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雄性不育(Male sterility)是存在于植物界中的一种普遍现象,它是指植物在有性繁殖过程中雄蕊非正常生长,自身不能产生有效花粉,但是雌蕊能正常发育受精[1]。目前研究人员已经在萝卜(Raphanus sativus L.)[2]、水稻(Oryza sativa L.)[3]、西瓜[Citrullus lanatus (Thunb.) Matsum. et Nakai][4]等多个品种或种间杂交品种中发现雄性不育现象,并开展了育种工作[5-6]。橡胶树存在雄性不育的现象,橡胶树育种主要采用杂交育种法,大面积推广种植的橡胶树品种几乎都是通过杂交选育获得的。杂交育种主要采用人工授粉的方式进行,普遍采用“雄蕊塞入法”。人工授粉耗费大,工序繁杂,加之橡胶树坐果率低(一般 5%~8%,最高不超过 10%),虽然每个杂交组合授粉数量超过 2 000 朵,但真正获得可用于后续选育的杂交种子非常少。因此,由于杂交种子少,选育范围狭窄,橡胶树选育效率不高[7-10]。云南省热带作物科学研究所利用亲本雄性不育和雌性不育的特性,通过建立橡胶树自然杂交授粉园获得了大量杂交种子,扩大了筛选范围,通过授粉园获得的橡胶树自然杂交种子培育出了‘云研 77-2 ’和‘云研 77-4 ’两个大面积推广的优良品种[11]。然而,由于橡胶树雄性不育相关机制方面的研究报道尚少,使得橡胶树雄性不育种质的有效利用受到了限制。
XTH木葡聚糖水解酶/内转糖苷酶(xyloglucan endotransglucosylase/hydrolase, XTH) 是重构细胞壁的重要物质,其机制是通过催化作用,使木葡聚糖分子断裂和重连,对植物的纤维素-木葡聚糖结构进行修饰从而影响细胞壁结构[12]研究表明,XTH也参与花的发育,在花药与花丝柱上发生作用;在转录水平上,XTH的表达受植物激素和环境因素的调控,在植物生长和发育的很多方面调节细胞生长发育,同时不同的XTH对激素响应存在明显的差异[13-14]。在拟南芥中研究发现XTH23基因参与盐胁迫下的侧根的发育,其突变体对盐胁迫有极高的敏感度,其次XTH23是通过关键的油菜素内酯信号通路转录因子BES1被盐诱导的,BES1直接作用于XTH23的上游以控制它们的表达,表明XTH23 通过BES1途径参与侧根发育,并有助于侧根对盐的适应[15]。据报道,在玉米中ZmXTH23表达具有组织特异性,在幼茎中的表达量最高,且受到脱落酸 、NaCl以及PEG6000的诱导,在应答非生物逆境胁迫中具有重要作用[16],GmXTH23在大豆各组织中皆有表达,在茎中的表达量最高;与野生型拟南芥相比,拟南芥中过表达GmXTH23基因使得拟南芥在干旱胁迫下的耐受性增强,同时在干旱胁迫下GmXTH23通过提高脯氨酸与超氧化物歧化酶含量,并降低植物细胞在胁迫下的损伤来提高植物的抗旱性[17]。在荔枝中花穗发育不同时期LcXTH 基因家族表达存在差异,LcXTH23表达量较高,推测其在花穗发育中起关键作用[18]。
笔者所在的课题组在前期转录组分析筛选出了一系列与橡胶树雄性不育相关的差异表达基因[19],其中包括XTH23基因。本研究利用qRT-PCR对HbXTH23基因在橡胶树不同的组织器官及雄花不同发育时期的表达进行分析,通过mRNA原位杂交(mRNA fluorescence in situ hybridization, mRNA-FISH)技术明确HbXTH23在橡胶树雄花中的特异性表达部位,以揭示HbXTH23基因在橡胶树雄性不育的功能与作用,从分子水平分析橡胶树雄性不育的机理,从而为橡胶树杂交育种提供科学的依据。
Expression analysis of candidate gene HbXTH23 related to male sterility in Hevea brasiliensis
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摘要: 为了揭示HbXTH23基因在橡胶树雄性不育的功能与作用,从分子水平分析橡胶树雄性不育的机制,利用qRT-PCR对HbXTH23基因在橡胶树不同的组织器官及雄花不同发育时期的表达进行分析,并通过mRNA原位杂交技术以探明HbXTH23在橡胶树雄花中的特异性表达部位。荧光定量结果表明,HbXTH23基因在不育品种的雄花表达量明显高于在可育品种雄花的表达量,且明显高于在其他组织的表达量,而在胶乳中几乎没有表达。在雄性可育品种小孢子母细胞时期、四分体和单核小孢子时期3个阶段的变化呈现平缓上升的趋势,在不育品种中,则表现为先升高后下降的趋势,在四分体时期的表达差异最大。利用mRNA-FISH技术表明HbXTH23基因主要是在花丝(柱)、花药壁中表达,推测其在花丝(柱)和花药壁上的异常表达可能是导致雄花不育发生的原因之一。HbXTH23开放阅读框为861 bp,编码286个氨基酸,蛋白分子量为31.95 kDa,理论等电点7.62,总平均亲水性GRAVY为−0.288,氨基酸序列密集分布在负值以下,为疏水性蛋白。亚细胞定位预测HbXTH23蛋白存在于细胞壁、细胞质中。具有信号肽,其剪切位点位于第26和第27号氨基酸之间,HbXTH23与刺毛黧豆、烟草蛋白亲缘关系较近。Abstract: Xyloglucan endotransglucosylase/hydrolase (XTH) is a key enzyme in plant cell wall remodeling and plays an important role in plant growth and development. Previously, we screened the differentially expressed gene XTH23 related to the male sterility in Hevea brasiliensis by transcriptome sequencing. The HbXTH23 gene expression in different organic tissues and male flowers at different development stages was then analyzed by using qRT-PCR, and the location of specific expression of HbXTH23 gene in the male flowers was analyzed by using mRNA fluorescence in situ hybridization (mRNA-FISH). The qRT-PCR results showed that HbXTH23 gene expression was significantly higher in male flowers of male sterile varieties (MS) than in male flowers of male fertile varieties (MF), and also significantly higher than in other tissues, and was almost not expressed in latex. HbXTH23 gene expression in MF at the three stages, microspore mother cell, tetrad and mononuclear microspore, presented a rising trend, and the expression in MS increased first and then decreased. And the highest difference in expression between MF and MS was at the tetrad stage. The results obtained by mRNA - FISH showed that HbXTH23 gene was mainly expressed in filaments (style) and the anther wall. It was speculated that the abnormal expression in the filaments (style) and anther wall may be one of the causes of male flower sterility. The open reading frame of HbXTH23 was 861 bp, and encoded a polypeptide of 286 amino acids with a relative molecular weight of 31.95 kDa and a theoretical isoelectric point of 7.62. The grand average hydropathy (GRAVY) score of the protein was −0.288, and the hydropathy values of amino acids in the sequence were mostly distributed below zero, which indicated that the protein was hydrophilic (hydrophobic). Subcellular localization predicted that HbXTH23 protein existed in cell wall and cytoplasm. It had a signal peptide, and the shear site of the signal peptide was located between the 26th and 27th amino acids. HbXTH23 was closely related to Mucuna pruriens and Nicotiana attenuata protein. Thes study can provide a scientific basis for the further study of the molecular mechanism of male sterility and the breeding of Hevea brasiliensis.
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Key words:
- Hevea brasiliensis /
- HbXTH23 /
- qRT- PCR /
- RNA - FISH /
- bioinformatics analysis
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