多花水仙类胡萝卜素生物合成途径部分酶基因的克隆与分析
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摘要
多花水仙(Narcissus tazettaL.)是石蒜科水仙属植物，由于多花水仙品种匮乏、花色单一，限制了产业的发展。本研究以金盏银台、白花水仙和黄花水仙为材料，利用RT-PCR和RACE技术克隆类胡萝卜素生物合成途径中八氢番茄红素合成酶基因(PSY)、八氢番茄红素脱氢酶基因(PDS)、ζ-胡萝卜素脱氢酶基因(ZDS)、番茄红素β-环化酶基因(LCY-β)和番茄红素ε-环化酶基因(LCY-ε)；并通过qRT-PCR分析该途径中PSY、PDS、ZDS和LCY-β四基因在花色形成中转录水平。本研究充实了多花水仙花色形成分子机理的背景资料，对多花水仙的花色育种探索具有重要意义。
1多花水仙PSY基因的克隆利用GenBank已登录的中国水仙PSY基因的cDNA序列，通过RT-PCR技
术获得三种花色类型PSY基因的ORF序列为1272-1275bp、编码423-424个氨基酸：分别命名为NtPSYJ、NtPSYW(登录号：JN991013)和NtPSYY(JN991014)。水仙属植物PSY推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异和缺失。以多花水仙基因组DNA为模板，获得三种花色类型PSY的基因组序列2667-2840bp：登录号为JN991017(金盏银台)、JN991016(白花水仙)和JN991015(黄花水仙)；均含有5个内含子(A-E)、变化范围在81-926bp。序列分析发现：A、B、D、E内含子的剪切位点均符合GT-AG规律，而C内含子符合GC-AG规律。
2多花水仙PDS基因的克隆利用GenBank已登录的中国水仙PDS基因的cDNA序列，通过RT-PCR技
术获得三种花色类型PDS基因的ORF序列都为1713bp、编码570个氨基酸：分别命名为NtPDSJ、NtPDSW(登录号：JQ797376)和NtPDSY(JQ797377)。水仙属植物PDS推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板，获得三种花色类型PDS的部分基因组序列7167-7199bp：登录号为JQ797382(金盏银台)、JQ797383(白花水仙)和JQ797384(黄花水仙)；都含有10个内含子(A-J)且符合GT-AG规律、变化范围在73-1489bp。
3多花水仙ZDS基因的克隆利用GenBank已登录的中国水仙ZDS基因的cDNA序列，通过RT-PCR技术获得三种花色类型ZDS基因的ORF序列都为1725bp、编码574个氨基酸：分
别命名为NtZDSJ、NtZDSW(登录号：JQ797378)和NtZDSY(JQ797379)。水仙属植物ZDS推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板，获得三种花色类型ZDS的部分基因组序列7382-9187bp：登录号为JQ797385(金盏银台)、JQ797386(黄花水仙)。序列分析发现：都含有11个内含子(A-K)且符合GT-AG规律、变化范围在87-2695bp，其中A、C、F内含子的大小存在较大差异。
4多花水仙LCY-β基因的克隆
利用GenBank已登录的中国水仙LCY-β基因的cDNA序列，通过RT-PCR技术获得三种花色类型LCY-β基因的ORF序列都为1715bp、编码504个氨基酸：分别命名为NtLCY-βJ、NtLCY-βW(登录号：JQ797380)和NtLCY-βY(JQ797381)。水仙属植物LCY-β推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板克隆LCY-β的基因组序列，发现三种花色类型LCY-β基因编码区内没有内含子；与cDNA序列比较同源率达98%以上。
5多花水仙LCY-ε基因的克隆
根据已知的中国水仙LCY-ε基因3′端序列，通过RACE技术获得其5′端序列，得到金盏银台LCY-ε基因cDNA全长1971bp，最大开放读码框为1599bp(GenBank登录号：JQ282903)，编码532个氨基酸，预测分子量为59771.1，等电点为6.15，5′端有153bp长的非翻译区，3′端包含111bp的非编码序列。根据金盏银台ORF设计引物，获得白花水仙和黄花水仙LCY-ε的ORF序列(JQ282902、JQ282904)。水仙属植物LCY-ε推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异；根据cDNA全长推导氨基酸序列与柑橘、玉米、番茄等高等植物LCY-ε基因的氨基酸序列进行同源性分析并构建系统进化树。
6qRT-PCR分析多花水仙花色形成中PSY、PDS、ZDS和LCY-B基因的表达
以多花水仙ACTIN基因为内参基因，检测三种花色PSY、PDS、ZDS和LCY-β四基因在花苞期、花蕾期、始花期和盛花期的表达水平，结果表明金盏银台与黄花水仙四基因在在花色形成中均有表达，表达量都呈先升后降的趋势，高峰都出现在始花期或者盛花期；而白花水仙四个基因在不同时期的转录趋势大体上都是减弱的，尤其是PSY几乎不表达的；虽然白花水仙PDS和ZDS在个别时期还有上升趋势，但是相对于其它花色相对表达量而言都不高。表明随着PSY、PDS、ZDS和LCY-β表达量的相应增加，多花水仙花色由花瓣白色到花瓣黄色的转变，因此调控这些基因可以改良多花水仙的花色。
Narcissus tazetta-L.is a member of the family Amaryllidaceae. The development of the narcissus industry was restricted for the critical problems of shortages in available resource and colorful varietie. Using Jinzhanyintaim, Baihua narcissus and Huanghua narcissus as test materials, the full-length cDNA and DNA sequence of phytoene synthase(PSY), phytoene desaturase(PDS), ζ-carotene desaturase(ZDS), lycopene-β-cyclase (LCY-β) and lycopene-ε-cyclase(LCY-e) were cloned by RACE-PCR and RT-PCR in this study. The gene expression patterns in the color formation of chinese narcissus were also analyzed by Real Time PCR. The study will provide a foundation to know more about the color formation of Narcissus tazetta L., as well as facilitate color-enriched and productivity-enhanced development of the narcissus.The major results obtained from the study were summarized as followed:
1Cloning and analysis of NtPSYgene from Narcissus tazetta L.
The open reading frame (ORF) of PSY were obtained from the three kinds of color type narcissus, the ORF length of NtPSYwas1272bp-1275bp encoding423-424amino acids. The genes were designated as NtPSYJ,NtPSYW(GenBank accession No.JN991013) and NtPSYY(JN991014) respectively. Comparing with other PSY genes in Narcissus genus, there were some replacements and missing amino acids. The genome DNA sequence of PSY genes in three color type narcissus were cloned, the length ranged from2667bp to2840bp, the GenBank accession numbers were No.JN991017(Jinzhanyintai), No.JN991016(Baihua) and No.JN991015(Huanghua). Five introns (A-E) were found in each DNA sequence, the length ranged from81bp to926bp. Sequence nalysis showed that the splicing sites of four introns (A、B、D、E) comformed to the GT-AG rule and introns C were consistented with GC-AG rule.
2Cloning and analysis of NtPDS from Narcissus tazetta L,.
The open reading frame (ORF) of PDS were obtained from the three kinds of color type narcissus, The ORF length of PDS were1713bp encoding570amino acids. The genes were designated as NtPDSJ, NtPDSW(accession number:JQ797376) and N/PDSY (JQ797377) respectively. Comparing with other PDS genes in Narcissus genus, there were some replacements and missing amino acids as well. The part of genome DNA sequence of PDSgenes in the three color type narcissus were cloned, a length ranged from7167bp to7199bp, and the GenBank accession number are No. JQ797382(Jinzhanyintai), No. JQ797383(Baihua) and No. JQ797384(Huanghua).Ten intons(A-J) were found in each DNA sequence, the length ranged from73bp to1489bp. Sequence analysis showed that the splicing sites of introns comformed to the GT-AG rule.
3Cloning and analysis of NtZDS from Narcissus tazetta L.
The open reading frame (ORF) of ZDSwas obtained from the three kinds of color type narcissus, the ORF length of Nt ZDS was1725bp encoding574amino acids. The genes were designated as NtZDSJ, NtZDSW(GenBank accession No. JQ797378) and NtZDSY(JQ797379) respectively. Comparing with other ZDS genes in Narcissus genus, there were some replacements and missing amino acids. The part of genome DNA sequence of ZDS genes in the three color type narcissus were cloned, the length ranged from7382bp to9187bp, the GenBank accession number are No. JQ797385(Jinzhanyintai), No. JQ797386(Huanghua). Eleven introns (A-K) were found in each DNA sequence, the length ranged from87bp to2695bp. Sequence analysis showed that the splicing sites of four introns comformed to the GT-AG rule and intron A, C, F size are quite different.
