Cloning and Functional Analysis of Pineapple AcZFP1 Gene Under Low Temperature Stress

doi:10.16420/j.issn.0513-353x.2023-0089

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (3): 495-508.doi: 10.16420/j.issn.0513-353x.2023-0089

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

Cloning and Functional Analysis of Pineapple AcZFP1 Gene Under Low Temperature Stress

LUO Jiandong¹, QIU Mengqing¹, ZHOU Huimin¹, XIE Weiwei¹, Huang Haixin¹, LIU Jieqi¹, ZHANG Zimin¹, XU Jian², CHEN Chengjie¹, HE Yehua¹, LIU Chaoyang¹^,^*()

¹ Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in South China，Ministry of Agriculture and Rural Affairs，Colleage of Horticulture，South China Agricultural University Guangzhou 510642，China
² Guangxi South Subtropical Agricultural Science Research Insititute，Chongzuo，Guangxi 532415，China

Received:2023-04-24 Revised:2023-11-30 Online:2024-03-25 Published:2024-03-22
Contact: LIU Chaoyang

Abstract

Abstract:

In this study，a C2H2 zinc finger proteins gene，named AcZFP1，was obtained from ‘Shenwan’pineapple. The open reading frame of AcZFP1 was 816 bp，which encoded 271 amino acids，and contained two typical zinc finger protein domains. The phylogenetic tree analysis showed that AcZFP1 shared the closest relationship with OsZFP252 and OsMSR15. Gene expression pattern analysis showed that AcZFP1 could be induced by several kinds of stress and hormonal treatment like cold，NaCl，PEG，ABA，SA and MeJA. Subcellular localization assays showed that AcZFP1 located in the nucleus. The AcZFP1-overexpressing（OE）Arabidopsis lines have higher survival rate and F_v/F_m under low temperature stress compared to wild type Arabidopsis. Moreover，the OE lines also showed higher level of soluble protein content，higher activity of superoxide dismutase（SOD）and peroxidase（POD），lower level of malondialdehyde（MDA）content. Further qRT-PCR analysis indicated that AcZFP1 could promote the expression of four typical Arabidopsis endogenous cold-responsive genes such as AtRD29A and AtCOR47，and play a positive regulatory role in low temperature stress.

Key words: pineapple, AcZFP1, low temperature stress, function analysis

LUO Jiandong, QIU Mengqing, ZHOU Huimin, XIE Weiwei, Huang Haixin, LIU Jieqi, ZHANG Zimin, XU Jian, CHEN Chengjie, HE Yehua, LIU Chaoyang. Cloning and Functional Analysis of Pineapple AcZFP1 Gene Under Low Temperature Stress[J]. Acta Horticulturae Sinica, 2024, 51(3): 495-508.

Figures/Tables 12

Table 1 Primers and sequences used in this study

用途 Usage		引物名称 Primer name	引物序列（5′-3′） Sequence
基因克隆 Gene cloning		AcZFP1-F AcZFP1-R	ATGGCAATTGATGCTTTAGA TCAAGCCGGGATTAGGAGCC
亚细胞定位 Subcellular localization	C18-AcZFP1-F C18-AcZFP1-R		cggtatcgataagcttATGGCAATTGATGCTTTAGA cgggctgcaggaattcAGCCGGGATTAGGAGCCGG
转化植株PCR验证 PCR assay of the transgenic plants		Det-AcZFP1-F Det-AcZFP1-R		CGCAGTACAAGTGCTCCGTA CGGGGCTTCTTGAAAGGGT
实时荧光定量PCR Fluorescent quantitative real-time PCR（qRT-PCR）	qAcZFP1-F qAcZFP1-R qAcActin-F qAcActin-R qAtActin-F qAtActin-R qAtKIN1-F qAtKIN1-R qAtKIN2-F qAtKIN2-R qAtCOR47-F qAtCOR47-R qAtRD29A-F qAtRD29A-R		GTGCCACTACGAAGGAACGA GTTTTGCACCCTCAAACCCT CTGGCCTACGTGGCACTTGACTT CACTTCTGGGCAGCGGAACCTTT CCTTCGTCTTGATCTTGCGG AGCGATGGCTGGAACAGAAC ACCAACAAGAATGCCTTCCA CCGCATCCGATACACTCTTT GTCAGAGACCAACAAGAATGCC TGACTCGGATCGCTACTTGTTC CAGTGTCGGAGAGTGTGGTG ACAGCTGGTGAATCCTCTGC GCCGAGAAACTTCAGATTGG CCATTCCTCCTCCTCCTTTC

