Cloning and Functional Analysis of NtMYB8 in Narcissus tazetta var. chinensis

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (10): 1895-1906.doi: 10.16420/j.issn.0513-353x.2021-0599

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Cloning and Functional Analysis of NtMYB8 in Narcissus tazetta var. chinensis

WU Lixuan, ZHOU Ping, FAN Yuxin, WANG Guiqing, ZENG Lihui^*()

College of Horticulture,Institute of Genetics and Breeding in Horticultural Plants,Fujian Agriculture and Forestry University,Fuzhou 350002,China

Received:2021-07-26 Revised:2021-09-23 Online:2021-10-25 Published:2021-11-01
Contact: ZENG Lihui E-mail:lhzeng@fafu.edu.cn

Abstract

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

CLC Number:

S682.21

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.

Figures/Tables 11

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

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

Fig. 2 Cloning of NtMYB8 in Chinese narcissus

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.

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

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.

Fig. 6 Transient expression analysis of NtMYB8 in Nicotiana tabacum

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

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

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

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

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Cloning and Functional Analysis of NtMYB8 in Narcissus tazetta var. chinensis

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