番木瓜类胡萝卜素生物合成途径部分酶基因cDNA的克隆与分析
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摘要
番木瓜(Carica papaya L.)是热带、亚热带地区广泛种植的果树,是类胡萝卜素的重要植物资源。类胡萝卜素是具有广泛生物功能的一大类重要的植物色素。本研究以红肉番木瓜‘马来10号’果肉为材料,利用RACE和RT-PCR技术克隆类胡萝卜素生物合成途径中八氢番茄红素脱氢酶基因(phytoene desaturase,PDS)、ζ-胡萝卜素脱氢酶基因(ζ-carotene desaturase,ZDS)和番茄红素β-环化酶基因(lycopene-β-cyclase,LCY-B)的cDNA序列,并采用半定量RT-PCR法分析该途径中八氢番茄红素合成酶基因(phytoene synthase,PSY)、PDS、ZDS和LCY-B四基因在果实不同发育时期的表达水平。本研究为阐明番木瓜果肉颜色形成的分子机理奠定了基础,也在分子水平充实了类胡萝卜素生物合成的理论研究。
1利用RACE和RT-PCR技术克隆番木瓜PDS基因cDNA全长
利用GenBank已登录的番木瓜PDS基因片段,通过RACE技术获得其5′端和3′端序列,从而得到番木瓜PDS基因cDNA(GenBank登录号为DQ666830)全长2202bp,最大开放读码框为1752bp,编码583个氨基酸,预测相对分子量为65110.0,等电点为6.26,5′端有223bp的非翻译区,3′端包含227bp的非编码序列;根据cDNA全长推导氨基酸序列与番茄、温州蜜柑、玉米等高等植物PDS基因的氨基酸序列进行同源性对比分析并构建系统进化树。
2利用RACE和RT-PCR技术克隆番木瓜ZDS基因cDNA全长
根据GenBank已登录的番木瓜ZDS基因3′端序列,通过RACE技术获得其5′端序列,得到番木瓜ZDS基因cDNA(GenBank登录号为DQ666829)全长1951bp,最大开放读码框为1659bp,编码552个氨基酸,预测分子量为60888.8,等电点为7.52,5′端有25bp长的非翻译区,3′端包含267bp的非编码序列;根据cDNA全长推导氨基酸序列与温州蜜柑、拟南芥、玉米等高等植物ZDS基因的氨基酸序列进行同源性分析并构建系统进化树。
3利用RT-PCR技术克隆番木瓜LCY-B基因的ORF
依据GenBank已登录的番木瓜LCY-B基因的DNA全长,通过RT-PCR技术获得其ORF序列(GenBank登录号为FJ599643)为1613bp,最大开放读码框为1512bp,编码503个氨基酸,预测分子量为56736.5,等电点为6.62,5′端有76bp长的非翻译区,3′端包含25bp的非编码序列;根据cDNA全长推导氨基酸序列与温州蜜柑、番茄、水仙等高等植物LCY-B基因的氨基酸序列进行同源性分析并构建系统进化树。
4利用半定量RT-PCR分析番木瓜PSY、PDS、ZDS和LCY-B基因的表达
以番木瓜Actin基因为阳性对照,检测番木瓜PSY、PDS、ZDS和LCY-B四基因在果实膨大结束期、果皮破色期、果皮50%转黄期和果皮全黄期四个时期的表达水平,结果表明四基因在四个时期均有表达,且在果实发育期表达量都呈先升后降的趋势,说明了这些酶基因在番木瓜类胡萝卜素代谢中不可缺少。四个酶基因的表达水平基本在同一时期达到最大且在果实成熟后期均趋于下降,说明它们有着密切的协作关系。
Papaya(Carica papaya L.) is a fruit crop cultivated in tropical and sub-tropical regions worldwide and rich in carotenoids. Carotenoids are a large class of plant pigments,which have a wide range of biological functions. In this study, the cDNA sequence of phytoene desaturase(PDS)、ζ-carotene desaturase(ZDS) and lycopene-β-cyclase(LCY-B) of carotenoid biosynthetic pathway in red flesh of‘NO.10 ma lai′papaya were cloned by RACE and RT-PCR, and the expresstion patterns of phytoene synthase(PSY)、PDS、ZDS and LCY-B in different developmental stages of papaya fruits were analyzed by semi quantitative RT-PCR.This study could provid the the theoretical basis for the molecular mechanism of carotenoids biosynthesis and of differnial flesh color in papaya.
