Cloning of the Promoter of MATE40 Gene from Prunus sibirica Seeds and Analysis of Gene Expression Driven by Promoter

doi:10.13560/j.cnki.biotech.bull.1985.2024-0423

Abstract

Abstract:

【Objective】 The transporter protein PsMATE40 has been identified with an important role in the transport of seed amygdalin of Prunus sibirica. The aim of this study is to clone the specific promoter of PsMATE40 gene from P. sibirica seeds and to explore PsMATE40 expression driven by its native promoter, which should provide an important foundation for the application of PsMATE40 and its specific promoter. 【Method】 Based on our recent obtained sequence of PsMATE40 gene from the P. sibirica seeds, the full-length promoter fragment（designated as Pro_PsMATE40）of PsMATE40 gene was cloned by genome walking technique with genomic DNA of P. sibirica seeds as a template, and the cis-acting elements were identified within its promoter region by online programs. The GUS staining analysis was conducted to examine transcriptional activity of PsMATE40 gene promoter. Importantly, the plant expression vectors ofPsMATE40 driven self-specific promoter（Pro_PsMATE40）and constitutive promoter（CaMV 35S）were constructed by the seamless cloning. Then it was transiently transformed into tobacco leaves by Agrobacterium-mediated method, and the transcriptional expression of PsMATE40 gene driven by them was comparatively analyzed. 【Result】 The full-length promoter sequence of PsMATE40 gene from P. sibirica seeds was 1 964 bp in length, containing two core promoter regulatory elements（CAAT-box and TATA-box）and several cis-acting regulatory elements in response to the hormone（auxin, jasmone acid and salicylic acid）, stress（light, drought and wounding）and seed development, and importantly, the transcriptional level of PsMATE40 driven by its self-specific promoter（Pro_PsMATE40）was significantly higher compared with under the constitutive promoter（CaMV35S）. 【Conclusion】 The expression of PsMATE40 gene from P. sibirica seeds is regulated by the complicated factors involved in plant hormones and biotic and abiotic stresses, and the self-specific promoter could effectively promote transcriptional expression of PsMATE40 gene.

Key words: Prunus sibirica seed, amygdalin, PsMATE40 gene, specific promoter, cloning, cis-acting regulatory elements, promoter-driven expression analysis

WANG Zi-rui, LIU Xiao-han, XIU Yu, LIN Shan-zhi. Cloning of the Promoter of MATE40 Gene from Prunus sibirica Seeds and Analysis of Gene Expression Driven by Promoter[J]. Biotechnology Bulletin, 2024, 40(11): 192-201.

Figures/Tables 9

References 29

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PCR扩增轮数Rounds of PCR amplification	PCR扩增程序PCR amplification procedure
第一轮PCR	94℃ 1 min; 98℃ 1 min; 94℃ 30 s, 65℃ 1 min, 72℃ 4 min, 5个循环; 94℃ 30 s, 25℃ 3 min, 72℃ 2 min; 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min
第二轮PCR	94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min
第三轮PCR	94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min

PCR扩增轮数Rounds of PCR amplification	PCR扩增程序PCR amplification procedure
第一轮PCR	94℃ 1 min; 98℃ 1 min; 94℃ 30 s, 65℃ 1 min, 72℃ 4 min, 5个循环; 94℃ 30 s, 25℃ 3 min, 72℃ 2 min; 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min
第二轮PCR	94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min
第三轮PCR	94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 98℃ 30 s, 65℃ 1 min, 72℃ 2 min, 94℃ 30 s, 65℃ 1 min, 72℃ 2 min, 15个循环; 72℃ 10 min

表达载体 Expression vector	引物Primer	引物序列Primer sequence（5'-3'）
pBI121-ProPsMATE40::GUS	Q121 F	TATGACCATGATTACGCCAAGCTT CACATCTTCCTCCAAATC
pBI121-ProPsMATE40::GUS	Q121 R	CTGACCACCCGGGGATCCTCTAGACATTTAGGGAAGAAGCCA
pBI121-35S::PsMATE40av	35S-121 F	TGGAGAGAACACGGGGGACTCTCCCTGCTTAATTTGCTCCACCCC
pBI121-35S::PsMATE40av	35S-121 R	TGGTGGCGACCGGTACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT
pBI121-ProPsMATE40::PsMATE40	MATE-121 F	GAACACGGGGGACTCTAGAGCTCCCTGCTTAATTTGCTCCACCCC
pBI121-ProPsMATE40::PsMATE40	MATE-121 R	TAAGGGACTGACCACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT

表达载体 Expression vector	引物Primer	引物序列Primer sequence（5'-3'）
pBI121-ProPsMATE40::GUS	Q121 F	TATGACCATGATTACGCCAAGCTT CACATCTTCCTCCAAATC
pBI121-ProPsMATE40::GUS	Q121 R	CTGACCACCCGGGGATCCTCTAGACATTTAGGGAAGAAGCCA
pBI121-35S::PsMATE40av	35S-121 F	TGGAGAGAACACGGGGGACTCTCCCTGCTTAATTTGCTCCACCCC
pBI121-35S::PsMATE40av	35S-121 R	TGGTGGCGACCGGTACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT
pBI121-ProPsMATE40::PsMATE40	MATE-121 F	GAACACGGGGGACTCTAGAGCTCCCTGCTTAATTTGCTCCACCCC
pBI121-ProPsMATE40::PsMATE40	MATE-121 R	TAAGGGACTGACCACCCGGGCGCTCAAGTTGCTGTCCATCTGTTT

基因 Gene	引物 Primer	序列 Sequence（5'-3'）
GUS	GUS-F	GAATACGGCGTGGATACGTTAG
GUS	GUS-R	GATCAAAGACGCGGTGATACA
PsMATE40	PsMATE40-F	AACAATGGCATCCAAGTTTTCGCCT
PsMATE40	PsMATE40-R	GGAGCACACCAGTCAAAGTTAAGAG
Actin	NtActin-F	CTGCTGGAATTCACGAAACA
Actin	NtActin-R	GCCACCACCTTGATCTTCAT