甘薯IbBADH基因家族全基因组鉴定与IbBADH1a上游转录调控因子筛选

doi:10.13560/j.cnki.biotech.bull.1985.2026-0119

摘要/Abstract

摘要：

目的对甘薯（Ipomoea batatas (L.) Lam.）甜菜碱醛脱氢酶（IbBADH）基因家族进行全基因组鉴定，分析其结构、进化及表达特征，并以IbBADH1a启动子为诱饵筛选逆境响应调控因子，为解析IbBADH介导的逆境响应网络奠定基础。方法基于甘薯基因组鉴定IbBADH家族成员，分析基因结构、系统发育关系及启动子顺式元件。结合转录组数据分析表达模式，克隆IbBADH1a启动子，构建pAbAi诱饵载体，利用酵母单杂交（Y1H）筛选甘薯块根cDNA文库，获得候选结合蛋白并进行点对点验证。结果鉴定到6个IbBADH成员。理化性质显示，IbBADH蛋白均呈酸性，不稳定指数均小于40，属亲水性蛋白，且预测主要定位于叶绿体。种内共线性分析检测到10对涉及IbBADH基因的片段重复基因对。种间共线性分析显示IbBADH基因与旋花科近缘种存在广泛的共线性关系。系统发育分析表明，IbBADH与旋花科BADH聚为4个类群。启动子分析显示，IbBADH家族成员启动子区域内富含逆境、激素响应顺式作用元件。表达模式分析表明，IbBADH1a在所有样本中表达水平最高。Y1H筛选获得4个逆境响应调控因子（BTF3、TSJT1、bHLH49、ERF073），点对点验证证实其均能与IbBADH1a启动子特异性结合。结论甘薯IbBADH家族成员在结构域与基因结构层面总体保守，其启动子富集逆境、激素响应相关顺式作用元件。IbBADH1a启动子受BTF3、TSJT1、bHLH49、ERF073调控，为解析IbBADH1a转录调控网络及抗逆相关机制提供了重要线索。

关键词: 甘薯, 甜菜碱醛脱氢酶, 顺式作用元件, 酵母单杂交, 互作蛋白

Abstract:

Objective The purpose of this study was to perform genome-wide identification of the betaine aldehyde dehydrogenase (IbBADH) gene family in sweetpotato (Ipomoea batatas (L.) Lam.), analyze its structure, evolution, and expression characteristics, and use the IbBADH1a promoter as bait to screen for stress response regulators, laying a foundation for analyzing the IbBADH-mediated stress response network. Method The IbBADH family members were identified based on the sweetpotato genome, and the gene structure, phylogenetic relationship, and promoter cis-elements were analyzed. The expression pattern was analyzed using transcriptome data, the IbBADH1a promoter was cloned, and the pAbAi bait vector was constructed. The sweetpotato root cDNA library was screened by yeast one-hybrid (Y1H) to obtain candidate binding proteins and perform point-to-point verification. Result Six IbBADH members were identified. The physicochemical properties showed that IbBADH protein was acidic, and the instability index was less than 40, indicating that the protein was hydrophilic, and it was predicted to be mainly located in chloroplasts. Intraspecific colinearity analysis detected 10 pairs of segmental duplication gene pairs involving the IbBADH gene. Interspecific collinearity analysis showed that the IbBADH gene showed extensive collinearity with related species of the Convolvulaceae family. Phylogenetic analysis showed that IbBADH and BADH proteins from Convolvulaceae clustered into four groups. Promoter analysis showed that the promoter regions of IbBADH family members were rich in stress and hormone response cis-acting elements. The expression pattern analysis indicates that IbBADH1a has the highest expression level among all the samples. Four stress response regulators (BTF3, TSJT1, bHLH49, ERF073) were obtained by Y1H screening, and point-to-point verification confirmed that they could specifically bind to the IbBADH1a promoter. Conclusion The IbBADH family members of sweetpotato are generally conserved at the level of domain and gene structure, and their promoters are rich in cis-acting elements related to stress and hormone response. The IbBADH1a was regulated by BTF3, TSJT1, bHLH49 and ERF073, and provide important clues for the analysis of IbBADH1a transcriptional regulatory network and stress resistance related mechanisms.

