【作者】 鲍碧丽；

【导师】 应铁进；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【摘要】 为了探索乙烯受体基因家族成员LeETR1或LeETR2对番茄(Lycopersicon esculetum)各表现型的调控规律,实验室前期构建了含有3’-端非编码区和35SCaMV启动子的反义LeETR1和LeETR2序列,以含反义LeETR1或LeETR2的表达载体转化番茄B1纯系,转化子经过多代筛选和鉴定,得到两个转基因株系ale1和ale2。将ale1与ale2杂交,得到杂交dale。本研究以ale1,ale2和dale作为对象,经过标记基因NPTⅡPCR、反义序列PCR和标记基因GUS活性鉴定证明转入反义序列的稳定遗传。对各株系生长发育、衰老脱落、果实成熟等乙烯相关表现型、以及组织的乙烯结合能力等进行了系统比较。结果如下:1.ale1表达使自然光照下生长的植株明显矮化,显著增强叶柄上位性生长,花序轴缩短,开花期和座果期都推迟,开花数和座果率下降,畸形果率上升。这些性状提示LeETR1在番茄营养生长到生殖生长过程都有重要的调控作用。ale2只表现出微弱的叶柄上位性生长等部分乙烯反应。dale表现型与ale1基本一致,没有特别增强的表现型,说明LeETR1与LeETR2同时缺失并没有比单一缺失产生更高的乙烯敏感性或更强的乙烯反应;LeETR2在番茄生长发育的调控上可能没有特别明确的功能分工,它可能只是对包括LeETR1在内的其它受体成员起辅助调控作用。2.无论是在叶子还是果实中,对照株系的乙烯结合能力要普遍高于转基因株系,说明转基因株系的乙烯受体表达数量要低于对照株系。在叶子的乙烯结合方面,各个株系叶子的乙烯结合能力没有体现差异性,而在果实乙烯结合能力方面,转基因株系间乙烯结合能力具有显著差异,说明乙烯受体LeETR1和LeETR2在叶子中的表达量占叶子总的受体的表达量比例不大,在果实中乙烯受体LeETR1和LeETR2对受体表达具有重要作用。另外,双突变体dale的乙烯结合能力与ale1和ale2类似,推测也存在乙烯受体家族成员间的特定时间和组织特异性地补偿作用。3.ale1表现出最明显的器官衰老和脱落加速,对乙烯作用抑制剂敏感性也更高,但ale2在花朵衰老上没有明显的表现,说明乙烯受体LeETRI和LeETR2对器官脱落的调控功能冗余,但也有作用程度的差异,在花朵衰老调控方面,LeETR1可能起了重要作用,而LeETR2可能没有直接的意义;dale叶柄脱落速度明显比ale1和ale2都快,说明dale在器官脱落区乙烯敏感性比ale1和ale2更强。这种结果提示LeETR1和LeETR2在调控器官脱落上既有功能冗余还可能存在功能叠加效果,两者同时缺失比单独缺失产生的乙烯敏感性更强,两者同时存在则可能对抑制乙烯诱导的脱落作用比单独存在时更强。4.转基因株系果实的单果重要比对照株系果实明显加重,果实形状要比对照株系更加宽扁。在成熟过程中,转基因株系果实的硬度、黏着性和弹性明显要低于对照株系。转基因株系果实的颜色要比对照株系更红,在BK+10以后趋于一致。在可溶性固形物和电导率上,ale1株系的含量要高于ale2株系,dale株系和对照株系。在酸含量方面,转基因株系的含量均要显著高于对照株系。在货架寿命上,转基因株系要比对照株系短。说明反义LeETR1和反义LeETR2果实及双突变体反义转基因果实成熟速度显著加快。这提示乙烯受体差异调控果实的成熟进程,除了前人已经明确的NR(LeETR3)外,LeETR1和LeETR2也与果实成熟有密切的关系,其中以LeETR1的调控效应较为明显。5.番茄乙烯受体LeETR1与LeETR2在不同组织中可能存在不同的相互关系。LeETR1在番茄生长发育过程中对平衡乙烯反应可能具有特别重要的功能地位。番茄乙烯受体家族各个成员都有其独特的表达特点,它们分别缺失或者表达减少都产生了明显的具有时空特异性的乙烯反应表现型。但是大量的证据表明,各个受体之间不是简单的独立存在关系,它们组成了由复杂的分工合作关系联接起来的统一体。更多还原

【Abstract】 In order to observe the regulation of two tomato ethylene recepors LeETR1 and LeETR2 on tomato phenotypes,two transformations from tomato(Lycopersicon esculentum) cultivar B1 with constructs containing the antisense sequence from the receiver domain and 3’-untranslated portion of the tomato ethylene receptor LeETR1 and LeETR2 separately under the control of an enhanced cauliflower mosaic virus 35S promoter were obtained by the former work of the group.The transgenic lines ale1 and ale2 and its crossed line dale were identified by PCR,GUS activity determination and PCR for the target genes,all the results were positive.Morphological and physiological changes of the transgenic lines,including growth and development,senescence and abscission,fruit ripening,ethylene binding capacity,were investigated or analyzed in this study.The results were presented as follows:1.The ale1 displayed the following significant characteristics,including shortened adult plant in natural light,epinastic petiole growth,shortened inflorescence stem,delayed anthesis and fruit set, decreased flower number and fruit set percent,and increased abnormality fruits.All