菠萝AcZFP1的克隆及其在低温胁迫下的功能分析

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

摘要/Abstract

摘要：

以‘神湾’菠萝为材料，克隆了1个C2H2型锌指蛋白基因，命名为AcZFP1，其开放阅读框为816 bp，编码271个氨基酸，含有2个典型的锌指蛋白结构域。进化树分析表明AcZFP1与水稻OsZFP252和OsMSR15的亲缘关系最近。基因表达分析发现，AcZFP1受低温、NaCl和聚乙二醇（PEG）干旱等多种逆境胁迫及外源ABA、SA和MeJA的诱导。试验表明，AcZFP1蛋白定位于细胞核中。AcZFP1过表达的拟南芥株系在低温处理后相比野生型对照有更高的成活率和F_v/F_m值，可溶性蛋白含量增加，SOD、POD酶活增强，MDA含量降低，植株的耐寒性显著增强。进一步的qRT-PCR分析显示，AcZFP1可以促进AtRD29A、AtCOR47等4个典型的拟南芥内源冷应答基因的表达，在低温胁迫中发挥正调控作用。

关键词: 菠萝, AcZFP1, 低温胁迫, 功能分析

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

罗健东, 邱梦青, 周慧敏, 解为玮, 黄海鑫, 刘洁琪, 张梓敏, 徐健, 陈程杰, 何业华, 刘朝阳. 菠萝AcZFP1的克隆及其在低温胁迫下的功能分析[J]. 园艺学报, 2024, 51(3): 495-508.

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.

图/表 12

表1 本研究中所用引物及序列

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

图1 ‘神湾’菠萝AcZFP1的PCR扩增

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

图2 菠萝 AcZFP1与其他物种中锌指蛋白的序列比对 Ph：矮牵牛；Sl：番茄；Ac：菠萝；Os：水稻；Th：小盐芥。红色框：植物锌指蛋白特有的高度保守序列。

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.

图3 菠萝AcZFP1与其他物种ZFP蛋白系统进化树进化树节点处的数字代表置信度。

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

表2 AcZFP1启动子顺式作用元件

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

图4 AcZFP1蛋白的亚细胞定位绿色荧光表示融合蛋白的位置，红色荧光表示细胞核所在位置。若两种荧光信号在同一处，叠加后荧光信号变为黄色。

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.

图5 AcZFP1的组织表达（A）和在低温、NaCl、PEG、ABA、SA、MeJA处理下的表达（B） * 表示与对照（0 h）有显著差异（P < 0.05）。

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）.

图6 拟南芥过表达AcZFP1株系（OE）PCR验证（A）与绿色荧光蛋白观测（B）

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

图7 冷处理条件下拟南芥野生型（WT）和过表达AcZFP1株系（OE）的表型（A）与Fv/Fm叶绿素荧光图像（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

图8 过表达 AcZFP1拟南芥（株系OE）与野生型（WT）在冷处理后的存活情况（A）和Fv/Fm比较（B） * 表示与野生型（WT）有显著差异（P < 0.05）。下同。

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.

图9 低温处理条件下野生型（WT）和过表达AcZFP1拟南芥（OE）的POD、SOD活性，可溶性蛋白及MDA含量

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

图10 低温处理条件下冷应答相关基因在野生型（WT）和转基因株系（OE）中的表达

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

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