4Cloning and analysis of NtLCY-βfrom Narcissus tazetta L.
The open reading frame (ORF) of LCY-βwere obtained from the three kinds of color type narcissus, the ORF length of NtLCY-β were1715bp encoding504amino acids. The genes were designated as NtLCY-βJ, NtLCY-βW(GenBank accession No. JQ797380) and MICY-βY (JQ797381) respectively. Comparing with other LCY-β genes in Narcissus genus, there were replacement and missing amino acid. Cloning from genomic DNA sequence of NtLCY-β showed that was no intron and the homologous rate are over98%compared with their cDNA.
5Cloning and analysis of NtLCY-ε
5'RACE primers were designed according to the known NtLCY-ε3'nucleotide seqence from GenBank, the5'nucleotide sequence was obtained by RACE from Jinzhanyintai narcissus. The full length cDNA of NtLCY-ε was1971bp with an opening reading frame of1659bp (GenBank accession No.JQ282903) encoding a protein with532amino acids(Mw=59771.1, pI=6.15). The5-terminal untranslated region (UTR) was153bp and the3-UTR was lllbp. The ORF sequence of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). According to the ORF sequence of LCY-ε gene in Jinzhanyintai, we obtained the ORF sequences of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). The nucleotide and amino acids from the three kinds of color type narcissus were similar. Replacements were found in the deduced amino acid sequence of LCY-ε gene in Narcissus genus plant with homologous comparison. The deduced amino acid sequence of NtLCY-ε was compared with that of other higher plants and phylogenetic tree was was constructed.
4Analysis the expression of PSY、PDS、ZDS and LCY-βin the color formation of Narcissus tazetta L. by qRT-PCR
NtACTIN was used as internal control gene, the expression patterns of NtPSY, NtPDS, NtZDS and NtLCY-β in the bud stage, alabastrum stage, early flowering stage and full-bloom stage were carried out. The results indicated that all of the four genes in Jinzhanyintai and Huanghua narcissus expressed in four stages, with higher expression level in the intermediate stages, while much lower in Baihua narcissus, especially the expression of PSY gene is almost undetectable. Additionally, the expression rate of PDS and ZDS in Baihua narcissus appears to be increasing at certain stage, but it still lower than that of other flowers. To sum up, the higher expression level of PSY、PDS、ZDS and LCY-β in Jinzhanyintai and Huanghua narcissus may cause the yellow color of the flower. Therefore, the regulation of these genes may regulate and control the color of Narcissus tazetta L. flower.
1多花水仙PSY基因的克隆利用GenBank已登录的中国水仙PSY基因的cDNA序列，通过RT-PCR技
术获得三种花色类型PSY基因的ORF序列为1272-1275bp、编码423-424个氨基酸：分别命名为NtPSYJ、NtPSYW(登录号：JN991013)和NtPSYY(JN991014)。水仙属植物PSY推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异和缺失。以多花水仙基因组DNA为模板，获得三种花色类型PSY的基因组序列2667-2840bp：登录号为JN991017(金盏银台)、JN991016(白花水仙)和JN991015(黄花水仙)；均含有5个内含子(A-E)、变化范围在81-926bp。序列分析发现：A、B、D、E内含子的剪切位点均符合GT-AG规律，而C内含子符合GC-AG规律。
2多花水仙PDS基因的克隆利用GenBank已登录的中国水仙PDS基因的cDNA序列，通过RT-PCR技
术获得三种花色类型PDS基因的ORF序列都为1713bp、编码570个氨基酸：分别命名为NtPDSJ、NtPDSW(登录号：JQ797376)和NtPDSY(JQ797377)。水仙属植物PDS推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板，获得三种花色类型PDS的部分基因组序列7167-7199bp：登录号为JQ797382(金盏银台)、JQ797383(白花水仙)和JQ797384(黄花水仙)；都含有10个内含子(A-J)且符合GT-AG规律、变化范围在73-1489bp。