1 Cloning and analysis of the full length cDNA of CpPDS by RACE and RT-PCR
Obtained the 5′and 3′end sequence by RACE,using CpPDS fragment from GenBank.And the full length cDNA of CpPDS(Genbank accession number DQ666830) is 2202bp, it contains a largest ORF that is 1752bp encoding 583 amino acids (Mw= 65110.0, pI=6.26),a 5′untranslated region of 223 bp and a 3′flanking sequence of 227 bp. The deduced amino acid sequence of CpPDS was compared with the sequences of other higher plants such as Solanum lycopersicum, Citrus unshiu, Zea mays,and so on.
2 Cloning and analysis of the full length cDNA of CpZDS by RACE and RT-PCR
Obtained the 5′nucleotide sequence by RACE, using CpZDS 3′nucleotide seqence from GenBank. And the full length cDNA of CpZDS(Genbank accession number DQ666829) is 1951bp, it contains a largest ORF that is 1659bp encoding 552 amino acids (Mw=60888.8, pI=7.52),a 5′untranslated region of 25 bp and a 3′flanking sequence of 267 bp. The deduced amino acid sequence of CpZDS was compared the sequences of other higher plants such as Citrus unshiu, Arabidopsis thaliana, Zea mays, and so on.
3 Cloning and analysis of the ORF of CpLCY-B by RT-PCR
Obtained the ORF(Genbank accession number FJ599643) by RT-PCR, using the full length DNA of CpLCY-B from GenBank.And the nucleotide sequence is 1613bp, it contains a largest ORF that is 1512bp encoding 503 amino acids (Mw=56736.5, pI=6.62),a 5′untranslated region of 76 bp and a 3′flanking sequence of 25 bp. The deduced amino acid sequence of CpLCY-B was compared with the sequences of other higher plants such as Citrus unshiu, Solanum lycopersicum, Narcissus pseudonarcissus, and so on.
4 Analysis the expression of CpPSY, CpPDS, CpZDS and CpLCY-B by sqRT-PCR
The expression patterns of CpPSY, CpPDS, CpZDS and CpLCY-B in the expansion stage, color break stage, 50% ripening stage and full ripening stage were carried out and CpActin was used as positive control. sqRT-PCR indicated that all of the four enzyme genes expressed in different developmental stages of papaya fruit with higher expression level the intermediate stages. So it shows that these enzyme genes are indispensable in carotenoids metabolism, and this is the manifestation of their importance. The levels of the four genes′expression reach the maximum during the same period and all in the downward trend in full ripening stage .It shows that they have close collaborative relationship.
1利用RACE和RT-PCR技术克隆番木瓜PDS基因cDNA全长
利用GenBank已登录的番木瓜PDS基因片段,通过RACE技术获得其5′端和3′端序列,从而得到番木瓜PDS基因cDNA(GenBank登录号为DQ666830)全长2202bp,最大开放读码框为1752bp,编码583个氨基酸,预测相对分子量为65110.0,等电点为6.26,5′端有223bp的非翻译区,3′端包含227bp的非编码序列;根据cDNA全长推导氨基酸序列与番茄、温州蜜柑、玉米等高等植物PDS基因的氨基酸序列进行同源性对比分析并构建系统进化树。
2利用RACE和RT-PCR技术克隆番木瓜ZDS基因cDNA全长
根据GenBank已登录的番木瓜ZDS基因3′端序列,通过RACE技术获得其5′端序列,得到番木瓜ZDS基因cDNA(GenBank登录号为DQ666829)全长1951bp,最大开放读码框为1659bp,编码552个氨基酸,预测分子量为60888.8,等电点为7.52,5′端有25bp长的非翻译区,3′端包含267bp的非编码序列;根据cDNA全长推导氨基酸序列与温州蜜柑、拟南芥、玉米等高等植物ZDS基因的氨基酸序列进行同源性分析并构建系统进化树。
3利用RT-PCR技术克隆番木瓜LCY-B基因的ORF
依据GenBank已登录的番木瓜LCY-B基因的DNA全长,通过RT-PCR技术获得其ORF序列(GenBank登录号为FJ599643)为1613bp,最大开放读码框为1512bp,编码503个氨基酸,预测分子量为56736.5,等电点为6.62,5′端有76bp长的非翻译区,3′端包含25bp的非编码序列;根据cDNA全长推导氨基酸序列与温州蜜柑、番茄、水仙等高等植物LCY-B基因的氨基酸序列进行同源性分析并构建系统进化树。
4利用半定量RT-PCR分析番木瓜PSY、PDS、ZDS和LCY-B基因的表达
