龙眼LFY同源基因表达及启动子克隆
摘要
龙眼(Dimocarpus longan L.)是我国具有重要经济价值的名优特色果树,也是我国在国际市场上极具竞争力的特色水果,但龙眼大小年结果、鲜果不能周年供应市场等问题尚未得到有效解决,这些都与龙眼花芽分化有关。
LFY基因在有花和无花植物中都存在,是决定花分生组织形成的必需基因,并在花形成中发挥着接连许多花诱导途径的输出信号和激活花器官决定基因的关键作用。因此,对LFY基因的研究成为目前关于成花基因研究中的一个热点。官磊(2007)采用染色体步移技术,克隆龙眼LFY同源基因编码区全长序列,并采用半定量RT-PCR技术对LLFY基因的功能进行了初步研究。本试验在此基础上细化了龙眼花芽分化采样过程,运用半定量RT-PCR技术进一步研究LLFY基因的表达。同时,进行LLFY在四季蜜龙眼中表达的研究。此外,本试验应用染色体步移技术,克隆了龙眼LLFY基因的启动子序列片段,并对启动子序列进行分析。主要研究结果如下:
1龙眼LLFY表达的研究
为了研究LLFY基因在龙眼花芽分化不同时期的表达情况,本实验分别选取“红核子”叶芽以及2月份到4月份不同时期的花序顶芽,运用半定量RT-PCR技术检测LLFY基因的表达情况。实验结果表明, LLFY的表达不仅与龙眼花序的分化有关,也与花芽的分化有关。同时,LLFY在“四季蜜”龙眼中的表达结果显示,LLFY表达量的增加可能与四季蜜龙眼早花以及四季开花的特性有关。
2龙眼LLFY启动子序列的克隆和分析
采用染色体步移技术,根据龙眼LLFY编码区基因组DNA序列设计特异性引物,进行LA-PCR扩增,获得龙眼LLFY基因启动子约800bp序列。运用Plant CARE、FootPriter等软件进行序列分析,结果表明,在此序列中除含有TATA-box和CAAT-box两个主要元件外,还含有多个潜在的顺式作用元件,如MYB结合位点、ABA响应元件(ABRE)和一些光响应元件如GA-box、GAG-motif、I-box等,说明龙眼LLFY的表达与干旱、光照、ABA有关。
Longan (Dimocarpus longan Lour.) is an important fruit in China, and very competitive in the international market, but there are some problems in longan production such as biennial bearing, the inadequate annual market supply of fresh fruit which are closely related to flower bud differentiation of in longan.
LFY gene exists in flowering and non-flowering plants, it is an essential gene that decides the form of floral meristem and play a key role in linking signals of flowering induction and activating floral organ determining genes during the flower formation.Guan Lei (2007) cloned the LFY homologous gene in longan(LLFY) and preliminary studied expression of LLFY by semi-quantitative RT-PCR analysis, the results showed LLFY genes were related to inflorescence differentiation and growth. On the basis of previous study, we detected the expression of LLFY gene further by RT-PCR in this article, meanwhile, LLFY‘s expression in“sijimi”longan was also studyed. In addition, the partial promoter sequence of longan LLFY was cloned by genome walking method, and the promoter sequence was analyzed by software. The main results showed as follows:
1 The expression research of longan LFY gene.
In order to study the expression of LLFY gene in different periods of flower bud differentiation, we selected“Honghezi”leaf buds and terminal inflorescence buds from February to April. The results showed that, the function of LLFY was related to not only the inflorescence differentiation, but also the flower bud differentiation. Meanwhile, the results showed the increasing expression of LLFY gene in“sijimi”may be related to its characteristics of early flowering and flowering throughout the growing season.
2 Cloning and analysing the promoter sequence of longan LFY gene.
Specific primers were designed and 800bp long sequence of LLFY promoter was cloned by genome walking method. Sequence analysis showed that LLFY’s promoter region contained not only two major components, the TATA-box and CAAT-box, but also several potential cis-acting elements such as MYB binding site、ABA responsive elements(ABRE) and some light responsive elements-GA-box、GAG-motif、I-box, etc. which means the expression of LLFY gene is related to drought, light, ABA.
