Cloning and Functional Analysis of CpTT8 Related to Anthocyanin Synthesis in Wintersweet（Chimonanthus praecox）

doi:10.16420/j.issn.0513-353x.2021-0581

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (10): 1945-1955.doi: 10.16420/j.issn.0513-353x.2021-0581

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Cloning and Functional Analysis of CpTT8 Related to Anthocyanin Synthesis in Wintersweet（Chimonanthus praecox）

ZHANG Qian¹, YANG Nan¹^,², SANG Haiyu¹, ZHAO Rong¹, SONG Xiaoxi¹, CHEN Longqing², XIANG Lin¹, ZHAO Kaige¹^,^*()

¹Key Laboratory of Horticltural Plant Biology,Ministy of Edtcation,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China
²College of Landscape Architecture and Horticulture Sciences,Southwest Forestry University,Kunming 650224,China

Received:2021-06-18 Revised:2021-09-09 Online:2021-10-25 Published:2021-11-01
Contact: ZHAO Kaige E-mail:zhaokaige@mail.hzau.edu.cn

Abstract

Abstract:

The full-length coding sequence of a bHLH transcription factor,named CpTT8,was cloned from Chimonanthus praecox‘H29’. Studies related to this transcription factor were carried out,including bioinformatics characteristics,spatiotemporal expression pattern analysis,gene function test,protein interactions,and protein-DNA interaction. Sequence analysis showed that the ORF of CpTT8 consists of 2 130 bp encoding 709 amino acids,and CpTT8 contains a bHLH domain. CpTT8 shows the highest sequence similarity to PpbHLH3 from peach and MdbHLH3 from apple. The results of quantitative RT-PCR revealed that the expression level of CpTT8 was highest in flower bud at the early stage of floral development. It was also expressed in roots,stems,leaves,and fruits,with the highest level in fruits. Overexpression of CpTT8 in Arabidopsis Col-0 and tt8 facilitated the accumulation of anthocyanins and proanthocyanidins,indicating that CpTT8 is a critical positive regulatory transcription factor in the anthocyanin biosynthesis pathway. Yeast two-hybrid assay and dual-luciferase analysis illustrated that CpTT8 needs to interact with MYB transcription factors to activate its downstream structural gene ANS.

Key words: Chimonanthus praecox, anthocyanin, CpTT8, functional identification, interaction

CLC Number:

S685.99

ZHANG Qian, YANG Nan, SANG Haiyu, ZHAO Rong, SONG Xiaoxi, CHEN Longqing, XIANG Lin, ZHAO Kaige. Cloning and Functional Analysis of CpTT8 Related to Anthocyanin Synthesis in Wintersweet（Chimonanthus praecox）[J]. Acta Horticulturae Sinica, 2021, 48(10): 1945-1955.

Figures/Tables 8

Table 1 Primers used in this study

作用 Funciton	引物名称 Primer name	引物序列（5′-3′） Primer sequence
qPCR	CpTT8qF	TCTGACTGAGTCCGAGTGGTTC
qPCR	CpTT8qR	TTGCTGTCAGCCTCATTTGC
内参Internal reference	CpRPL8qF	AAGATTGTACCAAGTGGTTGC
内参Internal reference	CpRPL8qR	TGCTCTACAGGGTTCATAGC
克隆Cloning	CpTT8-S1300F	CAAATCGACTCTAGAAAGCTTATGGCCTGCCCACCGAGCAG
克隆Cloning	CpTT8-S1300R	GCCCTTGCTCACCATGGTACCGTTCTGGGAAATGATTTGGT
酵母双杂交载体构建 Constructing yeast two-hybrid vectors	CpTT8-ADF	GCCATGGAGGCCAGT GAATTCATGGCCTGCCCACCGAGCAG
	CpTT8-ADR	AGCTCGAGCTCGATGGATCCCGTTCTGGGAAATGATTTGGT
	CpTT8-BDF	ATGGCCATGGAGGCCGAATTCATGGCCTGCCCACCGAGCAG
	CpTT8-BDR	CGCTGCAGGTCGACGGATCCCGTTCTGGGAAATGATTTGGT

Fig. 1 Phylogenetic tree of CpTT8 and other bHLH transcription factors that regulate anthocyanin synthesis

Fig. 2 Protein sequence alignment of CpTT8 and other bHLH transcription factors that regulate anthocyanin synthesis At: Arabidopsis thaliana;DK:Diospyros kaki;Aa:Artemisia annua;Lc:Litchi chinensis;Md:Malus × domestica;Nt:Nicotiana tabacum;Pa:Prunus avium;Ph:Petunia hybrida;Pp:Prunus persica;Vv:Vitis vinifera;Zm:Zea mays;Os:Oryza sativa.

Fig. 3 Spatiotemporal expression pattern of CpTT8 in Chimonanthus praecox

Fig. 4 The phenotype of transgenic Arabidopsis thaliana overexpressing CpTT8 induced by MeJA for 5 days（A）,total anthocyanins content（B）and semi-quantitative detection of gene expression（C） **:Represents a significant difference between transgenic lines and wild type（** P ≤ 0.01）.

Fig. 5 Seed phenotype ofCpTT8 overexpressed tt8 mutants

Fig. 6 The activation function of CpTT8 on the promoter of ANS A:The effect of CpTT8 on the promoter of CpANS;B:The effect of CpTT8 on the promoter of AtANS. Different letters indicate a significant difference（P ≤ 0.05）.

Fig. 7 Interactions between CpTT8 and CpMYB2 determined by Y2H assays

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Cloning and Functional Analysis of CpTT8 Related to Anthocyanin Synthesis in Wintersweet（Chimonanthus praecox）

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