以番木瓜Actin基因为阳性对照,检测番木瓜PSY、PDS、ZDS和LCY-B四基因在果实膨大结束期、果皮破色期、果皮50%转黄期和果皮全黄期四个时期的表达水平,结果表明四基因在四个时期均有表达,且在果实发育期表达量都呈先升后降的趋势,说明了这些酶基因在番木瓜类胡萝卜素代谢中不可缺少。四个酶基因的表达水平基本在同一时期达到最大且在果实成熟后期均趋于下降,说明它们有着密切的协作关系。
Papaya(Carica papaya L.) is a fruit crop cultivated in tropical and sub-tropical regions worldwide and rich in carotenoids. Carotenoids are a large class of plant pigments,which have a wide range of biological functions. In this study, the cDNA sequence of phytoene desaturase(PDS)、ζ-carotene desaturase(ZDS) and lycopene-β-cyclase(LCY-B) of carotenoid biosynthetic pathway in red flesh of‘NO.10 ma lai′papaya were cloned by RACE and RT-PCR, and the expresstion patterns of phytoene synthase(PSY)、PDS、ZDS and LCY-B in different developmental stages of papaya fruits were analyzed by semi quantitative RT-PCR.This study could provid the the theoretical basis for the molecular mechanism of carotenoids biosynthesis and of differnial flesh color in papaya.
1 Cloning and analysis of the full length cDNA of CpPDS by RACE and RT-PCR
Obtained the 5′and 3′end sequence by RACE,using CpPDS fragment from GenBank.And the full length cDNA of CpPDS(Genbank accession number DQ666830) is 2202bp, it contains a largest ORF that is 1752bp encoding 583 amino acids (Mw= 65110.0, pI=6.26),a 5′untranslated region of 223 bp and a 3′flanking sequence of 227 bp. The deduced amino acid sequence of CpPDS was compared with the sequences of other higher plants such as Solanum lycopersicum, Citrus unshiu, Zea mays,and so on.
2 Cloning and analysis of the full length cDNA of CpZDS by RACE and RT-PCR
Obtained the 5′nucleotide sequence by RACE, using CpZDS 3′nucleotide seqence from GenBank. And the full length cDNA of CpZDS(Genbank accession number DQ666829) is 1951bp, it contains a largest ORF that is 1659bp encoding 552 amino acids (Mw=60888.8, pI=7.52),a 5′untranslated region of 25 bp and a 3′flanking sequence of 267 bp. The deduced amino acid sequence of CpZDS was compared the sequences of other higher plants such as Citrus unshiu, Arabidopsis thaliana, Zea mays, and so on.
3 Cloning and analysis of the ORF of CpLCY-B by RT-PCR
Obtained the ORF(Genbank accession number FJ599643) by RT-PCR, using the full length DNA of CpLCY-B from GenBank.And the nucleotide sequence is 1613bp, it contains a largest ORF that is 1512bp encoding 503 amino acids (Mw=56736.5, pI=6.62),a 5′untranslated region of 76 bp and a 3′flanking sequence of 25 bp. The deduced amino acid sequence of CpLCY-B was compared with the sequences of other higher plants such as Citrus unshiu, Solanum lycopersicum, Narcissus pseudonarcissus, and so on.
4 Analysis the expression of CpPSY, CpPDS, CpZDS and CpLCY-B by sqRT-PCR
The expression patterns of CpPSY, CpPDS, CpZDS and CpLCY-B in the expansion stage, color break stage, 50% ripening stage and full ripening stage were carried out and CpActin was used as positive control. sqRT-PCR indicated that all of the four enzyme genes expressed in different developmental stages of papaya fruit with higher expression level the intermediate stages. So it shows that these enzyme genes are indispensable in carotenoids metabolism, and this is the manifestation of their importance. The levels of the four genes′expression reach the maximum during the same period and all in the downward trend in full ripening stage .It shows that they have close collaborative relationship.
引文
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