中国水仙NtMYB8的分离及功能研究

doi:10.16420/j.issn.0513-353x.2021-0599

摘要/Abstract

摘要：

从中国水仙（Narcissus tazetta var. chinensis）‘金盏银台’中分离获得1个MYB转录因子基因,命名为NtMYB8,GenBank登录号:MF5222090。通过多重序列比对和进化树分析发现,NtMYB8属R2R3-MYB家族,与原花青素激活因子亲缘关系最近;NtMYB8在中国水仙副冠中表达量最高;将NtMYB8在普通烟草瞬时转化,注射的叶片出现微弱红色斑点;转35S-NtMYB8烟草花瓣颜色变化不明显,qPCR检测显示NtMYB8促进转基因烟草类黄酮代谢途径大部分结构基因表达;酵母双杂试验表明NtMYB8能与中国水仙NtbHLH1互作;双荧光素酶瞬时表达试验显示NtMYB8与NtbHLH1互作能够显著促进水仙类黄酮代谢途径关键基因NtFLS的启动子活性。研究结果表明,NtMYB8是中国水仙类黄酮代谢途径的激活因子,可能促进副冠中黄酮醇的合成。

关键词: 水仙, R2R3-MYB, 类黄酮代谢, 激活因子, 结构基因

Abstract:

A MYB transcription factor gene,named NtMYB8,was obtained from Chinese narcissus （Narcissus tazetta var. chinensis）. GenBank accession number:MF5222090. Multiple protein sequence alignment and phylogenetic tree analysis showed that NtMYB8 contained R2 and R3 conserved domains,belonging to the R2R3-MYB family,and was closely related to proanthocyanidin activators. The expression level of NtMYB8 in the corona was the highest. In the transient transformation of common tobacco,the leaves injected with NtMYB8 had slight red spots. However,the petal color of NtMYB8- transgenic tobacco did not change significantly,and further analysis by qPCR showed that NtMYB8 promoted the expression levels of most tobacco structural genes in the flavonoid metabolic pathway. Yeast two-hybrid assay showed that NtMYB8 can interact with NtbHLH1,the bHLH protein of Chinese narcissus and significantly promote the Chinese narcissus FLS promoter activity by dual luciferase transient expression test. These results suggest that NtMYB8 is an activator of the flavonoid metabolism pathway in Chinese narcissus and may promoter the flavonol biosynthesis in corona.

Key words: Narcissus tazetta var. chinensis, R2R3-MYB, flavonoid metabolism, activator, structural gene

中图分类号:

S682.21

吴立璇, 周平, 范雨昕, 王桂青, 曾黎辉. 中国水仙NtMYB8的分离及功能研究[J]. 园艺学报, 2021, 48(10): 1895-1906.

WU Lixuan, ZHOU Ping, FAN Yuxin, WANG Guiqing, ZENG Lihui. Cloning and Functional Analysis of NtMYB8 in Narcissus tazetta var. chinensis[J]. Acta Horticulturae Sinica, 2021, 48(10): 1895-1906.

图/表 11

表1 引物及其序列

Table 1 The primers sequences used for PCR

用途 Description	引物名称 Primer name	引物序列（5′-3′） Primer sequence
RT-PCR	NtMYB8-1F	GTGCGAGAGAGACGAGAGAGTG
RT-PCR	NtMYB8-1R	GGCTAGTTGACATGCCTATTACCAG
qRT-PCR	NtMYB8-2F	AAACTCCGAGCCCTGGTATGG
qRT-PCR	NtMYB8-2R	TTTCAGCATCAGCACTGGTGTAAG
载体构建 Construction of vector	NtMYB8-3F	GGATCCAAAGAATTCGTGCGAGAGAGACGAGAGAGTG
载体构建 Construction of vector	NtMYB8-3R	ACTCTCGAGAAGCTTGGCTAGTTGACATGCCTATTACCAG
酵母双杂 Yeast double-hybrid assay	AD-MYB8F AD-MYB8R	GGAGGCCAGTGAATTCGGCTAGTTGACATGCCTATTACCAG CGAGCTCGATGGATCCGGCTAGTTGACATGCCTATTACCAG

图1 中国水仙‘金盏银台’花瓣和副冠的取样时期

Fig. 1 Different stages of the petal and corona of Narcissus tazetta var. chinensis

图2 中国水仙NtMYB8基因的克隆

Fig. 2 Cloning of NtMYB8 in Chinese narcissus

图3 NtMYB8与其他MYB转录因子氨基酸序列的多重序列比对 Nt:水仙;At:拟南芥;Dk:柿子;Pt:白杨;OG:文心兰;Vv:葡萄;Fa;草莓;Zm:玉米。

Fig.3 Multiple alignment of the NtMYB8 with other MYB proteins Nt:Narcissus tazetta var. chinensis;At:Arabidopsis thaliana;Dk:Diospyros kaki;Pt:Populus tremuloides;OG:Oncidium Gower Ramsey;Vv:Vitis vinifera;Fa:Fragaria ananassa;Zm:Zea mays.

图4 NtMYB8与类黄酮相关R2R3-MYB因子的系统进化树分析节点处数字代表亲缘关系的远近。

Fig. 4 Phylogenetic tree of NtMYB8 and other flavonoid biosynthesis R2R3-MYBs The numbers at node represent the distance of the kinship.

图5 水仙NtMYB8在不同发育阶段和不同组织的表达 1:花苞期;2:始花期;3:盛花期。不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 5 Relative expression of NtMYB8 gene at different development stages and organs in Chinese narcissus 1:Budding stage;2:Early flowering stage;3:Full-bloom stage. The different lowercase letters indicate significant differences in gene expression（P < 0.05）. The same below.

图6 NtMYB8在普通烟草叶片中的瞬时表达

Fig. 6 Transient expression analysis of NtMYB8 in Nicotiana tabacum

图7 烟草野生型（WT）与T1代NtMYB8转基因株系（M8-1、M8-2、M8-3）花瓣颜色比较

Fig. 7 Comparison of flower color in wild type（WT）and NtMYB8 transgenic（T1 generation M8-1,M8-2,M8-3）tobaccoes

图8 NtMYB8 转基因烟草（M8-1、M8-2、M8-3）花瓣中类黄酮代谢途径结构基因的表达

Fig. 8 Expression of structural genes in flavonoid metabolism pathway of NtMYB8 transgenic（M8-1,M8-2,M8-3）tobacco petals

图9 酵母双杂交验证 NtMYB8与NtbHLH1的互作关系

Fig. 9 Protein-protein interactions between NtMYB8 and NtbHLH1,studied by yeast two-hybrid assay

图10 双荧光素酶分析NtMYB8对NtLAR、NtDFR和NtFLS启动子的调控

Fig. 10 Analysis of the interaction of the NtMYB8 genes with the promoter sequences of NtLAR,NtDFR and NtFLS

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