Function Study of Peptide Coding Gene MhCLE8 in Regulating Anthocyanin Accumulation in Apple（Malus × domestica）

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (12): 2541-2550.doi: 10.16420/j.issn.0513-353x.2021-0717

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles

Function Study of Peptide Coding Gene MhCLE8 in Regulating Anthocyanin Accumulation in Apple（Malus × domestica）

SHI Yan, ZHANG Tian'en, ZHU Baihui, LI Tong, LI Xiuming^**(), YOU Chunxiang^**()

National Key Laboratory of Wheat Improvement，College of Horticultural Science and Engineering，Shandong Agricultural University，Tai'an，Shandong 271018，China

Received:2023-07-16 Revised:2023-10-10 Online:2023-12-25 Published:2023-12-29
Contact: LI Xiuming, YOU Chunxiang

Abstract

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

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.

Figures/Tables 10

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

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

Fig. 2 MhCLE8p treatment inhibit anthocyanin accumulation in apple calli

Fig. 3 Gel electrophoresis of the PCR product of MhCLE8

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

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.

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

Fig. 7 Overexpression of MhCLE8 in apple calli

Fig. 8 Anthocyanin accumulation in transgenic apple calli

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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Function Study of Peptide Coding Gene MhCLE8 in Regulating Anthocyanin Accumulation in Apple（Malus × domestica）

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