甘蓝型油菜玉米黄素环氧酶基因（BnZEP）的克隆与表达分析
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摘要
类胡萝卜素在植物体内具有非常重要的功能,一些有色的类胡萝卜素是许多植物花和果实的色素的主要来源,有的类胡萝卜素是脱落酸合成的前体物质,有些参与叶黄素循环,在植物消耗过剩光能、抗光氧化方面发挥作用。玉米黄素环氧酶基因(ZEP)是在叶黄素循环过程中起作用的基因,不仅参与类胡萝卜素的生物合成,也参与脱落酸的生物合成,是两个合成途径过程中的共同关键酶基因。
本研究以甘蓝型白花油菜品系HW243和黄花油菜中油821为实验材料,利用分子生物学相关技术,克隆出来BnZEP的cDNA序列和DNA序列,并对它们做了生物信息学方面的分析,其主要结果如下：
(1)根据NCBI数据库ZEP基因保守序列设计简并引物,利用PCR扩增获得了甘蓝型油菜白花材料BnZEP保守序列,其长度为582 bp,把此片段的核苷酸序列与拟南芥、盐芥、大白菜进行比对,其同源性分别达到89%、89%和96%,表明所扩增的片段为甘蓝型油菜BnZEP基因的保守序列,此片段与同属的白菜高度相近。
(2)采用cDNA末端快速扩增(RACE)技术获得了BnZEP的cDNA全长序列,其长度为2228 bp (Genbank登录号GU361616),包括一个完整的2010 bp开放阅读框(ORF),以及57 bp的5'UTR和161 bp的3'UTR区。ORF编码669个氨基酸残基。推导的氨基酸序列与拟南芥、大白菜、盐芥、龙胆和玉米等植物的氨基酸序列一致性分别达到89%、97%、92%、68%和65%。
(3)利用各种生物信息软件对ZEP蛋白的相关信息做了分析。ZEP蛋白分子量为73841.01 D,理论等电点IP=6.5292,20种基本的氨基酸全部包括,由于疏水性氨基酸比例远大于极性氨基酸,因此推测BnZEP编码的蛋白是一种疏水性多肽。在ZEP蛋白整体结构中,无规则卷曲、α-螺旋和延伸链是ZEP蛋白最大量的结构元件,而β-转角则散布于整个蛋白质中。
(4)根据cDNA全长序列设计了扩增全长DNA的引物,利用PCR扩增出了BnZEP的DNA完整序列。序列全长3268 bp (Genbank登录号GU561839),该基因包含16个外显子和15个内含子,与公布的拟南芥ZEP基因的外显子和内含子数量一致。
(5)利用半定量PCR检测到甘蓝型白花油菜和黄花油菜花瓣内的BnZEP表达量明显不同,白花的cDNA量明显高于黄花,推测BnZEP可能与白花性状存在一定的关联性,但有待进一步鉴定。
Carotenoids have important functions in plants. Some carotenoids provide the main source of the pigment in colored flowers and fruits, some carotenoids are precursors of abscisic acid synthesis, and some participate in xanthophyll cycle, drain the excess light energy, play a role of anti-photo-oxidation in plants. ZEP gene play a predominant role in xanthophyll cycle, not only participate in carotenoid biosynthesis, but also participate in abscisic acid biosynthesis pathway. It is the key enzyme gene of the two pathways.
We have experimented by using pure white flower HW243 and yellow flower Zhongyou 821 as materials, and cloned the cDNA sequence and DNA sequence of BnZEP based on molecular biology technologies. Also, we analyzed the relevant biological information of BnZEP and speculated ZEP protein. The main results were summarized as follows:
(1) We designed degenerate primers according to the conserved sequence of the ZEP gene published in NCBI database, and isolated the 582 bp products (conserved sequence) of the white flower Brassica napus using PCR amplification. Comparied with ZEP genes of other plants, the nucleotide sequence of this fragment shares, respectively,89%,89% and 96% identity with that of Arabidopsis thaliana, Thellungiella halophila, and Chinese cabbage. The results showed that the amplified fragment is a part of the BnZEP. This fragment showed a high degree of homology with the Chinese cabbage.
