黑莓花青素苷、原花色素的合成代谢及相关基因克隆和表达研究
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摘要
黑莓(Blackberry),又称黑刺莓,是悬钩子属(Rubus L.)植物中应用于园艺栽培使用的三大种群之一。黑莓果实含有丰富的花青素苷、原花色素以及微量矿质元素,具有较高的营养价值而风靡欧美,被列为世界第三代水果。其果实提取物的抗癌、消炎以及预防心血管疾病等保健功效显著,在病理学方面得到了广泛研究。但有关黑莓果实内含物,尤其是有关花青素苷以及原花色素的生物合成方面报道甚少。国外的黑莓品种选育主要来源于野生资源的筛选,而我国没有黑莓野生群体分布,主栽品种均引白国外。因此,应用分子生物学手段对引进的黑莓品种进行遗传改良以培育具有自主知识产权的黑莓品种,在我国黑莓育种工作中具有良好的发展前景。本课题从黑莓果实发育过程中的花青素苷和原花色素生理变化入手,克隆获得了类黄酮代谢途径中花青素苷和原花色素分支的6个关键结构基因和控制花青素苷合成的1个转录调节因子基因,并分析了这些基因在黑莓果实发育过程中的表达变化。主要研究内容和结果如下：
1分别采用了pH示差法和DMACA化学反应法对黑莓果实发育过程中的花青素苷和原花色素进行测定。结果表明：成熟的黑莓果实中含有丰富的花青素苷(1.426mg-g-1FW)。在果实发育的前期,花青素苷含量较低,而在果实发育的后期,即红色转为黑色过程中花青素苷大量积累。但幼嫩的果实中原花色素含量较高(>40mg-g-1FW),随着果实的成熟,原花色素含量逐渐减少,到果实成熟时原花色素含量达到最低值(4.12mg-g-1FW)。
2通过同源克隆的方式获得了黑莓类黄酮代谢的花青素苷和原花色素合成途径中的6个关键结构基因,包括查尔酮合成酶(Rubus Chalcone Synthase, RuCHS, Accession No. JN602374)、二氢黄酮醇4-还原酶(Rubus Dihydroflavonol4-reductase, RuDFR, Accession No. JF764809),无色花青素双加氧酶(也称花青素合成酶)(Rubus Leucoanthocyanidin dioxygenase, RuLDOX/ANS, Accession No. JF764807)、无色花青素还原酶(Rubus Leucoanthocyanidin Reductase, RuLAR, Accession No. JQ068826)和花青素还原酶(Rubus Anthocyanidin Reductase, RuANR, Accession No. JQ068825)以及一个糖基转移酶(Glycosyltransferase) RuGT1(Accession No. JF764808)编码基因。
3以黑莓基因组DNA为模板,同源克隆获得了RuMYB10基因片段,通过SON-PCR技术获得了该基因全长编码序列(Full Coding Sequences, CDS);以黑莓果实mRNA为模板,RT-PCR扩增获得了基因全长(Accession No.:JQ359611)。序列分析表明：该基因全长编码区共1837bp,编码216个氨基酸,分子量为24863Da。具有MYB转录因子家族特有的R1R2保守序列。与悬钩子属的欧洲红树莓(Rubus ideaus) RiMYB10相比,两者内含子区变异极大,但氨基酸具有较高的同源性(89.81%)。在RuMYB10基因的两个内含子中,第二个内含子长度为750bp,占全长40.8%；在R3重复单元中存在与bHLH因子互作的'[DE]Lx2[RK]x3Lx6Lx3R'序列;而促进花青素苷合成的MYB转录因子3个特征氨基酸残基中的丙氨酸(A)被丝氨酸(S)取代。
4采用改进的CTAB法获得了黑莓果实高质量RNA,用实时定量PCR技术对各结构基因以及调控基因RuMYB10在黑莓果实发育过程中的表达变化进行分析。结果表明：调控花青素苷合成的结构基因RuANS表达变化与花青素苷合成积累-致,在其果实红色到黑色转色过程中达到最高水平；而原花色素合成途径中的特异基因RuLAR和RuANR在幼果期表达量最高,伴随着原花色素的减少,转录水平也呈下降趋势；而作为类黄酮代谢前期以及中期的结构基因RuCHS和RuDFR,其前期有一个转录增加的过程,到果实变红之前达到最大值；而后转录水平逐渐降低,直到花青素苷大量积累的果实变黑阶段又有一个转录增加的过程。作为花青素苷合成代谢的特异调控因子,RuMYB10基因表达模式与RuANS基因类似,在花青素积累高峰来临之前达到最高表达量,从而验证了该因子调控作用的特异性。相关性分析表明,RuANS是黑莓花青素苷合成途径中的特异酶,而相对于RuANR, RuLAR和黑莓原花色素的积累关系更为密切。
5采用简并引物扩增所获得的RuGT糖基转移酶可能是一个原花色素合成相关的糖基转移酶(Glycosyltransferases, GTs)家族成员。同已报道的其他类黄酮糖基转移酶具有较高的同源性。其转录水平在幼果期最高,随着果实的发育而逐渐降低,直到果实大量积累花青素苷时才有小幅回升。此模式与以往报道的类黄酮糖基转移酶截然不同,而与新近报道的原花色素特异糖基转移酶基因(?)JGT72L1表达模式类似。其底物特异性及酶促动力学特征正在作进一步研究。
