西瓜脱落酸受体蛋白基因<i>ClPYL8</i>的克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20196279

摘要/Abstract

摘要：

脱落酸(ABA)受体蛋白PYL作为ABA信号转导的起始组分积极参与植物体内ABA介导的信号转导,在调控植株抵御逆境胁迫中具有重要功能。前期研究依据转录组数据筛选出西瓜对盐胁迫负响应的基因Cla97C07G133100.1,命名为ClPYL8。为进一步探讨西瓜脱落酸受体基因ClPYL8的生物学功能,以西瓜TN07011为试验材料,采用生物信息学、亚细胞定位及RT-qPCR等方法对ClPYL8的结构和功能进行解析。结果表明:ClPYL8基因的开放阅读框为702 bp,编码233个氨基酸。蛋白保守结构域预测ClPYL8含有PYR/PYL/RCAR-like保守结构域,属于SPRBCC superfamily超家族。启动子顺式作用元件分析表明,ClPYL8启动子中含有逆境响应、生长素响应、光响应等多种元件。采用无缝克隆技术构建EGFP融合表达载体并转入烟草,结果显示,ClPYL8定位于细胞核和细胞膜。组织特异性(RT-qPCR)分析显示,该基因在种子中的表达量最高,在果实和雌花中的表达量最低;在盐胁迫下,ClPYL8的表达量呈现下调表达趋势;在干旱胁迫下,该基因的表达量呈现先下降后上升的表达趋势;在低温胁迫下,该基因的表达量呈现先上升后下降的表达趋势。综上,ClPYL8积极响应非生物胁迫,可能通过脱落酸信号转导途径调控西瓜的逆境响应,推测其在盐胁迫中可能起负调控作用,在干旱和低温胁迫下可能起正调控作用。

关键词: 西瓜, ClPYL8, 载体构建, 亚细胞定位, 非生物胁迫

Abstract:

The abscisic acid(ABA)receptor protein PYL,as the initial component of ABA signaling transduction,actively participated in ABA-mediated signaling transduction in plants and played a crucial role in regulating plant resistance to stress.In our previous study,based on transcriptome data,the gene Cla97C07G133100.1,which was negatively responsive to salt stress in watermelon,was screened out and named ClPYL8.To further explore the biological function of the watermelon ABA receptor ClPYL8,using watermelon TN07011 as the experimental material,the structure and function of ClPYL8 were analyzed using bioinformatics,subcellular localization,and RT-qPCR methods.The results showed that the open reading frame of ClPYL8 gene was 702 bp,encoding 233 amino acids.Protein conserved domain prediction revealed that ClPYL8 contained the PYR/PYL/RCAR-like conserved domain,belonging to the SPRBCC superfamily.The promoter cis-acting element analysis indicated that the ClPYL8 promoter contained various elements responsive to stress,auxin,and light.The seamless cloning technique was used to construct the EGFP fusion expression vector,which was then transformed into tobacco.The results showed that ClPYL8 was localized in the nucleus and cell membrane.Tissue-specific(RT-qPCR)analysis revealed that the gene exhibited the highest expression level in seeds and the lowest in fruits and female flowers;under salt stress,the expression of ClPYL8 showed a downregulation trend;under drought stress,the gene expression first decreased and then increased;under low-temperature stress,the gene expression first increased and then decreased.In conclusion,ClPYL8 actively responded to abiotic stress and might regulate watermelon's stress response through the ABA signaling pathway,suggesting a potential negative regulatory role in salt stress and positive regulatory roles in drought and low-temperature stress.

Key words: Watermelon, ClPYL8, Vector construction, Subcellular localization, Abiotic stress

中图分类号:

邓满, 高英, 杨柏明, 张卫华, 蔡泽宇, 周歆茗, 吴颖. 西瓜脱落酸受体蛋白基因ClPYL8的克隆、亚细胞定位及表达分析[J]. 华北农学报, 2026, 41(2): 21-27. doi: 10.7668/hbnxb.20196279.

DENG Man, GAO Ying, YANG Baiming, ZHANG Weihua, CAI Zeyu, ZHOU Xinming, WU Ying. Cloning,Subcellular Localization,and Expression Analysis of Watermelon Abscisic Acid Receptor Protein Gene ClPYL8[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 21-27. doi: 10.7668/hbnxb.20196279.

http://www.hbnxb.net/CN/Y2026/V41/I2/21

图/表 7

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
ClPYL8-F	ATGCCTACTTCCATTCAACTT
ClPYL8-R	TTAGGAAGTTTTAGCCATGTTTTC
ClPYL8-MQ-F	acgggggacgagctcggtaccATGCCTACTTCCATTCAACTTCATC
ClPYL8-MQ-R	gctcaccatgtcgacggatccGGAAGTTTTAGCCATGTTTTCAGC
ClPYL8-Q-F	CATCGCCTACGAAACTAC
ClPYL8-Q-R	TAAGATTCCACCACAACC
ClACTIN-F	CCATGTATGTTGCCATCCAG
ClACTIN-R	GGATAGCATGGGGTAGAGCA

图1 ClPYL8基因克隆(A)和EGFP融合表达载体双酶切验证(B) A:M.DL2000 DNA Marker,1.ClPYL8的片段;B:M.DL10000 DNA Marker,1.重组质粒的双酶切验证。

Fig.1 Cloning of ClPYL8 gene(A)and double enzyme digestion validation of EGFP fluorescent expression vector(B) A:M.DL2000 DNA Marker,1.Fragment of ClPYL8;B:M.DL10000 DNA Marker,1.Double digestion verification of the recombinant plasmid.

图2 ClPYL8蛋白序列特征及高级结构预测 A.ClPYL8蛋白保守结构域;B.蛋白二级结构;C.蛋白三级结构;D.顺式作用元件;E.蛋白互作预测。

Fig.2 Sequence characteristics and advanced structure prediction of ClPYL8 protein A.ClPYL8 protein conserved domain;B.Protein secondary structure;C.Protein tertiary structure;D.cis-acting element;E.Protein-protein interaction prediction.

图3 西瓜ClPYL8蛋白和其他物种PYL蛋白的多序列比对

Fig.3 Multiple sequence alignment of watermelon ClPYL8 protein and other species PYL protein

图4 PYL的进化树分析

Fig.4 Phylogenetic tree analysis of PYL

图5 ClPYL8蛋白亚细胞定位

Fig.5 Subcellular localization of ClPYL8 protein

图6 ClPYL8在不同组织(A)、盐(B)、干旱(C)和低温(D)胁迫下的表达分析不同字母表示在P<0.05水平差异显著。

Fig.6 Expression analysis of ClPYL8 under different tissues(A),salt(B),drought(C),and low temperature(D)stresses Different letters indicate significant differences at the 0.05 level.

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西瓜脱落酸受体蛋白基因ClPYL8的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization,and Expression Analysis of Watermelon Abscisic Acid Receptor Protein Gene ClPYL8

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西瓜脱落酸受体蛋白基因ClPYL8的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization,and Expression Analysis of Watermelon Abscisic Acid Receptor Protein Gene ClPYL8

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