黄瓜性别分化的研究
摘要
黄瓜是研究植物性别分化的经典材料，对黄瓜进行性别分化研究，具有重要的理论价值和实践意义。本研究以强雄性系黄瓜ZH-20和全雌性系黄瓜GY1为材料，对黄瓜性别分化和表达机理进行了研究。
通过石蜡切片对黄瓜花芽分化和雌、雄花的发育进行了详细的形态学观察。结果表明黄瓜雌、雄花的发育过程可细分为10个时期。黄瓜单性花的分化和发育过程经历了无性期、两性期和单性期。两种性器官原基在发育的起始都出现，在两性期(时期5)之前雌、雄花之间不存在明显的形态学上的差异，花芽从两性期开始进一步分化，雌花花芽中心凹陷形成下位子房，雄花雄蕊体积快速增大，最终发育形成单性的雌、雄花。
在黄瓜性别分化形态学研究的基础上，进一步考察了单性花中未形成有功能器官的相反性别原基的发育。通过形态学，组织化学，同工酶和蛋白质电泳等方法分析了黄瓜雄花中心皮的发育状况，结果发现黄瓜雄花心皮能不断地进行细胞分裂，体积也不断增大，心皮中RNA含量较为丰富；雄花心皮在发育后期出现特化的组织结构，至雄花开花期时，形成类似雌花胎座的结构，但没有分化出胚珠；雄花心皮在不同发育时期过氧化物同工酶和酯酶同工酶谱带和表达量均存在差异，发育后期仍能合成新的蛋白质，进一步表明在成熟的雄花中心皮不再处于原基状态。这些结果都表明雄花中的心皮处于不断的发育之中，但其发育模式与雌花中的心皮有着明显的差异。
采用RT-PCR技术克隆到黄瓜亚精胺合成酶基因长为297的cDNA片断，结合3-RACE，5-RACE技术，拼接后获得了全长为1398bp的cDNA(CsSPDS)，该基因具有一个951个碱基的开放阅读框(ORF)，编码长为317个氨基酸残基的蛋白质。CsSPDS的氨基酸序列与其它已知的亚精胺合成酶具有高度的同源性，其与咖啡亚精胺合成酶同源性最高(identity，87％)。不同器官杂交表达分析表明CsSPDS主要在幼根、幼叶和花芽等快速生长发育的组织中表达。我们还分析了乙烯利处理后从雄向雌的性逆转过程中CsSPDS的表达状况，结果表明三叶期黄瓜茎尖在处理后2小时CsSPDS表达量降低，处理后4小时开始增加，到8小时时达到最高，之后一直高于未处理组。并讨论了乙烯与多胺的相互作用及其与黄瓜性别分化的关系。
Cucumber is a unique model plant for sex expression. Studies on sex differentiation of cucumber has both practical and theoretical significance. The mechanism of sex differentiation and expression in cucumber was investigated using the androecious line (ZH-20)and full female line (GYl)as material in our study..
Morphological analyses were conducted on differentiation and development of female and male sex organs during the floral bud initiation and sex differentiation process in cucumber, and the results showed that the development of female and male flowers could be divided into 10 stages, and the formation of cucumber unisexual flowers first underwent a hermaphoroditic stage, then developed forward maleness or femaleness. No significant morphological difference existed hi female and male flowers at stages earlier than hermaphoroditic stage (stage 5), after which the flower bud developed unisexually. The center of female floral bud is concave, then the hypogynous ovary appeared and the unisexual female flower was formed finally; while the anther of male floral bud enlarged rapidly and the unisexual male flower was formed finally.
Development of carpel in cucumber male flower was studied by morphological, histochemical and isoenzyme electrophoretic analysis. The results showed that: (1) cell number of the carpel in male flowers increased continuously during the development of male flower, and the carpel in male flowers was abundant in RNA content; (2) the carpel in male flower at latter developmental stage was differentiated, and a placenta-like structure was formed in the carpel of male flower during anthesis, while ovule did not appear as that of mature female flower; (3) The POD and esterase isoenzyme electrophoretic profile of carpel varied with the development of male flower and SDS-PAGE analysis of carpel indicated that the carpel in male flowers at latter development stages could synthesize new proteins, which further
indicated that the carpel in mature male flower was no longer at the stage of primordium. It could be deduced from the results that carpel of cucumber male flowers develops continuously in a distinct pattern with that of female flowers during sex expression process.
A partial cDNA fragment of cucumber spermidine synthase gene was isolated by RT-PCR, and the full-length cDNA was obtained by 5 -RACE and 3 -RACE techniques. Sequences analysis showed that the size of full-length cDNA was 1398 bp which contains a 951 bp coding region encoding 317 amino acid residues. CsSPDS amino acid sequences showed high degree identity compared with known SPDS from other species. And SPDS from Coffea arabica has the highest similarity to CsSPDS. Organ-specific expression analysis indicated that the CsSPDS gene expression was predominant in rapid growing tissues such as young roots, flower buds and young leaves. The response of CsSPDS expression to the sex reversal from male to female by the application of ethephon to seedlings at 3 leaf stage was also examined, and the results showed that the CsSPDS transcript level in shoot apices decreased 2h after treatment, increased 4h after treatment, reached the peak 8h after treatment, and the CsSPDS transcript level of treatment was always higher
than that of control. The relationship between ethylene and polyamine metabolism and its role in sex expression of cucumber were also discussed.
