龙胆八氢番茄红素脱氢酶（PDS）和番茄红素ε-环化酶（LYCE）基因启动子的功能分析
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摘要
高等植物类胡萝卜素是在细胞质体内通过类异戊二烯途径合成而呈现黄色、橙红色和红色的一大类色素物质。类胡萝卜素是人类饮食结构中不可缺少的重要成分,它对人体健康有重要的生物功能,它不仅是维生素A合成的前体物质,而且在视觉保护、降低癌症和心血管疾病的发病率、提高人体免疫力等方面有重要作用。
目前,通过类胡萝卜素基因工程获得“金水稻”、“金油菜”和“金马铃薯”等极大地增强了人们开展植物类胡萝卜素基因工程的信心。然而,人们对植物体内类胡萝卜素生物合成的调控机制以及类胡萝卜素生物合成基因的表达调控机制还不甚清楚。八氢番茄红素脱氢酶(PDS)和番茄红素ε-环化酶(LYCE)都是类胡萝卜素生物合成途径的关键调节酶,解析PDS和LYCE基因启动子的功能,不仅为阐明类胡萝卜素基因表达调控机制提供了理论依据,而且获得的组织或器官特异性表达启动子可应用于植物品质改良及园艺植物花颜色改变的分子育种等。因此对龙胆PDS和LYCE基因启动子的功能分析具有重要的理论意义和潜在的应用价值。
朱长甫老师利用反向PCR技术,成功地从龙胆基因组中分离到PDS和LYCE基因启动子序列。应用PLACE和PlantCare数据库对这两个基因启动子进行顺式作用元件分析,结果表明:龙胆PDS基因启动子序列长1077 bp,在启动子上游-68 bp处有一个预测的TATA盒;龙胆LYCE基因启动子序列长938 bp,在启动子上游-69 bp处有一个预测的TATA盒;此外通过对比这两个启动子的顺式作用元件发现,二者都具有增强基因转录作用的CAAT盒,光调控顺式作用元件盒I、G盒、I盒、ATC元件,脱落酸(ABA)调控顺式作用元件ABRE等。推测这两个启动子相似的顺式作用元件可能对基因调控有相似的功能。
应用双元载体pBI101系统,分别将龙胆PDS和LYCE基因启动子与GUS基因融合构建成重组质粒。应用农杆菌介导法,使重组质粒在番茄果实和叶组织中瞬时表达,研究结果表明:在叶片和含有大量叶绿体的未成熟的绿色番茄果实中,PDS和LYCE基因启动子驱动的GUS基因几乎不表达,伴随着果实的发育,果实中的叶绿体逐渐转化发育成有色体,PDS和LYCE基因启动子驱动的GUS基因表达量逐渐增强,在含有有色体的成熟番茄果实中表达量最大。推测PDS和LYCE基因启动子的表达与有色体的形成及果实的发育密切相关。为进一步探究PDS基因启动子在基因表达调控中的作用,本实验应用农杆菌介导法,使融合基因GlPDSPro:GUS在番茄植株中稳定表达,通过对转基因番茄基因组DNA的PCR检测,证实已经获得了转基因植物。
Carotenoids are a large class of yellow, orange and red pigments, which are synthesized within plastids in higher plants through isoprenoid biosynthetic pathway. Carotenoids are important nutritional composition in human diets and have important biological functions in human health. They are not only the precursors of vitamin A, but also playing important roles in protecting vision, decreasing the incidence of varieties of cancers and cardiovascular diseases, enhancing immune ability and so on.
At present,“golden rice”,“golden canola”and“golden potato”have already been abtained through carotenoid genetic engineering. These achievements greatly enhanced the confidence in developing carotenoid genetic engineering in higher plants. However, a major limitation is our poor knowledge of the regulation mechanisms of carotenoid biosynthetic pathway and the expression regulation mechanisms of carotenogenic genes in higher plants.