Key words: sweetpotato, betaine aldehyde dehydrogenase (BADH), cis-acting element, yeast one-hybrid, interacting proteins

周广立, 陈锦钗, 李鑫鑫, 武小平, 贾峥嵘, 阎昊, 贺立恒, 贾小云. 甘薯IbBADH基因家族全基因组鉴定与IbBADH1a上游转录调控因子筛选[J]. 生物技术通报, 2026, 42(11): 113-123.

ZHOU Guang-li, CHEN Jin-chai, LI Xin-xin, WU Xiao-ping, JIA Zheng-rong, Yan Hao, HE Li-heng, JIA Xiao-yun. Genome-wide Identification of the IbBADH Gene Family and Screening of Upstream Transcriptional Regulators of IbBADH1a in Sweetpotato[J]. Biotechnology Bulletin, 2026, 42(11): 113-123.

图/表 8

图1 不同物种BADH蛋白的系统发育分析★甘薯（Ipomoea batatas）；▲小薯（Ipomoea cordatotriloba）；●三浅裂野牵牛（Ipomoea trifida）；○三裂叶薯（Ipomoea triloba）；■水稻（Oryza sativa）；□拟南芥（Arabidopsis thaliana）。系统树采用Neighbor-Joining方法构建，Bootstrap重复1 000次；不同符号表示不同物种

Fig. 1 Phylogenetic analysis of BADH proteins from different species★ Ipomoea batatas;▲ Ipomoea cordatotriloba;● Ipomoea trifida;○ Ipomoea triloba;■ Oryza sativa;□ Arabidopsis thaliana.The phylogenetic tree was constructed using the Neighbor-Joining method with 1 000 bootstrap replicates, and symbols indicate different species

图2 IbBADH家族成员系统发育、保守基序与基因结构分析A：IbBADH蛋白系统发育树；B：保守基序分布（不同颜色框代表不同Motif；红色外框表示BADH/ALDH保守结构域PLN02467）；C：外显子-内含子结构（黄色框表示外显子，黑线表示内含子）

Fig. 2 Phylogeny, conserved motifs, and gene structures of IbBADH family membersA: Phylogenetic tree of IbBADH proteins; B: distribution of conserved motifs (colored boxes indicate motifs; the red frame indicates the conserved BADH/ALDH domain PLN02467); C: exon-intron structures (yellow boxes, exons; black lines, introns)

图3 IbBADH基因家族启动子区顺式调控元件的分布

Fig. 3 Distribution of cis-acting elements in the promoter regions of the IbBADH gene family

图4 IbBADH基因家族在甘薯不同组织及胁迫/激素处理下的表达热图ABA：脱落酸；MeJA：茉莉酸甲酯；SA：水杨酸；FR：纤维根；ISR：初始贮藏根；RB：根体；RS：根茎；DE：块根远端；PE：块根近端

Fig. 4 Heatmap of IbBADH expression profiles in different sweetpotato tissues following treatment with various abiotic stresses and hormonesABA: Abscisic acid; MeJA: methyl jasmonate; SA: salicylic acid; FR: fibrous roots; ISR: initiative storage roots; RB: root bodies; RS: root stalks; DE: distal ends of the tuber; PE: proximal ends of the tuber

图5 IbBADH1a启动子的PCR产物电泳图M：DL2 000 marker，1： IbBADH1a扩增产物

Fig. 5 Agarose gel electrophoresis of PCR products of the IbBADH1a promoterM: DL2 000 marker, 1: PCR product of IbBADH1a

图6 不同AbA浓度下诱饵菌株的自激活抑制检测

Fig. 6 Suppression of bait autoactivation at different AbA concentrations

图7 筛库阳性克隆插入片段PCR鉴定M：DL2 000 marker；1-133：不同阳性克隆的PCR扩增产物

Fig. 7 PCR identification of inserts from positive Y1H library clonesM: DL2 000 marker; 1-133: PCR products amplified from different positive clones

图8 IbBADH1a启动子A段候选调控因子的点对点Y1H验证AD为空载体阴性对照；BTF3、TSJT1、bHLH49和ERF073为候选调控因子；在SD/-Leu与SD/-Leu/AbA（100 ng/mL）平板上点滴培养并观察生长

Fig. 8 Point-to-point Y1H verification of candidate regulatory factors of IbBADH1a promoter A segmentAD empty vector served as a negative control; BTF3, TSJT1, bHLH49, and ERF073 were candidate regulatory factors. Transformants were spotted on SD/-Leu and SD/-Leu/AbA (100 ng/mL) plates for growth assessment

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