珠芽魔芋<i>AbAIL5</i>基因克隆、表达特性及促进愈伤再生功能解析

doi:10.7668/hbnxb.20196232

摘要/Abstract

摘要：

体胚发生过程中AIL5是控制植物生长发育的关键基因之一,为了挖掘AbAIL5基因在珠芽魔芋体细胞胚胎发生过程中的功能,加快魔芋分子育种的进程,用珠芽魔芋叶面球为材料,克隆该基因并进行生物信息学分析,解析其核苷酸序列与蛋白结构模式。珠芽魔芋AbAIL5基因的ORF长度654 bp,编码217个氨基酸,为包含一个保守AP2超家族结构域的亲水性蛋白,不含跨膜结构域或信号肽。进化树分析结果表明,珠芽魔芋AbAIL5与紫金舌唇兰的亲缘关系最近。亚细胞定位显示其位于细胞核中。通过qRT-PCR分析,AbAIL5在魔芋地上部(叶片、叶柄和叶面球)中都有表达,叶面球发育初期表达量较高,叶柄、叶与叶面球相对表达量分别为1.00,0.55,1.42。为进行功能验证,成功获得AbAIL5基因,构建过表达载体,通过农杆菌介导法转入珠芽魔芋愈伤组织,AbAIL5过表达后30 d生长速率及发芽数明显提高,且总体成活率提高,70 d后植株长势明显优于对照,初步判断其对促进遗传转化有一定促进作用。qRT-PCR结果显示,AbAIL5过表达组表达水平是对照组表达水平的5.34倍,说明AbAIL5基因对不定芽分化及愈伤组织增殖有一定促进作用,为揭示珠芽魔芋AbAIL5基因在发育过程的功能研究及提高珠芽魔芋遗传转化率提供依据。

关键词: 珠芽魔芋, AbAIL5基因, 生物信息学分析, 基因克隆, 分子特征

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

中图分类号:

刘亚昕, 田震, 何金娣, 杨梁蕊, 李泽美, 吴学尉. 珠芽魔芋AbAIL5基因克隆、表达特性及促进愈伤再生功能解析[J]. 华北农学报, 2026, 41(3): 82-90. doi: 10.7668/hbnxb.20196232.

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.

http://www.hbnxb.net/CN/Y2026/V41/I3/82

图/表 14

表1 生物信息分析在线网站

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/

图1 AbAIL5基因预测cDNA序列及氨基酸序列

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

图2 AbAIL5基因的PCR产物

Fig.2 PCR products of AbAIL5 gene

图3 AbAIL5蛋白亲水/疏水性分析

Fig.3 Hydrophilic/Hydrophobic analysis of AbAIL5 protein

图4 AbAIL5蛋白跨膜结构域预测

Fig.4 Predicted transmembrane segments of AbAIL5

图5 AbAIL5氨基酸序列的信号肽预测

Fig.5 Predicted signal peptide of AbAIL5 amino acid sequences

图6 AbAIL5蛋白的氨基酸磷酸化位点预测

Fig.6 Prediction of AbAIL5 potential phosphorylation sites

图7 AbAIL5蛋白的二级结构预测不同颜色代表不同的二级结构元件:蓝色代表α-螺旋,紫色代表延伸链,绿色代表β-转角,黄色代表无规则卷曲。

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.

图8 AbAIL5蛋白的三级结构预测

Fig.8 Tertiary structure of AbAIL5 protein

图9 珠芽魔芋AbAIL5蛋白结构域分析

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

图10 珠芽魔芋与其他物种AbAIL5蛋白的系统进化树分析

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

图11 珠芽魔芋AbAIL5基因在不同部位表达模式情况(A)与叶面球4个时期表达模式情况(B) 不同大写字母表示在0.01水平上差异极显著;相同小写字母表示差异不显著。

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.

图12 珠芽魔芋AbAIL5蛋白亚细胞定位结果

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

图13 AbAIL5过表达愈伤组织的荧光观察、表型分析及分子鉴定 A.空载与AbAIL5过表达EGFP荧光信号;B.30 d与70 d后愈伤组织生长情况:a,b.空载与过表达30 d后的表型,e,f.空载与过表达70 d后的表型;C.转基因愈伤组织的分子水平验证:a.30 d后空载与AbAIL5过表达qPCR结果,b.阳性愈伤组织PCR验证(M.DL2000 DNA Marker,1.AbAIL5过表达,2.空载CK,3.野生型,4.AbAIL5过表达农杆菌AGL1,5.空载农杆菌AGL1)。

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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珠芽魔芋AbAIL5基因克隆、表达特性及促进愈伤再生功能解析

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

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珠芽魔芋AbAIL5基因克隆、表达特性及促进愈伤再生功能解析

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

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