蜡梅花青素苷合成相关基因CpTT8的克隆和功能分析

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

摘要/Abstract

摘要：

以蜡梅（Chimonanthus praecox L.）‘H29’为试材克隆得到1个bHLH类转录因子的全长编码序列,命名为CpTT8。对其生物信息学特征、时空表达特性、基因功能、蛋白互作、蛋白-DNA互作等进行了分析。CpTT8的ORF为2 130 bp,编码709个氨基酸,含有典型的bHLH结构域;蜡梅CpTT8与桃PpbHLH3、苹果MdbHLH3同源性最高。实时荧光定量PCR结果表明CpTT8在早期花芽中表达量最高;在根、茎、叶、果实中也有表达,以果实中的表达量最高。在拟南芥Col-0和tt8突变体中超量表达CpTT8有利于花青素苷和原花青素的积累,说明CpTT8是花青素苷合成过程中的正调控因子。酵母双杂交实验和双荧光素酶分析揭示了CpTT8需与MYB转录因子互作才能激活下游结构基因ANS的表达。

关键词: 蜡梅, 花青素苷, CpTT8, 功能鉴定, 互作

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

中图分类号:

S685.99

张倩, 杨楠, 桑海煜, 赵荣, 宋晓惜, 陈龙清, 向林, 赵凯歌. 蜡梅花青素苷合成相关基因CpTT8的克隆和功能分析[J]. 园艺学报, 2021, 48(10): 1945-1955.

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.

图/表 8

表1 本研究中所使用引物

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

图1 CpTT8与其他植物调控花青素苷合成的bHLH转录因子的进化树

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

图2 CpTT8与其他调控花青素苷合成的bHLH蛋白的序列比对 At:拟南芥;DK:柿;Aa:红掌;Lc:荔枝;Md:苹果;Nt:烟草;Pa:甜樱桃;Ph:矮牵牛;Pp:桃;Vv:葡萄;Zm:玉米;Os:水稻。

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.

图3 CpTT8在蜡梅‘H29’中的时空表达分析

Fig. 3 Spatiotemporal expression pattern of CpTT8 in Chimonanthus praecox

图4 MeJA诱导5 d的超量表达CpTT8的转基因拟南芥的表型（A）、花青素苷含量（B）和基因表达半定量检测（C） **表示转基因株系与对照的花青素苷含量存在极显著差异（** P ≤ 0.01）。

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）.

图5 超量表达CpTT8的转基因拟南芥tt8突变体种子表型

Fig. 5 Seed phenotype ofCpTT8 overexpressed tt8 mutants

图6 CpTT8对结构基因ANS启动子的激活特性分析 A:对蜡梅CpANS启动子活性的影响;B:对拟南芥AtANS启动子活性的影响。不同字母表示差异显著（P ≤ 0.05）。

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）.

图7 CpTT8和CpMYB2的酵母双杂交分析

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

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