Genome-wide Identification of the IbBADH Gene Family and Screening of Upstream Transcriptional Regulators of IbBADH1a in Sweetpotato

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

Abstract

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. Methods 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. Results 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

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

Figures/Tables 8

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

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)

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

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

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

Fig. 6 Suppression of bait autoactivation at different AbA concentrations

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

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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