Blackberry (Rubus L.) belongs to the Rubus genus and is among one of the three Rubus populations which were used in horticultural productions. It is included in the thrived'third generation'fruits, due to the fact that blackberry fruits are rich in anthocyanin, proanthocyanidin and several mineral element contents. It has become most popular in both European and America, so does in China in the recent years. Fruit extractions of blackberry exhibited a lot of health positive effects in the aspects of anti-cancer, anti-inflammation, and against cardiovascular disease. Hence, studies involved in chemistry and pharmacy were widely carried out around the world. However, biosynthesis of the extraction fractions, especially the anthocyanin and proanthocyanidins, was seldom reported. It has been demonstrated that there are not any wild blackberry resources distributing in China. Most of our cultivars were imported from abroad, in which most excellent blackberry traits were selected. In such a background, molecular manipulations aiming to improve or create new germplasm are necessary here in China. In the present study, we initially investigated and determined the anthocyanin and proanthocyanidin content changes in blackberry fruits. Based on the conserved sequences of other plants, we successfully isolated6key structural genes in the flavonoid biosynthesis pathway. The transcription factor, RuMYb10, which specially regulates the anthocyanin branch, was also obtained. Finally, expression patterns of all these genes were assayed by quantitative RT-PCR. The major results were as follows:
(1) Total anthocyanin content was estimated using the pH differential assay. Proanthocyanidins were determined using a standard method based on the specificity reaction between flvanol and DMACA reagent. Our results indicated that blackberry fruits were rich in both anthocyanin and proanthocyanidin contents. Anthocyanin in fruits could count1.426mg·g-1fresh weight fruits. The major anthocyanin was synthesized in the late developing stage, when fruits turn black from red in the outter appearance. On the contrary, proanthocyanidins peaked in the early green fruits. When the fruits get mature, PA gradually diminished and reached a level that could not be detected.
(2) Based on the knowledge that genes were similar in the phylogenetic relatives, degenerate primers were designed and used to amplify the key structural genes in the pathway. We managed to characterize6genes from blackberry fruits, including genes encoding chalcone synthase, RuCHS, dihydroflavonol4-reductase, RuDFR, leucoanthocyanidin dioxygenase, RuANS, leucoanthocyanidin reductase, RuLAR, anthocyanidin reductase, RuANR, and a glycosyltransferase gene RuGTl. All these genes were deposited in Genbank with accession number JN602374, JF764809, JF764807, JQ068826, JQ068825and JF764808, respectively.
