苹果多肽编码基因MhCLE8调控花青苷积累的功能研究

doi:10.16420/j.issn.0513-353x.2021-0717

摘要/Abstract

摘要：

利用拟南芥和苹果愈伤组织等植物材料，研究多肽MhCLE8（CLAVATA3/EMBRYO SURROUNDING REGION 8）调控花青苷积累的作用。结果显示，体外合成MhCLE8p多肽处理拟南芥幼苗和苹果愈伤组织材料显著抑制了花青苷的积累。多序列比对和系统分析结果表明，MhCLE8与多个物种中的同源序列均存在高度保守的CLE基序，其中MhCLE8与杏（CAB4276204.1）和山荆子（TQE01035.1）存在较高的同源关系。在平邑甜茶中克隆MhCLE8，连接双元载体pRI，通过农杆菌介导的转基因技术获得了MhCLE8在拟南芥和苹果愈伤组织中的稳定过表达材料。表型分析表明，MhCLE8转基因拟南芥幼苗和苹果愈伤组织的花青苷积累量显著低于野生型。同时，qRT-PCR结果表明，MhCLE8转基因植物材料中花青苷合成结构基因的表达受到了不同程度的抑制。综上，多肽编码基因MhCLE8是苹果花青苷积累的负调控因子。

关键词: 苹果, 花青苷, 多肽激素, MhCLE8

Abstract:

In this study，the role of MhCLE8（CLAVATA3/EMBRYO SURRSURROUNDING REGION 8）peptide hormone gene in regulating anthocyanin accumulation was investigated using the meterials Arabidopsis and apple calli. The results showed that the accumulation of anthocyanin in Arabidopsis seedlings and apple calli was significantly inhibited when treated with MhCLE8 peptide （referred to as MhCLE8p）which was synthesised in vitro. Phylogenetic analysis and multiple protein sequences alignment showed that MhCLE8 and its homologues in other plant species contain a highly conserved CLE-motif，and MhCLE8 showed high homology with Prunus armeniaca（CAB4276204.1）and Malus baccata（TQE01035.1）. The MhCLE8 gene was cloned to binary vector pRI，and transferred to Arabidopsis and apple calli to obtain the stable overexpression materials by Agrobacterium-mediated transgenic technology. The transgenic Arabidopsis seedlings and apple calli showed decresed accumulation of anthocyanin. The qRT-PCR analysis showed that the expression of anthocyanin biosynthesis structural genes in MhCLE8 overexpressed plant materials was inhibited to varying degrees. In summary，all the results suggested that the peptide encoding gene MhCLE8 was a negative regulator of anthocyanin accumulation.

Key words: apple, anthocyanin, peptide hormone, MhCLE8

石岩, 张天恩, 朱百慧, 李彤, 李秀明, 由春香. 苹果多肽编码基因MhCLE8调控花青苷积累的功能研究[J]. 园艺学报, 2023, 50(12): 2541-2550.

SHI Yan, ZHANG Tian'en, ZHU Baihui, LI Tong, LI Xiuming, YOU Chunxiang. Function Study of Peptide Coding Gene MhCLE8 in Regulating Anthocyanin Accumulation in Apple（Malus × domestica）[J]. Acta Horticulturae Sinica, 2023, 50(12): 2541-2550.

图/表 10

表1 本研究中所用的引物

Table 1 Primer sequences used in this study

引物名称 Primer name	基因序列号 Gene ID	引物序列（5′-3′）Primer sequence
引物名称 Primer name	基因序列号 Gene ID	F	R
RT-AtCHS	AT5G13930	ATCACAGCCGTTACCTTCCG	AAAAGAGCCTGACCGACGAG
RT-AtF3H	AT3G51240	CCACACGTGACAATGGCAAG	TCGCCGAGATTGACGACAAA
RT-AtDFR	AT5G42800	TGGACGACTTATGCAACGCT	ATATAACGTCCCTTGGCGGC
RT-AtANS	AT4G22880	ACACAACATGGTTCCGGGTT	GTGACCCATTTGCCCTCGTA
RT-AtUFGT	AT5G54060	TGGATTCAGCAACCGTTGGT	ACTCCCACATCGACCCAAAC
RT-MdCHS	MDP0000686666	GCTACAAATGCGGTGATAG	CGCCTCCACTACAACCTCC
RT-MdF3H	MDP0000323864	GCCGATCACCTACACCGAG	GTACAAGAAGTGGGAAGGC
RT-MdDFR	MDP0000494976	GGTAAATGTAAAACAATAGAG	GGAAGTGGTTTTGTCGCCTG
RT-MdANS1	MDP0000271553	CACCTTCATCCTCCACAACAT	ATGTGCTCAGCAAAAGTTCGT
RT-MdUFGT	MDP0000405936	GGAAGTGGTTTTGTCGCCTG	CATTATTATTGAGCAACGAAC
RT-MdCLE8	MDP0000240955	AAGAGGAACGTAACCGGGTC	ACTCAATCGCTTCGGGTTGT

图1 MhCLE8p处理抑制拟南芥花青苷积累 ** 与对照差异极显著（Tukey 检验，P < 0.01）。下同。

Fig. 1 MhCLE8p treatment inhibit anthocyanin accumulation in Arabidopsis ** indicate extremely significant difference between control（Tukey's test，P < 0.01）. The same below.

图2 MhCLE8p处理抑制苹果愈伤组织花青苷积累

Fig. 2 MhCLE8p treatment inhibit anthocyanin accumulation in apple calli

图3 MhCLE8基因PCR扩增产物的凝胶电泳

Fig. 3 Gel electrophoresis of the PCR product of MhCLE8

图4 MhCLE8及其同源基因的进化树（A）分析和CLE基序比对（B）

Fig. 4 Phylogenetic tree of MhCLE8 and its homologous（A）and sequence alignment of CLE-motif（B）

图5 拟南芥异源过表达MhCLE8基因抑制花青苷积累 OE-1、OE-2和OE-3为3个转基因株系，下同。AtCHS、AtF3H、AtDFR、AtANS和AtUFGT为花青苷合成结构基因。

Fig. 5 Ectopic expression of MhCLE8 inhibit anthocyanin accumulation in Arabidopsis OE-1，OE-2 and OE-3 were three transgenic lines. The same below. AtCHS，AtF3H，AtDFR，AtANS and AtUFGT were anthocyanin synthesis structural genes.

图6 MhCLE8转基因苹果愈伤组织（OE-1、OE-2和OE-3）的PCR鉴定与定量检测

Fig.6 PCR identification and qRT-PCR detection of MhCLE8 expression in transgenic apple calli（OE-1，OE-2 and OE-3）

图7 过表达MhCLE8的苹果愈伤组织

Fig. 7 Overexpression of MhCLE8 in apple calli

图8 过表达MhCLE8的苹果愈伤组织花青苷积累

Fig. 8 Anthocyanin accumulation in transgenic apple calli

图9 过表达MhCLE8苹果愈伤组织中花青苷合成结构基因的相对表达量 MdCHS、MdF3H、MdDFR、MdANS1和MdUFGT为花青苷合成结构基因。

Fig.9 The relative expression of anthocyanin synthesis structural genes in transgenic apple calli MdCHS，MdF3H，MdDFR，MdANS1 and MdUFGT were anthocyanin synthesis structural genes.

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