Phytoene desaturase (PDS) and Lycopene epsilon-cyclase (LYCE) are key regulatory enzymes in carotenoid biosynthetic pathway. Characterizing the functions of PDS and LYCE promoters could provide theoretic evidence in elucidating the molecular control mechanisms of carotenogenic gene expression. The abtained tissue or organ specific promoters could be used in the molecule breeding of nutritional improvement in major staple crops and flower colour modification in horticultural plants. Therefore, functional characterization of gentian (Gentiana lutea) PDS and LYCE gene promoters not only has great theoretical significance, but also has potential practical application value.
The promoter fragments for gentian PDS and LYCE genes were cloned from genomic DNA by the inverse polymerase chain reaction (Zhu C., unpublished data). Using PLACE and PlantCare databases to analysis promoter cis-acting elements indicated that the full-length of PDS promoter (1077 bp) contained a putative TATA box located in the up-stream (-68 bp) of 3′end of promoter, the full-length of LYCE promoter (938 bp) contained a putative TATA box located in the up-stream (-69 bp) of 3′end of promoter. Furthermore, both of these two promoters had CAAT boxes whose function was enhancing gene transcription, light regulation cis-acting element box I, G box, I box, ATC motif, ABA regulation cis-acting element ABRE and so on. The similar cis-acting elements existed in PDS and LYCE gene promoters indicated that these two promoters might have similar functions in regulating gene expression.
The full-length promoter fragments were fused to a gene encoding beta-glucuronidase (GUS) and the fusion constructs were introduced into tomato fruits and leaves by Agrobacterium-mediated transient transformation. The results of GUS transient expression indicated that PDS and LYCE gene promoters did not drive the GUS gene expression in chloroplasts-containing leaves and immature green fruits of tomato. During the development of tomato fruit, the transition from chloroplasts to chromoplasts occur, expression levels of the GUS gene driven by PDS and LYCE gene promoters gradually increased, the highest expression levels of GUS were found in mature chromoplast-containing fruits. This revealed that the expression of PDS and LYCE gene promoters was associated with chromoplast formation and the development of fruits of tomato. In order to extensively study the function of PDS gene promoter in tomato, fusion gene of GlPDSPro:GUS was introduced into tomato by Agrobacterium-mediated stable transformation. We extracted genomic DNA from transgenic tomato plants. Genomic DNA PCR analysis confirmed that stable transgenic tomato plants were obtained.
目前,通过类胡萝卜素基因工程获得“金水稻”、“金油菜”和“金马铃薯”等极大地增强了人们开展植物类胡萝卜素基因工程的信心。然而,人们对植物体内类胡萝卜素生物合成的调控机制以及类胡萝卜素生物合成基因的表达调控机制还不甚清楚。八氢番茄红素脱氢酶(PDS)和番茄红素ε-环化酶(LYCE)都是类胡萝卜素生物合成途径的关键调节酶,解析PDS和LYCE基因启动子的功能,不仅为阐明类胡萝卜素基因表达调控机制提供了理论依据,而且获得的组织或器官特异性表达启动子可应用于植物品质改良及园艺植物花颜色改变的分子育种等。因此对龙胆PDS和LYCE基因启动子的功能分析具有重要的理论意义和潜在的应用价值。
朱长甫老师利用反向PCR技术,成功地从龙胆基因组中分离到PDS和LYCE基因启动子序列。应用PLACE和PlantCare数据库对这两个基因启动子进行顺式作用元件分析,结果表明:龙胆PDS基因启动子序列长1077 bp,在启动子上游-68 bp处有一个预测的TATA盒;龙胆LYCE基因启动子序列长938 bp,在启动子上游-69 bp处有一个预测的TATA盒;此外通过对比这两个启动子的顺式作用元件发现,二者都具有增强基因转录作用的CAAT盒,光调控顺式作用元件盒I、G盒、I盒、ATC元件,脱落酸(ABA)调控顺式作用元件ABRE等。推测这两个启动子相似的顺式作用元件可能对基因调控有相似的功能。
应用双元载体pBI101系统,分别将龙胆PDS和LYCE基因启动子与GUS基因融合构建成重组质粒。应用农杆菌介导法,使重组质粒在番茄果实和叶组织中瞬时表达,研究结果表明:在叶片和含有大量叶绿体的未成熟的绿色番茄果实中,PDS和LYCE基因启动子驱动的GUS基因几乎不表达,伴随着果实的发育,果实中的叶绿体逐渐转化发育成有色体,PDS和LYCE基因启动子驱动的GUS基因表达量逐渐增强,在含有有色体的成熟番茄果实中表达量最大。推测PDS和LYCE基因启动子的表达与有色体的形成及果实的发育密切相关。为进一步探究PDS基因启动子在基因表达调控中的作用,本实验应用农杆菌介导法,使融合基因GlPDSPro:GUS在番茄植株中稳定表达,通过对转基因番茄基因组DNA的PCR检测,证实已经获得了转基因植物。