(3) A fragment sequence encoding the conserved domain of MYB transcription factor was amplified using genomic DNA as templates. Then SON-PCR technique was used to amplify both flanking sequences of the gene. The full coding sequences of RuMYB10were obtained and tested in cDNA templates. Sequence analysis suggested that the gene,1837bp in length, encoding a polypeptide consisting of216amino acids,24863Da in molecular weight. The peptide possessed the R2R3domain of MYB transcription factors. When compared with Rubus ideaus RiMYB10, the most striking diversities were found in the noncoding regions, including the intron length and the intron sequence divergence. The second intron of RuMYB10ranked750bp, constitute of as much as40.8%of the full length sequence. In the R3repeat region, the motif '[DE]Lx2[RK]x3Lx6Lx3R' which was implicated in its bHLH cofactors was perfectly matched. Of the three deduced amino acid residues in anthocyanin-promoting MYBs, the alanine was substitute by a serine residue.
(4) Total RNA of high quality was isolated from blackberry fruits at different maturation stage using our newly adopted CTAB-based protocol. Expression levels of all the above isolated genes were investigated by quantitative RT-PCR. RuANS, which channels materials into anthocyanin and proanthocyanidin branch, showed coordination with anthocyanin production. The highest level was observed in the turning stage, from red to black fruits. RuLAR and RuANR, presenting the specificity enzymes in the PA pathway, were strictly concomitant to the accumulation of PAs in the fruits. The most abundant transcriptions of RuLAR and RuANR were present in the initial green fruits or even earlier in the flowers. Along with the degradation of PA in the developing process, their expression levels gradually decreased. RuCHS, as one of the early genes in the pathway, and RuDFR, which acted as the key step in the middle of anthocyanin or PA synthesis, had two expression peaks. The first peak appeared just before the time when fruits got red, and the second one existed in the last stage, when fruits were black, accumulating large quantitative of anthocyanin content. Expression of RuMYB10was similar to that of RuANS gene. It was coordinating to the synthesis of anthocyanin in the blackberry fruits, indicating its specific role in the anthocyanin branch. Pearson's coefficient analysis indicated that the RuANS was the most important enzyme in blackberry anthocyanin biosynthesis. As for PA production, RuLAR was more crucial when compared with RuANR.
(5) The isolated RuGT1might be a candidate proanthocyanidin-related glycosyltransferase. The expression patterns of the isolated RuGTl were totally different to that of the previous reported flavonoid glycosyltransferase gene. The highest expression levels appeared in the fruits of green stage, when PA was the dominant metabolites. Afterward, transcripts decreased till the last stage with a slight increasing in the black berries. This was much similar to that of the UGT72L1in the Medicago truncatula, which catalyze the glycosylation of epicatechin. However, the subtract specificity and enzymatic kinetics need further investigation.
1分别采用了pH示差法和DMACA化学反应法对黑莓果实发育过程中的花青素苷和原花色素进行测定。结果表明：成熟的黑莓果实中含有丰富的花青素苷(1.426mg-g-1FW)。在果实发育的前期,花青素苷含量较低,而在果实发育的后期,即红色转为黑色过程中花青素苷大量积累。但幼嫩的果实中原花色素含量较高(>40mg-g-1FW),随着果实的成熟,原花色素含量逐渐减少,到果实成熟时原花色素含量达到最低值(4.12mg-g-1FW)。