of these characteristics suggest that LeETR1 possibly play an important role in regulating tomato vegetative and reproductive growth and development.ale2 only displayed shortened epicoty1,accelerated abscission and slightly epinastic petiole,the phenotype of dale was similar with ale1,which suggest LeETR2 possibly act in limited tissues.2.In fruits,ale1,ale2 and dale showed higher ethylene binding capacity.In leaves,there was no significant difference between the transgenic lines and the control line,which indicates that the ratio of tomato ethylene receptor LeETR1 and LeETR2 expression level is low compared with total level of receptor expression in tomato leaves;ethylene receptor LeETR1 and LeETR2 play an important role in receptor expression in tomato fruits.The ethylene binding capacity of dale was similar with ale1 and ale2,which may indicate that there exists a compensation expression among the receptor gene family.3.ale1 showed an accelerated organ senescence and abscission,and higher sensitivity to ethylene inhibitors.ale2 did not show obvious difference with B1 in flower senescence.dale showed an accelerated petiole abscission than ale1 and ale2,which indicates that LeETR1 and LeETR2 play an important role in negative regulating organ senescence and abscission,they have functionally redundancy,but have different effect degree.4.The individual fruit weight of the transgenic lines was significantly heavier than wild type B1, while the shape of the fruits was much flatter than B1.The firmness,viscosity and elasticity of the transgenic lines were lower than B1(P<0.05) at different stages of ripening.The transgenic lines had a deeper red color compared with B1(P<0.05),but after BK+10,the color of the four lines came to identical.The content of the total soluble solids and relative electrical conductivity of ale1 were significantly higher than ale2,dale and B1(P<0.05).The content of titratable acidity of transgenic lines were much higher than B1(P<0.05).The shelf lives of the transgenic lines were shorter than that of B1. All the results indicate that ethylene receptors differentially regulate fuirts ripening.Besides NR (LeETR3),LeETR1 and LeETR2 may have a close relationship with fruit ripening,and LeETR1 have an obvious regulation effect.5.Through the research it was found that different relation of LeETR1 with LeETR2 possibly exist in different tissue.LeETR1 possibly play a very important role in balancing ethylene response during tomato growth and development.Each member of tomato ethylene receptor family has its unique expression pattern,the loss-of-function or less expression of the receptor would result in obvious spatial and temporal specificity in ethylene responses.Enough evidence has shown that each member of tomato ethylene receptor family do not exist separately,but form an entia including complicated division and cooperation.更多还原