LFY基因在有花和无花植物中都存在,是决定花分生组织形成的必需基因,并在花形成中发挥着接连许多花诱导途径的输出信号和激活花器官决定基因的关键作用。因此,对LFY基因的研究成为目前关于成花基因研究中的一个热点。官磊(2007)采用染色体步移技术,克隆龙眼LFY同源基因编码区全长序列,并采用半定量RT-PCR技术对LLFY基因的功能进行了初步研究。本试验在此基础上细化了龙眼花芽分化采样过程,运用半定量RT-PCR技术进一步研究LLFY基因的表达。同时,进行LLFY在四季蜜龙眼中表达的研究。此外,本试验应用染色体步移技术,克隆了龙眼LLFY基因的启动子序列片段,并对启动子序列进行分析。主要研究结果如下:
1龙眼LLFY表达的研究
为了研究LLFY基因在龙眼花芽分化不同时期的表达情况,本实验分别选取“红核子”叶芽以及2月份到4月份不同时期的花序顶芽,运用半定量RT-PCR技术检测LLFY基因的表达情况。实验结果表明, LLFY的表达不仅与龙眼花序的分化有关,也与花芽的分化有关。同时,LLFY在“四季蜜”龙眼中的表达结果显示,LLFY表达量的增加可能与四季蜜龙眼早花以及四季开花的特性有关。
2龙眼LLFY启动子序列的克隆和分析
采用染色体步移技术,根据龙眼LLFY编码区基因组DNA序列设计特异性引物,进行LA-PCR扩增,获得龙眼LLFY基因启动子约800bp序列。运用Plant CARE、FootPriter等软件进行序列分析,结果表明,在此序列中除含有TATA-box和CAAT-box两个主要元件外,还含有多个潜在的顺式作用元件,如MYB结合位点、ABA响应元件(ABRE)和一些光响应元件如GA-box、GAG-motif、I-box等,说明龙眼LLFY的表达与干旱、光照、ABA有关。
Longan (Dimocarpus longan Lour.) is an important fruit in China, and very competitive in the international market, but there are some problems in longan production such as biennial bearing, the inadequate annual market supply of fresh fruit which are closely related to flower bud differentiation of in longan.
LFY gene exists in flowering and non-flowering plants, it is an essential gene that decides the form of floral meristem and play a key role in linking signals of flowering induction and activating floral organ determining genes during the flower formation.Guan Lei (2007) cloned the LFY homologous gene in longan(LLFY) and preliminary studied expression of LLFY by semi-quantitative RT-PCR analysis, the results showed LLFY genes were related to inflorescence differentiation and growth. On the basis of previous study, we detected the expression of LLFY gene further by RT-PCR in this article, meanwhile, LLFY‘s expression in“sijimi”longan was also studyed. In addition, the partial promoter sequence of longan LLFY was cloned by genome walking method, and the promoter sequence was analyzed by software. The main results showed as follows:
1 The expression research of longan LFY gene.
In order to study the expression of LLFY gene in different periods of flower bud differentiation, we selected“Honghezi”leaf buds and terminal inflorescence buds from February to April. The results showed that, the function of LLFY was related to not only the inflorescence differentiation, but also the flower bud differentiation. Meanwhile, the results showed the increasing expression of LLFY gene in“sijimi”may be related to its characteristics of early flowering and flowering throughout the growing season.
2 Cloning and analysing the promoter sequence of longan LFY gene.
Specific primers were designed and 800bp long sequence of LLFY promoter was cloned by genome walking method. Sequence analysis showed that LLFY’s promoter region contained not only two major components, the TATA-box and CAAT-box, but also several potential cis-acting elements such as MYB binding site、ABA responsive elements(ABRE) and some light responsive elements-GA-box、GAG-motif、I-box, etc. which means the expression of LLFY gene is related to drought, light, ABA.
引文
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