通过石蜡切片对黄瓜花芽分化和雌、雄花的发育进行了详细的形态学观察。结果表明黄瓜雌、雄花的发育过程可细分为10个时期。黄瓜单性花的分化和发育过程经历了无性期、两性期和单性期。两种性器官原基在发育的起始都出现，在两性期(时期5)之前雌、雄花之间不存在明显的形态学上的差异，花芽从两性期开始进一步分化，雌花花芽中心凹陷形成下位子房，雄花雄蕊体积快速增大，最终发育形成单性的雌、雄花。
在黄瓜性别分化形态学研究的基础上，进一步考察了单性花中未形成有功能器官的相反性别原基的发育。通过形态学，组织化学，同工酶和蛋白质电泳等方法分析了黄瓜雄花中心皮的发育状况，结果发现黄瓜雄花心皮能不断地进行细胞分裂，体积也不断增大，心皮中RNA含量较为丰富；雄花心皮在发育后期出现特化的组织结构，至雄花开花期时，形成类似雌花胎座的结构，但没有分化出胚珠；雄花心皮在不同发育时期过氧化物同工酶和酯酶同工酶谱带和表达量均存在差异，发育后期仍能合成新的蛋白质，进一步表明在成熟的雄花中心皮不再处于原基状态。这些结果都表明雄花中的心皮处于不断的发育之中，但其发育模式与雌花中的心皮有着明显的差异。
采用RT-PCR技术克隆到黄瓜亚精胺合成酶基因长为297的cDNA片断，结合3-RACE，5-RACE技术，拼接后获得了全长为1398bp的cDNA(CsSPDS)，该基因具有一个951个碱基的开放阅读框(ORF)，编码长为317个氨基酸残基的蛋白质。CsSPDS的氨基酸序列与其它已知的亚精胺合成酶具有高度的同源性，其与咖啡亚精胺合成酶同源性最高(identity，87％)。不同器官杂交表达分析表明CsSPDS主要在幼根、幼叶和花芽等快速生长发育的组织中表达。我们还分析了乙烯利处理后从雄向雌的性逆转过程中CsSPDS的表达状况，结果表明三叶期黄瓜茎尖在处理后2小时CsSPDS表达量降低，处理后4小时开始增加，到8小时时达到最高，之后一直高于未处理组。并讨论了乙烯与多胺的相互作用及其与黄瓜性别分化的关系。
Cucumber is a unique model plant for sex expression. Studies on sex differentiation of cucumber has both practical and theoretical significance. The mechanism of sex differentiation and expression in cucumber was investigated using the androecious line (ZH-20)and full female line (GYl)as material in our study..
Morphological analyses were conducted on differentiation and development of female and male sex organs during the floral bud initiation and sex differentiation process in cucumber, and the results showed that the development of female and male flowers could be divided into 10 stages, and the formation of cucumber unisexual flowers first underwent a hermaphoroditic stage, then developed forward maleness or femaleness. No significant morphological difference existed hi female and male flowers at stages earlier than hermaphoroditic stage (stage 5), after which the flower bud developed unisexually. The center of female floral bud is concave, then the hypogynous ovary appeared and the unisexual female flower was formed finally; while the anther of male floral bud enlarged rapidly and the unisexual male flower was formed finally.
Development of carpel in cucumber male flower was studied by morphological, histochemical and isoenzyme electrophoretic analysis. The results showed that: (1) cell number of the carpel in male flowers increased continuously during the development of male flower, and the carpel in male flowers was abundant in RNA content; (2) the carpel in male flower at latter developmental stage was differentiated, and a placenta-like structure was formed in the carpel of male flower during anthesis, while ovule did not appear as that of mature female flower; (3) The POD and esterase isoenzyme electrophoretic profile of carpel varied with the development of male flower and SDS-PAGE analysis of carpel indicated that the carpel in male flowers at latter development stages could synthesize new proteins, which further
indicated that the carpel in mature male flower was no longer at the stage of primordium. It could be deduced from the results that carpel of cucumber male flowers develops continuously in a distinct pattern with that of female flowers during sex expression process.
A partial cDNA fragment of cucumber spermidine synthase gene was isolated by RT-PCR, and the full-length cDNA was obtained by 5 -RACE and 3 -RACE techniques. Sequences analysis showed that the size of full-length cDNA was 1398 bp which contains a 951 bp coding region encoding 317 amino acid residues. CsSPDS amino acid sequences showed high degree identity compared with known SPDS from other species. And SPDS from Coffea arabica has the highest similarity to CsSPDS. Organ-specific expression analysis indicated that the CsSPDS gene expression was predominant in rapid growing tissues such as young roots, flower buds and young leaves. The response of CsSPDS expression to the sex reversal from male to female by the application of ethephon to seedlings at 3 leaf stage was also examined, and the results showed that the CsSPDS transcript level in shoot apices decreased 2h after treatment, increased 4h after treatment, reached the peak 8h after treatment, and the CsSPDS transcript level of treatment was always higher
than that of control. The relationship between ethylene and polyamine metabolism and its role in sex expression of cucumber were also discussed.
引文
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2. Irish EE and Nelson T. 1989, Sex determination in monoecious and dioecious plants. Plant Cell, 1: 737-744
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