Fig. 1 PCR amplification results of the AcZFP1 gene in pineapple‘Shenwan’

Fig. 2 Protein sequence alignment of AcZFP1 and ZFP proteins in other species Ph：Petunia hybrida；Sl：Solanum lycopersicum；Ac：Ananas comosus；Os：Oryza sativa；Th：Thellungiella halophila. The red box indicates the highly conserved sequences peculiar to zinc finger proteins in plants.

Fig. 3 Phylogenetic tree analysis of pineapple AcZFP1 and ZFP proteins in other plant species Numbers at node represent bootstrap support values.

Table 2 Analysis of cis-elments in the promoter of AcZFP1

作用元件 Functional element	序列 Sequence	位点功能 Function of site	位置/bp Location
LTRE	CCGAC	低温响应元件Low temperature responsive element	+627，+194
MBS	CAACTG	与干旱诱导相关的MYB绑定位点 MYB binding site involved in drought-inducibility	-316
ABRE	ACGTC	参与ABA响应cis-Acting element involved in the abscisic acid responsiveness	-838，-1 495， +1 366
CGTCA-motif	CGTCA	参与MeJA响应 cis-Acting element involved in the MeJA-responsiveness	+876，+1509
TGACG-motif	TGACG	参与MeJA响应cis-Acting element involved in the MeJA-responsiveness	-876，-1 509
P-box	CCTTTTG	赤霉素应答元件Gibberellin-responsive element	+219，-1 054
SARE	TGACG	水杨酸响应元件Salicylic acid -responsive promoter element	-881，-1 514
G-box	CACGTG	参与光响应cis-Acting regulatory element involved in light responsiveness	-1 365
TCT-motif	TCTTAC	光响应元件的一部分Part of a light responsive element	-722
TCCC-motif	TCTCCCT	光响应元件的一部分Part of a light responsive element	+1 830，+1 858

Fig. 4 The subcellular localization of AcZFP1 The green fluorescence indicates the location of the fusion protein，and the red fluorescence indicates the location of the nucleus. If the two fluorescence signals are in the same place，the fluorescence signal will be yellow after superposition.

Fig. 5 Expression of AcZFP1 in different tissues（A）and expression under low temperature，NaCl，PEG，ABA，SA and MeJA treated（B） Asterisks indicate significant differences from control（0 h）（P < 0.05）.

Fig. 6 PCR verification of Arabidopsis thaliana overexpressing AcZFP1（OE）（A）and observation of green fluorescent protein（B）

Fig. 7 Morphological characteristics（A）and Fv/Fm chlorophyll fluorescence image（B）of wild type（WT）and overexpressing AcZFP1 Arabidopsis thaliana lines（OE）under cold treatment

Fig. 8 Comparison of survival rate（A）and Fv/Fm（B）in wild type（WT）and overexpressing AcZFP1 Arabidopsis thaliana lines（OE）under cold treatment Asterisks indicate significant differences from wild type（WT）（P < 0.05）. The same below.

Fig. 9 POD and SOD activities，soluble protein and MDA content of wild-type（WT）and overexpressing AcZFP1 Arabidopsis thaliana lines（OE）under low temperature treatment conditions

Fig. 10 Expression of cold-responsive genes in wild type（WT）and the transgenic lines（OE）under low temperature treatment

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Cloning and Functional Analysis of Pineapple AcZFP1 Gene Under Low Temperature Stress

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