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作 者 ：赵会芳1,2,巩振辉1*,赵利民1,柯桂兰1

期 刊 ：西北植物学报 2013年 7期 页码：1317~1324

关键词：大白菜；小孢子；细胞质雄性不育；cDNA-AFLP；基因表达；

Keywords:Chinese cabbage, microspore, cytoplasmic-genic male sterility, cDNA-AFLP, gene expression,

摘 要 ：以大白菜雄性不育材料RC₇及其保持系B₇为材料,通过对两材料花药发育过程进行细胞学观察,并利用cDNA-AFLP技术分析两材料花蕾的基因表达差异,以明确RC₇发生雄性败育的时期和方式,为大白菜CMS的分子机理研究提供依据。结果显示:（1）细胞学观察发现,大白菜雄性不育材料RC₇及其保持系B₇的小孢子母细胞减数分裂属同时型,小孢子在四分体中的排列属四面体型,保持系B₇花药的绒毡层属于腺质型;不育材料RC₇在四分体时期绒毡层细胞内出现大的液泡,呈现败育征兆,单核期绒毡层解体,中层退化,小孢子核开始解体、败育,属于单核花粉败育型。（2）对两材料蕾期基因的cDNA-AFLP差显比较分析表明,共获得了23条阳性差异片段,其中在不育系RC₇花蕾中特异表达的有8条,在保持系B₇花蕾中特异表达有10条,两系中共有条带5条。（3）对不育系中特异表达的8条谱带进行Blast搜索发现,H1与拟南芥光合反应蛋白基因有较高的一致性,H26与拟南芥钙调磷酸酶类磷酸酯酶家族蛋白质的部分序列存在90%的一致性,属于细胞信号转导基因;对来自保持系的10条谱带的差异片段功能以及一致性进行比对分类发现,它们包括糖代谢基因、蛋白质的合成与运输基因、乙烯诱导基因、电子传递和能量途径基因、未知或假定蛋白等。研究表明,大白菜雄性不育材料RC₇可能是由于绒毡层细胞液泡化和径向肥大,小孢子受挤压后破裂降解,不能形成正常的成熟花粉粒而败育;乙烯诱导基因(H29)在保持系中特异表达,在不育系中沉默,表明不育系中缺乏乙烯相关基因。

Abstract:Morphology of cytoplasmic-genic male sterility line RC₇ and its maintainer line of Chinese cabbage were microscopically studied to make clear RC₇ abortive period and mode,analysis of gene expression in bud by using cDNA-AFLP technology,which will provide a scientific basis for Chinese cabbage on molecular mechanism of CMS.（1）The microspore mother cells perform the simultaneous type of meiosis.The microspores are arranged in a tetrahedral shape in the tetrad,and the tapetum of the maintainer line belongs to glandular type.The male sterile RC₇ shows larger vacuoles in the tapetum cell,which is abortion signs.In uninucleate stage,tapetal disintegration,middle cell disappeared and microspore nucleus began to collapse,which belongs to uninucleate abortion type.（2）The gene expression in flower buds of CMS line and its maintainer line was compared by means of cDNA-AFLP,a total of 23 differential transcript-derived fragments (TDFs) were identified and sequenced.8 TDFs were expressed specifically in CMS line,whereas 10 could be detected only in the maintainer line,moreover 5 TDFs were expressed both two lines.（3）The result of GenBank BLAST showed that H1 TDFs coming from CMS line shared higher identity with photoassimilate-responsive protein of Arabidopsis thaliana,H29 TDFs shared higher identity with calcineurin-like phosphoesterase family of A.thaliana.10 TDFs from maintainer line were highly identical to known sequences.These genes might be related to glucose metabolism,protein synthesis and transport,ethylene-inducible gene,electron transport and energy pathway,unknown or putative protein and so on.The results showed that the reason for the abortion was possibly that the tapetum cells were subjected to hypertrophy via vacuolation so that the microspores were extruded and broken into pieces,that makes the bicellular pollen grains could not form.Ehylene induced genes (H29) specifically expressed in the maintainer line,silenced in the sterile lines,which indicated that lack of sterile lines of ethylene related genes.

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