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

doi:10.7668/hbnxb.20196279

Abstract

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

CLC Number:

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.

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Figures/Tables 7

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

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.

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.

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

Fig.4 Phylogenetic tree analysis of PYL

Fig.5 Subcellular localization of ClPYL8 protein

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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