2通过同源克隆的方式获得了黑莓类黄酮代谢的花青素苷和原花色素合成途径中的6个关键结构基因,包括查尔酮合成酶(Rubus Chalcone Synthase, RuCHS, Accession No. JN602374)、二氢黄酮醇4-还原酶(Rubus Dihydroflavonol4-reductase, RuDFR, Accession No. JF764809),无色花青素双加氧酶(也称花青素合成酶)(Rubus Leucoanthocyanidin dioxygenase, RuLDOX/ANS, Accession No. JF764807)、无色花青素还原酶(Rubus Leucoanthocyanidin Reductase, RuLAR, Accession No. JQ068826)和花青素还原酶(Rubus Anthocyanidin Reductase, RuANR, Accession No. JQ068825)以及一个糖基转移酶(Glycosyltransferase) RuGT1(Accession No. JF764808)编码基因。
3以黑莓基因组DNA为模板,同源克隆获得了RuMYB10基因片段,通过SON-PCR技术获得了该基因全长编码序列(Full Coding Sequences, CDS);以黑莓果实mRNA为模板,RT-PCR扩增获得了基因全长(Accession No.:JQ359611)。序列分析表明：该基因全长编码区共1837bp,编码216个氨基酸,分子量为24863Da。具有MYB转录因子家族特有的R1R2保守序列。与悬钩子属的欧洲红树莓(Rubus ideaus) RiMYB10相比,两者内含子区变异极大,但氨基酸具有较高的同源性(89.81%)。在RuMYB10基因的两个内含子中,第二个内含子长度为750bp,占全长40.8%；在R3重复单元中存在与bHLH因子互作的'[DE]Lx2[RK]x3Lx6Lx3R'序列;而促进花青素苷合成的MYB转录因子3个特征氨基酸残基中的丙氨酸(A)被丝氨酸(S)取代。
4采用改进的CTAB法获得了黑莓果实高质量RNA,用实时定量PCR技术对各结构基因以及调控基因RuMYB10在黑莓果实发育过程中的表达变化进行分析。结果表明：调控花青素苷合成的结构基因RuANS表达变化与花青素苷合成积累-致,在其果实红色到黑色转色过程中达到最高水平；而原花色素合成途径中的特异基因RuLAR和RuANR在幼果期表达量最高,伴随着原花色素的减少,转录水平也呈下降趋势；而作为类黄酮代谢前期以及中期的结构基因RuCHS和RuDFR,其前期有一个转录增加的过程,到果实变红之前达到最大值；而后转录水平逐渐降低,直到花青素苷大量积累的果实变黑阶段又有一个转录增加的过程。作为花青素苷合成代谢的特异调控因子,RuMYB10基因表达模式与RuANS基因类似,在花青素积累高峰来临之前达到最高表达量,从而验证了该因子调控作用的特异性。相关性分析表明,RuANS是黑莓花青素苷合成途径中的特异酶,而相对于RuANR, RuLAR和黑莓原花色素的积累关系更为密切。
5采用简并引物扩增所获得的RuGT糖基转移酶可能是一个原花色素合成相关的糖基转移酶(Glycosyltransferases, GTs)家族成员。同已报道的其他类黄酮糖基转移酶具有较高的同源性。其转录水平在幼果期最高,随着果实的发育而逐渐降低,直到果实大量积累花青素苷时才有小幅回升。此模式与以往报道的类黄酮糖基转移酶截然不同,而与新近报道的原花色素特异糖基转移酶基因(?)JGT72L1表达模式类似。其底物特异性及酶促动力学特征正在作进一步研究。
Blackberry (Rubus L.) belongs to the Rubus genus and is among one of the three Rubus populations which were used in horticultural productions. It is included in the thrived'third generation'fruits, due to the fact that blackberry fruits are rich in anthocyanin, proanthocyanidin and several mineral element contents. It has become most popular in both European and America, so does in China in the recent years. Fruit extractions of blackberry exhibited a lot of health positive effects in the aspects of anti-cancer, anti-inflammation, and against cardiovascular disease. Hence, studies involved in chemistry and pharmacy were widely carried out around the world. However, biosynthesis of the extraction fractions, especially the anthocyanin and proanthocyanidins, was seldom reported. It has been demonstrated that there are not any wild blackberry resources distributing in China. Most of our cultivars were imported from abroad, in which most excellent blackberry traits were selected. In such a background, molecular manipulations aiming to improve or create new germplasm are necessary here in China. In the present study, we initially investigated and determined the anthocyanin and proanthocyanidin content changes in blackberry fruits. Based on the conserved sequences of other plants, we successfully isolated6key structural genes in the flavonoid biosynthesis pathway. The transcription factor, RuMYb10, which specially regulates the anthocyanin branch, was also obtained. Finally, expression patterns of all these genes were assayed by quantitative RT-PCR. The major results were as follows:
(1) Total anthocyanin content was estimated using the pH differential assay. Proanthocyanidins were determined using a standard method based on the specificity reaction between flvanol and DMACA reagent. Our results indicated that blackberry fruits were rich in both anthocyanin and proanthocyanidin contents. Anthocyanin in fruits could count1.426mg·g-1fresh weight fruits. The major anthocyanin was synthesized in the late developing stage, when fruits turn black from red in the outter appearance. On the contrary, proanthocyanidins peaked in the early green fruits. When the fruits get mature, PA gradually diminished and reached a level that could not be detected.