(2) Using rapid amplification of cDNA ends (RACE) technology, we got the full-length cDNA of BnZEP. The cDNA has the sequence length of 2228 bp (Genbank accession number GU361616). It contains an 2010 bp open reading frame (ORF) encoding 669 amino acid residues, a 57 bp 5'untranslated region and a 161 bp 3'untranslated region. Comparied with ZEP proteins, the deduced amino acid sequence of this protein shares 89%,97%,92%,68% and 65% identity with that of Arabidopsis thaliana, Chinese cabbage,Thellungiella halophila, lutea and corn respectively.
(3) In addition, we analyzed the relevant information of ZEP protein by some bioinformatics softwares. The molecular weight of ZEP protein was 73841.01 D, and the theoretical isoelectic point was 6.5292. The protein included twenty kinds of basic amino acids. As the proportion of hydrophobic amino acids was much greater than polar amino acids, we suggested that ZEP protein was a hydrophobic polypetide. From the integral structure of ZEP protein, we found that random coils,α-helixes and extended chains were the maximum amount of ZEP protein structural elements, while theβ-turns were scattered throughout the whole protein.
(4) According to the full length cDNA, we designed the specific primers and amplified the complete sequence of DNA using PCR.. The DNA has the sequence length of 3268 bp (Genbank accession number GU561839), contain 16 exons and 15 introns. The number of exons and introns are same with that of published Arabidopsis thaliana.
(5) Using semi-quantitative PCR, we found that the BnZEP expression products wereas significantly different between white flower and yellow flower. The BnZEP expression products in white flower were more than that of in yellow flower. After analysis, we speculated that the BnZEP may has some relativity with the white flower character.
本研究以甘蓝型白花油菜品系HW243和黄花油菜中油821为实验材料,利用分子生物学相关技术,克隆出来BnZEP的cDNA序列和DNA序列,并对它们做了生物信息学方面的分析,其主要结果如下：
(1)根据NCBI数据库ZEP基因保守序列设计简并引物,利用PCR扩增获得了甘蓝型油菜白花材料BnZEP保守序列,其长度为582 bp,把此片段的核苷酸序列与拟南芥、盐芥、大白菜进行比对,其同源性分别达到89%、89%和96%,表明所扩增的片段为甘蓝型油菜BnZEP基因的保守序列,此片段与同属的白菜高度相近。
(2)采用cDNA末端快速扩增(RACE)技术获得了BnZEP的cDNA全长序列,其长度为2228 bp (Genbank登录号GU361616),包括一个完整的2010 bp开放阅读框(ORF),以及57 bp的5'UTR和161 bp的3'UTR区。ORF编码669个氨基酸残基。推导的氨基酸序列与拟南芥、大白菜、盐芥、龙胆和玉米等植物的氨基酸序列一致性分别达到89%、97%、92%、68%和65%。
(3)利用各种生物信息软件对ZEP蛋白的相关信息做了分析。ZEP蛋白分子量为73841.01 D,理论等电点IP=6.5292,20种基本的氨基酸全部包括,由于疏水性氨基酸比例远大于极性氨基酸,因此推测BnZEP编码的蛋白是一种疏水性多肽。在ZEP蛋白整体结构中,无规则卷曲、α-螺旋和延伸链是ZEP蛋白最大量的结构元件,而β-转角则散布于整个蛋白质中。
(4)根据cDNA全长序列设计了扩增全长DNA的引物,利用PCR扩增出了BnZEP的DNA完整序列。序列全长3268 bp (Genbank登录号GU561839),该基因包含16个外显子和15个内含子,与公布的拟南芥ZEP基因的外显子和内含子数量一致。
(5)利用半定量PCR检测到甘蓝型白花油菜和黄花油菜花瓣内的BnZEP表达量明显不同,白花的cDNA量明显高于黄花,推测BnZEP可能与白花性状存在一定的关联性,但有待进一步鉴定。
Carotenoids have important functions in plants. Some carotenoids provide the main source of the pigment in colored flowers and fruits, some carotenoids are precursors of abscisic acid synthesis, and some participate in xanthophyll cycle, drain the excess light energy, play a role of anti-photo-oxidation in plants. ZEP gene play a predominant role in xanthophyll cycle, not only participate in carotenoid biosynthesis, but also participate in abscisic acid biosynthesis pathway. It is the key enzyme gene of the two pathways.
