
Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display
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DOI：
10.1007/s00299-007-0492-7
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发表时间：
2008-03
影响因子：
6.2
通讯作者：
Jinfa Zhang;R. Turley;J. Stewart
中科院分区：
文献类型：
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作者：
Jinfa Zhang;R. Turley;J. Stewart
将野生二倍体三叶棉（Gossypium trilobum, D8）的细胞质和细胞核generf2导入栽培的四倍体陆地棉（Gossypium hirsutum）中，培育出CMS-D8及其恢复系。目前还没有关于therf2基因如何与cms相关基因相互作用以及CMS-D8细胞质如何影响核基因表达的信息。本研究旨在通过mRNA差异显示（mRNA differential display， DD）的方法，鉴定非恢复性可育维持系ARK8518 （rf2rf2）与其细胞质为D8的等基因杂合恢复系ARK8518R （rf2rf2）在花药组织中的差异表达基因。在由12个锚定引物和8个任意十聚体引物组成的31个引物组合扩增的3000多个DDRT-PCR条带中，约有100个条带被鉴定为定性差异显示。共分离、克隆和测序了38个cDNA片段，其中包括花药中12个优先表达的cDNA条带。反向northern blot分析显示，杂合恢复体ARK8518R中只有编码Cys-3-His锌指蛋白和氨基肽酶的4个基因表达上调，而编码磷酸核糖酰氨基甲酸转移酶（PAT）、淀粉合成酶（SS）、4-香豆酸-辅酶a连接酶、电子转运蛋白、钙连联蛋白、精氨酸脱羧酶和多泛素的22个基因表达下调。SS的下调解释了杂合恢复植株不育花粉粒中淀粉积累不足的原因。本文讨论了CMS及其恢复的分子机制，特别是SS和PAT基因在rf2向CMS- d8恢复中的可能作用。这项调查是首次对棉花进行这样的分析。
CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear geneRf2from the wild diploidGossypium trilobum(D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how theRf2gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf2rf2) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf2rf2) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterilerf2pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration ofRf2to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.