摘要
目前,小麦杀雄剂途径是小麦杂种优势利用的一条重要而简捷的途径,大量实践表明,它可以把杂交小麦育种的起点建立在小麦常规育种的最新成果上。在众多小麦杀雄剂中,杀雄剂SQ-1有杀雄彻底、品种间互作效应小,且对植株农艺性状、雌蕊育性和杂交种籽粒品质无负面影响等特点,是目前较为优良的化学杂交剂之一。然而,其诱导小麦雄性不育的机理还尚不清楚。为揭示小麦生理型雄性不育的分子机理,更好的为小麦杂种优势利用提供理论依据和技术支撑,本研究以杀雄剂SQ-1诱导的小麦生理型雄性不育和对等可育系在转录水平上的表达差异,所构建的AFLP-cDNA小麦生理型雄性不育文库为基础,经EST序列的比对分析,从中挑选了9个候选基因片段采用了RT-PCR半定量表达分析技术及基因克隆技术,对其候选基因进行了生理型雄性不育相关分子机理的研究,获得如下重要结果：
1、以小麦品种西农2611和西农9766为材料,研究比较了9个候选基因在不育和可育等生理系不同时期花药中的表达丰度。结果显示,SQ-1诱导的雄性不育花药中,糖酵解、三羧酸循环及电子传递链过程的相关基因：烯醇酶、丙酮酸脱氢酶E1亚基(E1-PDHc)、线粒体NAD7基因受到抑制；而细胞色素C氧化酶Ⅶ亚基、嘧啶核酸二硫化物氧化还原酶和抗坏血酸过氧化物酶(APX),在不育花药中则表现为基因表达丰度最高时期提前。由此推测SQ-1可抑制花药中的糖酵解、三羧酸循环及电子传递链过程,使不育株花药的有氧代谢过程受阻,能量供应不足,导致花药细胞大量败育。在不育花药中,小孢子发育前期由于电子传递链过程受抑制,花药细胞内积累了过量活性氧,为消除过量的活性氧花药中抗氧胁迫相关基因则大量表达。
2、在SQ-1处理的生理型雄性不育花药中ARF-GTP蛋白的表达受到抑制,可能使膜运输作用受阻,引起小孢子正常发育所需物质供应不足,与雄性不育有一定关系；而泛素途径相关基因：泛素-核糖体融合蛋白S27a和F-box蛋白,在不育花药中基因表达丰度最高时期提前,推测在小孢子发育关键期,不育花药中泛素相关基因大量表达,加速有用蛋白的降解而与雄性不育密切相关。
3、以小麦品种西农2611可育组二核期花药的cDNA一链为模板,扩增获得了普通小麦E1-PDHc基因和ARF-GTP基因的cDNA序列,并向NCBI提交,登陆号分别为563379和GU563378。经结构域分析及同源序列比对,GU563379序列片段长1206bp,编码330个氨基酸,具有典型的植物E1-PDHc基因保守结构域,包含E1-PDHc基因ORF编码框的完整3′端；而GU563378,经结构域分析及同源序列比对,具有典型的植物ARF-GTP基因保守结构域,并包含完整的ORF开放阅读框,ORF序列长1380bp,编码460个氨基酸。
Nowadays, Chemical hybrid agent (CHA) is an important and convenient way of heterosis。A lot of facts suggest that it can build hybrid breeding on the latest fruit of wheat conventional breeding。SQ-1 is a new type of wheat Gametocide which can lead to male complete sterility, and without any side effects on the pistil fertility and agronomic traits, However, its mechanism of inducing wheat male sterility is not clear yet. In order to reveal the molecular mechanism and provide theoretical and technical support for utilization of heterosis, nine candidate genes were selected from AFLP-cDNA library of Physiological male Sterile lines induced by gametocide SQ-1 and cloned and analyzed using semi-quantitative RT-PCR in this study. The results were as follows:
1. wheat XiNong 2611 and XiNong 9766 induced by Chemical hybrid agent (CHA) SQ-1 and their near fertile lines were used as materials the expression pattem.of nine candidate genes were compared in sterile and fertile anthers. The results showed Enolase,Pyruvate dehydrogenase E1 subunit (E1-PDHc), Mitochondrial NAD7 gene in male sterile anthers, were inhibited; but Cytochrome C oxidase subunitⅦ, Pyrimidine nucleotide disulfide oxidoreductase and the Ascorbate peroxidase (APX), expressed highest ahead in sterile anthers Speculated that glycolysis, Krebs cycle and electron transport process was inhibited in anthers, so the energy shortage of anthers developvent leaded to the anther abortion in early microspore development that is, the early uninucleate stage.
SQ-1 treatment in the physiological male sterile anthers ARF-GTP protein was inhibited membrane transport function may be blocked, causing material required for the normal development of microspores in short supply, there is a certain relationship with male sterility; the pan-Pathway related gene:ubiquitin-ribosomal protein S27a fusion and F-box protein, in the sterile anther highest abundance of gene expression in the period ahead, suggesting that the critical period in the development of small spores, sterile anthers large number of ubiquitin-related gene expression to accelerate the degradation of a useful protein is closely related with male sterility.
2. Using the first strand cDNA Xinong 2611 as template, E1-PDHc gene (GenBank accession GU563379) and ARF-GTP gene (GenBank accession GU563378) was cloned..GU563379 sequence fragment length 1206bp, encoding 330 amino acids, with a typical plant El-PDHc gene conserved domain, and through domain analysis and homology alignment with the typical plant ARF-GTP gene conserved domain, and GU563378contains a complete ORF, ORF sequence was 1380bp in lengthencoding 460 amino acids.
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