(2) Based on the knowledge that genes were similar in the phylogenetic relatives, degenerate primers were designed and used to amplify the key structural genes in the pathway. We managed to characterize6genes from blackberry fruits, including genes encoding chalcone synthase, RuCHS, dihydroflavonol4-reductase, RuDFR, leucoanthocyanidin dioxygenase, RuANS, leucoanthocyanidin reductase, RuLAR, anthocyanidin reductase, RuANR, and a glycosyltransferase gene RuGTl. All these genes were deposited in Genbank with accession number JN602374, JF764809, JF764807, JQ068826, JQ068825and JF764808, respectively.
(3) A fragment sequence encoding the conserved domain of MYB transcription factor was amplified using genomic DNA as templates. Then SON-PCR technique was used to amplify both flanking sequences of the gene. The full coding sequences of RuMYB10were obtained and tested in cDNA templates. Sequence analysis suggested that the gene,1837bp in length, encoding a polypeptide consisting of216amino acids,24863Da in molecular weight. The peptide possessed the R2R3domain of MYB transcription factors. When compared with Rubus ideaus RiMYB10, the most striking diversities were found in the noncoding regions, including the intron length and the intron sequence divergence. The second intron of RuMYB10ranked750bp, constitute of as much as40.8%of the full length sequence. In the R3repeat region, the motif '[DE]Lx2[RK]x3Lx6Lx3R' which was implicated in its bHLH cofactors was perfectly matched. Of the three deduced amino acid residues in anthocyanin-promoting MYBs, the alanine was substitute by a serine residue.
(4) Total RNA of high quality was isolated from blackberry fruits at different maturation stage using our newly adopted CTAB-based protocol. Expression levels of all the above isolated genes were investigated by quantitative RT-PCR. RuANS, which channels materials into anthocyanin and proanthocyanidin branch, showed coordination with anthocyanin production. The highest level was observed in the turning stage, from red to black fruits. RuLAR and RuANR, presenting the specificity enzymes in the PA pathway, were strictly concomitant to the accumulation of PAs in the fruits. The most abundant transcriptions of RuLAR and RuANR were present in the initial green fruits or even earlier in the flowers. Along with the degradation of PA in the developing process, their expression levels gradually decreased. RuCHS, as one of the early genes in the pathway, and RuDFR, which acted as the key step in the middle of anthocyanin or PA synthesis, had two expression peaks. The first peak appeared just before the time when fruits got red, and the second one existed in the last stage, when fruits were black, accumulating large quantitative of anthocyanin content. Expression of RuMYB10was similar to that of RuANS gene. It was coordinating to the synthesis of anthocyanin in the blackberry fruits, indicating its specific role in the anthocyanin branch. Pearson's coefficient analysis indicated that the RuANS was the most important enzyme in blackberry anthocyanin biosynthesis. As for PA production, RuLAR was more crucial when compared with RuANR.
(5) The isolated RuGT1might be a candidate proanthocyanidin-related glycosyltransferase. The expression patterns of the isolated RuGTl were totally different to that of the previous reported flavonoid glycosyltransferase gene. The highest expression levels appeared in the fruits of green stage, when PA was the dominant metabolites. Afterward, transcripts decreased till the last stage with a slight increasing in the black berries. This was much similar to that of the UGT72L1in the Medicago truncatula, which catalyze the glycosylation of epicatechin. However, the subtract specificity and enzymatic kinetics need further investigation.
引文
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