3多花水仙ZDS基因的克隆利用GenBank已登录的中国水仙ZDS基因的cDNA序列，通过RT-PCR技术获得三种花色类型ZDS基因的ORF序列都为1725bp、编码574个氨基酸：分
别命名为NtZDSJ、NtZDSW(登录号：JQ797378)和NtZDSY(JQ797379)。水仙属植物ZDS推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板，获得三种花色类型ZDS的部分基因组序列7382-9187bp：登录号为JQ797385(金盏银台)、JQ797386(黄花水仙)。序列分析发现：都含有11个内含子(A-K)且符合GT-AG规律、变化范围在87-2695bp，其中A、C、F内含子的大小存在较大差异。
4多花水仙LCY-β基因的克隆
利用GenBank已登录的中国水仙LCY-β基因的cDNA序列，通过RT-PCR技术获得三种花色类型LCY-β基因的ORF序列都为1715bp、编码504个氨基酸：分别命名为NtLCY-βJ、NtLCY-βW(登录号：JQ797380)和NtLCY-βY(JQ797381)。水仙属植物LCY-β推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异。以多花水仙基因组DNA为模板克隆LCY-β的基因组序列，发现三种花色类型LCY-β基因编码区内没有内含子；与cDNA序列比较同源率达98%以上。
5多花水仙LCY-ε基因的克隆
根据已知的中国水仙LCY-ε基因3′端序列，通过RACE技术获得其5′端序列，得到金盏银台LCY-ε基因cDNA全长1971bp，最大开放读码框为1599bp(GenBank登录号：JQ282903)，编码532个氨基酸，预测分子量为59771.1，等电点为6.15，5′端有153bp长的非翻译区，3′端包含111bp的非编码序列。根据金盏银台ORF设计引物，获得白花水仙和黄花水仙LCY-ε的ORF序列(JQ282902、JQ282904)。水仙属植物LCY-ε推导的氨基酸序列同源比对发现，氨基酸存在个别位置的差异；根据cDNA全长推导氨基酸序列与柑橘、玉米、番茄等高等植物LCY-ε基因的氨基酸序列进行同源性分析并构建系统进化树。
6qRT-PCR分析多花水仙花色形成中PSY、PDS、ZDS和LCY-B基因的表达
以多花水仙ACTIN基因为内参基因，检测三种花色PSY、PDS、ZDS和LCY-β四基因在花苞期、花蕾期、始花期和盛花期的表达水平，结果表明金盏银台与黄花水仙四基因在在花色形成中均有表达，表达量都呈先升后降的趋势，高峰都出现在始花期或者盛花期；而白花水仙四个基因在不同时期的转录趋势大体上都是减弱的，尤其是PSY几乎不表达的；虽然白花水仙PDS和ZDS在个别时期还有上升趋势，但是相对于其它花色相对表达量而言都不高。表明随着PSY、PDS、ZDS和LCY-β表达量的相应增加，多花水仙花色由花瓣白色到花瓣黄色的转变，因此调控这些基因可以改良多花水仙的花色。
Narcissus tazetta-L.is a member of the family Amaryllidaceae. The development of the narcissus industry was restricted for the critical problems of shortages in available resource and colorful varietie. Using Jinzhanyintaim, Baihua narcissus and Huanghua narcissus as test materials, the full-length cDNA and DNA sequence of phytoene synthase(PSY), phytoene desaturase(PDS), ζ-carotene desaturase(ZDS), lycopene-β-cyclase (LCY-β) and lycopene-ε-cyclase(LCY-e) were cloned by RACE-PCR and RT-PCR in this study. The gene expression patterns in the color formation of chinese narcissus were also analyzed by Real Time PCR. The study will provide a foundation to know more about the color formation of Narcissus tazetta L., as well as facilitate color-enriched and productivity-enhanced development of the narcissus.The major results obtained from the study were summarized as followed:
1Cloning and analysis of NtPSYgene from Narcissus tazetta L.
The open reading frame (ORF) of PSY were obtained from the three kinds of color type narcissus, the ORF length of NtPSYwas1272bp-1275bp encoding423-424amino acids. The genes were designated as NtPSYJ,NtPSYW(GenBank accession No.JN991013) and NtPSYY(JN991014) respectively. Comparing with other PSY genes in Narcissus genus, there were some replacements and missing amino acids. The genome DNA sequence of PSY genes in three color type narcissus were cloned, the length ranged from2667bp to2840bp, the GenBank accession numbers were No.JN991017(Jinzhanyintai), No.JN991016(Baihua) and No.JN991015(Huanghua). Five introns (A-E) were found in each DNA sequence, the length ranged from81bp to926bp. Sequence nalysis showed that the splicing sites of four introns (A、B、D、E) comformed to the GT-AG rule and introns C were consistented with GC-AG rule.
