郁金香花青素合成酶基因TgANS的克隆与功能鉴定

doi:10.16420/j.issn.0513-353x.2021-0638

摘要/Abstract

摘要：

以红色的郁金香品种‘纯火’为材料,采用高效液相色谱技术对其4个花期的花青素苷含量进行了测定,结果表明,随着花朵的发育,花青素苷的含量逐渐升高,在盛开期含量最高,达3 002.75 μg · g^-1 FW。从花中克隆出结构基因TgANS,GenBank登录号为MZ442383,开放阅读框长1 071 bp,编码356个氨基酸,TgANS基因表达水平在不同花期存在显著差异,随着花发育逐渐升高,表达趋势与花青素苷的积累趋势基本一致。将TgANS导入烟草进行异源表达,转基因株系花色明显变红,TgANS表达量显著增加,花青素苷含量增加,进一步对这些株系内源的NtCHS、NtCHI、NtFLS、NtF3H、NtF3’H和NtDFR表达进行分析,NtF3H、NtF3’H和NtDFR表达量显著上升,NtCHS和NtCHI表达量下降。表明郁金香TgANS参与了花青素苷的合成。

关键词: 郁金香, 花青素合成酶（ANS）, 花青素苷, 基因功能

Abstract:

In this study,the contents of anthocyanins in the tepals were determined from flower of red Tulipa gesneriana‘Strong Fire’at four developmental stages by high performance liquid chromatography（HPLC）. The results showed that the content of anthocyanins continued to increase along with the development of flowers,and the highest content was up to 3 002.75 μg · g^-1 FW in fully flowering stage flower. The sequence of TgANS was cloned,and the GenBank accession number was MZ442383. TgANS has 1 071 bp ORF region,encoding 356 amino acids. The expression level of TgANS differed significantly at different flower stages,and showed a trend of rising during the flower development. The expression pattern was consistent with the accumulation of anthocyanin in the four stages of flower development. Ectopic expression of TgANS in transgenic tobacco plants showed that the flowers of transgenic plants obviously turned red and TgANS was expressed at high levels in transgenic plants. The anthocyanin content in the tepals of the transgenic plants was slightly higher than that in empty vector control. Further analysis of the flower color related genes indicated that the expression of genes NtF3H,NtF3’H and NtDFR were increased significantly,and NtCHS,NtCHI were decreased in transgenic tobacco plants. These results indicated that tulip TgANS gene is involved in the biosynthesis of anthocyanin.

Key words: Tulipa gesneriana, anthocyanidin synthase（ANS）, anthocyanin, gene function

中图分类号:

S682.263

黄玲, 胡先梅, 梁泽慧, 王艳平, 产祝龙, 向林. 郁金香花青素合成酶基因TgANS的克隆与功能鉴定[J]. 园艺学报, 2022, 49(9): 1935-1944.

HUANG Ling, HU Xianmei, LIANG Zehui, WANG Yanping, CHAN Zhulong, XIANG Lin. Cloning and Function Identification of Anthocyanidin Synthase Gene TgANS in Tulipa gesneriana[J]. Acta Horticulturae Sinica, 2022, 49(9): 1935-1944.

图/表 6

表1 qRT-PCR表达的引物信息

Table 1 The primers used for qRT-PCR

引物 Primer	正向引物序列（5′-3′） Forward sequence	反向引物序列（5′-3′） Reverse sequence
TgANS	CGCTCGAGATCCTCAGCAAT	GTCCGGGGAGGAAACTTAGC
TgUBQ10	GTCGCACGTTGGCTGATTAC	GGGCTTAAAGACCACCACGA
NtCHS	AAGCAAGAGAAACTAAAGGCTACAAG	AAATCCAAAAAGCACACCCCAT
NtCHI	CGGGTGCCTCCATTCTTTTTACT	CCTGACACTCTTTCGGCGATACTAC
NtF3H	CCAGACAAACCAGATGGATGGATAG	CAAGGGTAAGGTCGGGCTGTG
NtF3’H	TGGCTATTTCATTCCAAAAGGCTCA	CTTCAAAGTCATTTCCTCGCACATC
NtDFR	GCAGTTGCTTCCCTTTTCTACC	TTCCCCATTGGTTGACTTTCC
NtFLS	CTTGAAGGGAAAAGGGGTTGG	CGCAACTTCTCGCAGCCTCT
NtEF1α	TGGTTGTGACTTTTGGTCCCA	ACAAACCCACGCTTGAGATCC

图1 郁金香花朵发育不同阶段花青素苷含量和TgANS表达量数据表示3次独立试验的平均值和标准误,P < 0.05,Tukey’s method。

Fig. 1 Analysis of anthocyanins content and relative expression levels of TgANS in tulip flower development The values indicates averages of three independent replicates and SEs. P < 0.05,Tukey’s method.

图2 不同植物ANS氨基酸同源序列比对同源比对的各序列为福斯特郁金香TfANS（AGJ50591）、松叶百合的LcANS（AGC92011）、百合的LhANS（ALA55544）、美丽百合的LsANS（BAS69334）、溪荪的IsANS（QCI56004）、海枣的PdANS（XP_008783582）。黑色线条表示2-酮戊二酸双加氧酶家族保守结构域,*和#分别表示结合亚铁离子的保守氨基酸和2-酮戊二酸结合位点氨基酸。

Fig. 2 Alignment of the deduced amino acid sequences of ANS from other plant species TgANS sequence was aligned with ANS from Tulipa fosteriana（TfANS,AGJ50591）;Lilium cernuum（LcANS,AGC92011）;L. hybrida（LhANS,ALA55544）;L. speciosum（LsANS,BAS69334）;Iris sanguinea（IsANS,QCI56004）;Phoenix dactylifera（PdANS,XP_008783582）. The black underline shows the conserved domain 2OG-Fe II_Oxy. * and # indicate conserved residues which are required for ferrous-iron coordination,and 2-oxoglutarat binding,respectively.

图3 ANS系统进化树各节点处数字表示bootstrap值,重复1 000次。

Fig. 3 Phylogenetic analysis of ANS The numbers at node represent the bootstrap values,with 1 000 replicates.

图4 转基因烟草花瓣中TgANS的表达量及花青素苷含量 EV：空载株系。*** P < 0.001,student’s t test。

Fig. 4 Analysis of flower color phenotype of transgenic tobacco EV：The controls that only transformed empty vetor. *** P < 0.001,student’s t test.

图5 TgANS转基因烟草中类黄酮合成相关基因表达分析 * P < 0.05,*** P < 0.001,student’s t test.

Fig. 5 qRT-PCR analysis of flavonoid-related genes expression in TgANS transgenic tobacco

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