Cloning,Expression Characterization and Functional Analysis of AbAIL5 in Promoting Callus Regeneration in Amorphophallus bulbifer

doi:10.7668/hbnxb.20196232

Abstract

Abstract:

AINTEGUMENTA-LIKE 5(AIL5)is one of the key genes regulating plant growth and development during somatic embryogenesis.To explore the function of the AbAIL5 gene in the somatic embryogenesis of Amorphophallus bulbifer and to accelerate its molecular breeding process,the AbAIL5 gene was cloned from the bulbils of A.bulbifer,and its nucleotide sequence and protein structure were analyzed using bioinformatics tools.The open reading frame(ORF)of AbAIL5 was 654 bp in length,encoding 217 amino acids.Bioinformatics analysis indicated that the AbAIL5 protein was a hydrophilic protein containing a conserved AP2 superfamily domain,without transmembrane domains or signal peptides.Phylogenetic analysis showed that AbAIL5 was most closely related to that of Platanthera zijinensis.Subcellular localization analysis revealed that the protein resides specifically within the cell nucleus.Regarding expression patterns,qRT-PCR analysis revealed that AbAIL5 was expressed in the aerial parts(leaves,petioles,and bulbils)of A.bulbifer,with higher expression levels observed in the early stages of bulbil development.The relative expression levels in petioles,leaves,and bulbils were 1.00,0.55,and 1.42,respectively.For functional verification,an AbAIL5 overexpression vector was constructed and transformed into A.bulbifer callus mediated by Agrobacterium tumefaciens.After 30 days of culture,the AbAIL5 overexpression group showed significantly increased growth rates and germination numbers,as well as an improved overall survival rate compared with the control group.At 70 days,plant growth in the overexpression group was significantly superior to that of the control.qRT-PCR results confirmed that the expression level in the overexpression group was 5.34-fold higher than that of the control.These results indicated that AbAIL5 facilitates adventitious bud differentiation and callus proliferation,providing a theoretical basis for revealing the function of AbAIL5 in the development of A.bulbifer and for improving its genetic transformation efficiency.

Key words: Amorphophallus bulbife, AbAIL5 gene, Bioinformatics analysis, Gene cloning, Molecular characteristics

CLC Number:

LIU Yaxin, TIAN Zhen, HE Jindi, YANG Liangrui, LI Zemei, WU Xuewei. Cloning,Expression Characterization and Functional Analysis of AbAIL5 in Promoting Callus Regeneration in Amorphophallus bulbifer[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 82-90. doi: 10.7668/hbnxb.20196232.

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Figures/Tables 14

Tab.1 Online websites for bioinformatics analysis

分析工具 Analysis tools	网址 URL
Expasy	https://www.expasy.org/
TMHMM-2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
SignalP-5.0	https://services.healthtech.dtu.dk/services/SignalP-5.0/
NetPhos-3.1	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
Sopma	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html
Swiss-model	https://swissmodel.expasy.org/

Fig.1 The prediction of cDNA sequence deduced and amino acid sequence of AbAIL5 overall length

Fig.2 PCR products of AbAIL5 gene

Fig.3 Hydrophilic/Hydrophobic analysis of AbAIL5 protein

Fig.4 Predicted transmembrane segments of AbAIL5

Fig.5 Predicted signal peptide of AbAIL5 amino acid sequences

Fig.6 Prediction of AbAIL5 potential phosphorylation sites

Fig.7 Secondary structure of AbAIL5 protein Different colors indicate distinct secondary structural elements:blue represents alpha helix,purple represents extended strand,green represents beta turn,and yellow represents random coil.

Fig.8 Tertiary structure of AbAIL5 protein

Fig.9 Analysis of AbAIL5 protein domain of A.bulbifer

Fig.10 Analysis of the phylogenetic tree of A.bulbifer AbAIL5 protein in other species

Fig.11 Expression patterns of the AbAIL5 gene in different tissues of A.bulbifer (A) and expression patterns of the AbAIL5 gene during four developmental stages of leaf bulbils in A.bulbifer (B) Capital letters indicate differences and significance at the 0.01 level;lowercase letters indicate no significant difference.

Fig.12 Results of subcellular localization of AbAIL5 protein in A.bulbifer

Fig.13 Fluorescence observation,phenotypic analysis,and molecular identification of AbAIL5 overexpression callus A.EGFP fluorescence signals of empty vector and AbAIL5 overexpression;B.Growth status of callus after 30 d and 70 d:a,b.Phenotypes of empty vector and overexpression after 30 d,e,f.Phenotypes of empty vector and overexpression after 70 d;C.Molecular verification of transgenic callus:a.qPCR results of empty vector and AbAIL5 overexpression after 30 d,b.PCR verification of positive callus,lane descriptions in the electrophoresis gel are as follows(M.DL2000 DNA Marker,1.AbAIL5 overexpression,2.Empty vector CK,3.Wild type,4.AbAIL5 overexpression Agrobacterium AGL1,5.Empty vector Agrobacterium AGL1).

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