Cloning and Expression Pattern Analysis of ZmPAP26b Gene in Maize under Drought Stress

doi:10.7668/hbnxb.20194777

Abstract

Abstract:

Drought stress has a serious effect on the growth and development of maize,which leads to a decrease of maize yield.Purple acid phosphatase is a phospholipase protein involved in many physiological and biochemical functions of plants.In order to further study the role of purple acid phosphatase family genes in the process of stress resistance of maize,this paper explored the response mode of ZmPAP26b gene under drought stress,and Real-time fluorescence Quantitavive analysis was used to analyze the relative gene expression in different maize inbreeding lines under simulated drought conditions;ZmPAP26b(GenBank:NC_050104.1)was cloned from maize,and PAP genes in Zea mays,Arabidopsis thaliana,Oryza sativa L.,Triticum aestivum L.,Sorghum bicolor and Brachypodium distachyon were identified and bioinformatic analysis was performed.Meanwhile,prokaryotic overexpression strains were constructed for functional verification.The results showed that the expression of this gene decreased in drought tolerant materials and increased in drought sensitive materials under drought stress.The CDS length of this gene was 1 431 bp,encoding 476 amino acids.A total of 228 PAP genes were found in six species,divided into 4 subfamilies by phylogenetic analysis.The 19 PAP genes in maize were distributed on 9 chromosomes and had similar conserved domains.Analysis of promoter cis-acting elements showed that they contained elements responding to drought and hormones.Prokaryotic expression experiments showed that the growth of strains containing the recombinant plasmid pET28a-ZmPAP26b was inhibited compared with non-loaded strains under 10% PEG-6000 and 15%PEG-6000 simulated drought stress.In summary,it is speculated that ZmPAP26b is negatively regulated under drought stress.

Key words: Maize, ZmPAP, Drought stress, Prokaryotic expression, Bioinformatics

GUO Zhaoyang, YIN Yuhang, LIU Yu, XIE Yitong, PEI Yuhe, SONG Xiyun, ZHAO Meiai. Cloning and Expression Pattern Analysis of ZmPAP26b Gene in Maize under Drought Stress[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 10-19. doi: 10.7668/hbnxb.20194777.

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Figures/Tables 11

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
ZmPAP26b-qPCR-F	TTCCTGGGGTCGATTCGTTG
ZmPAP26b-qPCR-R	CCACAGCCATTGAGGAGTGT
ZmPAP26b-F	ATGAGGGGCTGGGGTTTG
ZmPAP26b-R	CTATAATAGGGAGATCAAATCCTCAAGG
BamH I-ZmPAP26b-F	CAGCAAATGGGTCGCGGATCCATGAGGGGCTGGGGTTTG
Sac I-ZmPAP26b-R	GCAAGCTTGTCGACGGAGCTCCTATAATAGGGAGATCAAATCCTCAAGG
Actin1-F	GTCCATGAGGCCACGTACAA
Actin1-R	CCGGACCAGTTTCGTCATA

Fig.1 The relative expression of ZmPAP26b A.Tissue-specific analysis of ZmPAP26b;B.Relative expression of ZmPAP26b in different maize under drought stress. The different lowercase letters indicate the significant differences at P<0.05.

Fig.2 Comparison of ZmPAP26b partial homologous amino acid sequences in six species

Fig.3 Phylogenetic tree of ZmPAP homologous genes in six species

Fig.4 Chromosome localization of PAP gene family members in maize

Fig.5 Analysis of conserved motif,conserved domain and gene structure of maize PAP gene family

Fig.6 Analysis of promoter cis-elements in maize PAP gene family

Tab.2 Cis-acting elements of PAP genes promoter in maize

顺式作用元件Cis-element	序列Sequence	功能Function
TC-rich repeats	GTTTTCTTAC/ATTCTCTAAC	参与防御和应激反应
LTR	CCGAAA	参与低温反应
MBS	CAACTG	MYB结合位点参与干旱诱导
TATC-box	TATCCCA	参与赤霉素反应
TCA-element	CCATCTTTTT/TCAGAAGAGG	参与水杨酸反应
ABRE	ACGTG/CACGTG	参与脱落酸反应
CGTCA-motif	CGTCA	参与MeJA反应
AuxRR-core	GGTCCAT	参与生长素反应
RY-element	CATGCATG	参与种子特异性调控
CAT-box	GCCACT	分生组织表达
motif I	gGTACGTGGCG	根特异性表达
GCN4_motif	TGAGTCA	参与胚乳表达
HD-Zip 1	CAAT(A/T)ATTG	参与栅栏叶肉细胞分化
ACE	GCGACGTACC	光响应性

Fig.7 Cloning of ZmPAP26b and vector construction A.ZmPAP26b gene amplification(M.DL2000 DNA Marker);B.pCE2-ZmPAP26b bacterial liquid PCR;C.pET28a-ZmPAP26b bacterial liquid PCR;D.Double digestion of prokaryotic expression vector pET28a-ZmPAP26b (M1.DL15000 DNA Marker;M2.DL2000 DNA Marker).

Fig.8 Sequencing results of intermediate vector pCE2-ZmPAP26b

Fig.9 Growth curves of recombinant plasmid strain and pET28a strain under drought treatment

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