Carotenoids are a large class of yellow, orange and red pigments, which are synthesized within plastids in higher plants through isoprenoid biosynthetic pathway. Carotenoids are important nutritional composition in human diets and have important biological functions in human health. They are not only the precursors of vitamin A, but also playing important roles in protecting vision, decreasing the incidence of varieties of cancers and cardiovascular diseases, enhancing immune ability and so on.
At present,“golden rice”,“golden canola”and“golden potato”have already been abtained through carotenoid genetic engineering. These achievements greatly enhanced the confidence in developing carotenoid genetic engineering in higher plants. However, a major limitation is our poor knowledge of the regulation mechanisms of carotenoid biosynthetic pathway and the expression regulation mechanisms of carotenogenic genes in higher plants.
Phytoene desaturase (PDS) and Lycopene epsilon-cyclase (LYCE) are key regulatory enzymes in carotenoid biosynthetic pathway. Characterizing the functions of PDS and LYCE promoters could provide theoretic evidence in elucidating the molecular control mechanisms of carotenogenic gene expression. The abtained tissue or organ specific promoters could be used in the molecule breeding of nutritional improvement in major staple crops and flower colour modification in horticultural plants. Therefore, functional characterization of gentian (Gentiana lutea) PDS and LYCE gene promoters not only has great theoretical significance, but also has potential practical application value.
The promoter fragments for gentian PDS and LYCE genes were cloned from genomic DNA by the inverse polymerase chain reaction (Zhu C., unpublished data). Using PLACE and PlantCare databases to analysis promoter cis-acting elements indicated that the full-length of PDS promoter (1077 bp) contained a putative TATA box located in the up-stream (-68 bp) of 3′end of promoter, the full-length of LYCE promoter (938 bp) contained a putative TATA box located in the up-stream (-69 bp) of 3′end of promoter. Furthermore, both of these two promoters had CAAT boxes whose function was enhancing gene transcription, light regulation cis-acting element box I, G box, I box, ATC motif, ABA regulation cis-acting element ABRE and so on. The similar cis-acting elements existed in PDS and LYCE gene promoters indicated that these two promoters might have similar functions in regulating gene expression.
The full-length promoter fragments were fused to a gene encoding beta-glucuronidase (GUS) and the fusion constructs were introduced into tomato fruits and leaves by Agrobacterium-mediated transient transformation. The results of GUS transient expression indicated that PDS and LYCE gene promoters did not drive the GUS gene expression in chloroplasts-containing leaves and immature green fruits of tomato. During the development of tomato fruit, the transition from chloroplasts to chromoplasts occur, expression levels of the GUS gene driven by PDS and LYCE gene promoters gradually increased, the highest expression levels of GUS were found in mature chromoplast-containing fruits. This revealed that the expression of PDS and LYCE gene promoters was associated with chromoplast formation and the development of fruits of tomato. In order to extensively study the function of PDS gene promoter in tomato, fusion gene of GlPDSPro:GUS was introduced into tomato by Agrobacterium-mediated stable transformation. We extracted genomic DNA from transgenic tomato plants. Genomic DNA PCR analysis confirmed that stable transgenic tomato plants were obtained.
引文
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