2Cloning and analysis of NtPDS from Narcissus tazetta L,.
The open reading frame (ORF) of PDS were obtained from the three kinds of color type narcissus, The ORF length of PDS were1713bp encoding570amino acids. The genes were designated as NtPDSJ, NtPDSW(accession number:JQ797376) and N/PDSY (JQ797377) respectively. Comparing with other PDS genes in Narcissus genus, there were some replacements and missing amino acids as well. The part of genome DNA sequence of PDSgenes in the three color type narcissus were cloned, a length ranged from7167bp to7199bp, and the GenBank accession number are No. JQ797382(Jinzhanyintai), No. JQ797383(Baihua) and No. JQ797384(Huanghua).Ten intons(A-J) were found in each DNA sequence, the length ranged from73bp to1489bp. Sequence analysis showed that the splicing sites of introns comformed to the GT-AG rule.
3Cloning and analysis of NtZDS from Narcissus tazetta L.
The open reading frame (ORF) of ZDSwas obtained from the three kinds of color type narcissus, the ORF length of Nt ZDS was1725bp encoding574amino acids. The genes were designated as NtZDSJ, NtZDSW(GenBank accession No. JQ797378) and NtZDSY(JQ797379) respectively. Comparing with other ZDS genes in Narcissus genus, there were some replacements and missing amino acids. The part of genome DNA sequence of ZDS genes in the three color type narcissus were cloned, the length ranged from7382bp to9187bp, the GenBank accession number are No. JQ797385(Jinzhanyintai), No. JQ797386(Huanghua). Eleven introns (A-K) were found in each DNA sequence, the length ranged from87bp to2695bp. Sequence analysis showed that the splicing sites of four introns comformed to the GT-AG rule and intron A, C, F size are quite different.
4Cloning and analysis of NtLCY-βfrom Narcissus tazetta L.
The open reading frame (ORF) of LCY-βwere obtained from the three kinds of color type narcissus, the ORF length of NtLCY-β were1715bp encoding504amino acids. The genes were designated as NtLCY-βJ, NtLCY-βW(GenBank accession No. JQ797380) and MICY-βY (JQ797381) respectively. Comparing with other LCY-β genes in Narcissus genus, there were replacement and missing amino acid. Cloning from genomic DNA sequence of NtLCY-β showed that was no intron and the homologous rate are over98%compared with their cDNA.
5Cloning and analysis of NtLCY-ε
5'RACE primers were designed according to the known NtLCY-ε3'nucleotide seqence from GenBank, the5'nucleotide sequence was obtained by RACE from Jinzhanyintai narcissus. The full length cDNA of NtLCY-ε was1971bp with an opening reading frame of1659bp (GenBank accession No.JQ282903) encoding a protein with532amino acids(Mw=59771.1, pI=6.15). The5-terminal untranslated region (UTR) was153bp and the3-UTR was lllbp. The ORF sequence of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). According to the ORF sequence of LCY-ε gene in Jinzhanyintai, we obtained the ORF sequences of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). The nucleotide and amino acids from the three kinds of color type narcissus were similar. Replacements were found in the deduced amino acid sequence of LCY-ε gene in Narcissus genus plant with homologous comparison. The deduced amino acid sequence of NtLCY-ε was compared with that of other higher plants and phylogenetic tree was was constructed.
4Analysis the expression of PSY、PDS、ZDS and LCY-βin the color formation of Narcissus tazetta L. by qRT-PCR
NtACTIN was used as internal control gene, the expression patterns of NtPSY, NtPDS, NtZDS and NtLCY-β in the bud stage, alabastrum stage, early flowering stage and full-bloom stage were carried out. The results indicated that all of the four genes in Jinzhanyintai and Huanghua narcissus expressed in four stages, with higher expression level in the intermediate stages, while much lower in Baihua narcissus, especially the expression of PSY gene is almost undetectable. Additionally, the expression rate of PDS and ZDS in Baihua narcissus appears to be increasing at certain stage, but it still lower than that of other flowers. To sum up, the higher expression level of PSY、PDS、ZDS and LCY-β in Jinzhanyintai and Huanghua narcissus may cause the yellow color of the flower. Therefore, the regulation of these genes may regulate and control the color of Narcissus tazetta L. flower.
引文
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