We have experimented by using pure white flower HW243 and yellow flower Zhongyou 821 as materials, and cloned the cDNA sequence and DNA sequence of BnZEP based on molecular biology technologies. Also, we analyzed the relevant biological information of BnZEP and speculated ZEP protein. The main results were summarized as follows:
(1) We designed degenerate primers according to the conserved sequence of the ZEP gene published in NCBI database, and isolated the 582 bp products (conserved sequence) of the white flower Brassica napus using PCR amplification. Comparied with ZEP genes of other plants, the nucleotide sequence of this fragment shares, respectively,89%,89% and 96% identity with that of Arabidopsis thaliana, Thellungiella halophila, and Chinese cabbage. The results showed that the amplified fragment is a part of the BnZEP. This fragment showed a high degree of homology with the Chinese cabbage.
(2) Using rapid amplification of cDNA ends (RACE) technology, we got the full-length cDNA of BnZEP. The cDNA has the sequence length of 2228 bp (Genbank accession number GU361616). It contains an 2010 bp open reading frame (ORF) encoding 669 amino acid residues, a 57 bp 5'untranslated region and a 161 bp 3'untranslated region. Comparied with ZEP proteins, the deduced amino acid sequence of this protein shares 89%,97%,92%,68% and 65% identity with that of Arabidopsis thaliana, Chinese cabbage,Thellungiella halophila, lutea and corn respectively.
(3) In addition, we analyzed the relevant information of ZEP protein by some bioinformatics softwares. The molecular weight of ZEP protein was 73841.01 D, and the theoretical isoelectic point was 6.5292. The protein included twenty kinds of basic amino acids. As the proportion of hydrophobic amino acids was much greater than polar amino acids, we suggested that ZEP protein was a hydrophobic polypetide. From the integral structure of ZEP protein, we found that random coils,α-helixes and extended chains were the maximum amount of ZEP protein structural elements, while theβ-turns were scattered throughout the whole protein.
(4) According to the full length cDNA, we designed the specific primers and amplified the complete sequence of DNA using PCR.. The DNA has the sequence length of 3268 bp (Genbank accession number GU561839), contain 16 exons and 15 introns. The number of exons and introns are same with that of published Arabidopsis thaliana.
(5) Using semi-quantitative PCR, we found that the BnZEP expression products wereas significantly different between white flower and yellow flower. The BnZEP expression products in white flower were more than that of in yellow flower. After analysis, we speculated that the BnZEP may has some relativity with the white flower character.
引文
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[3]Bartleyg E, Scolnik PA. Plant carotenoids:Pigments for photoprotection, visual attraction and human health [J]. Plant Cell,1995, (7):1027-1038.
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[6]Rock CD, Zeevaart JAD. The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proc Natl Acad Sci,1991,88:7496-7499.
[7]Mayne ST. Beta-carotene, carotenoids, and disease prevention in humans. FASEB J. 1996,10:690-701.
[8]惠伯棣.类胡萝卜素化学及生物化学[M].中国轻工业出版社.2004.
[9]Lange BM, Rujan T, Martin W and Croteau R. Isoprenoid biosynthesis:the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci,2000,97:13172-13177.
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