Cloning and Function Analysis of the ScDHNS Gene of Crotonase/Enoyl-CoA Superfamily from a Wild Potato Species

doi:10.13560/j.cnki.biotech.bull.1985.2024-0528

Abstract

Abstract:

【Objective】 The DHNS（1,4-dihydroxy-2-naphthoyl-CoA synthase）gene in potatoes is a potentially important in the biosynthesis of glycoalkaloid metabolism in Solanaceae plants. Researching and verifying of the function of the potato DHNS gene can provide a source of genes and materials for the selection of low-glycoalkaloid potato varieties（lines）. 【Method】 The full-length sequence of ScDHNS cDNA was cloned from the wild potato species Solanum chacoense using the RACE technique. This sequence was analyzed for bioinformatics and subcellular localization, and its function was verified by constructing an overexpression vector pBWA（V）HS-DHNS to transform cultivated potato. 【Result】 The read open frame of ScDHNS cDNA sequence is 1 023 bp, encodes 340 amino acids, has a molecular weight of 37.34 kD and an isoelectric point of 8.592, and contains a typical ECH domain. It belongs to the enoyl hydratase/acetyl-CoA superfamily and is present in the genomes of plants such as Brachypodium distachyon and Medicago truncatula with homologous genes, showing gene expansion and contraction events. After the overexpression of the ScDHNS gene, it was found that the expressions of the transformed plants ScDHNS and SGT1 were significantly upregulated, and the expressions were notably higher than those in wild-type（WT）potato plants. Additionally, the total glycoalkaloid content in the corresponding transformed plants was significantly higher than that in WT potato plants, reaching a maximum of 364.3 mg/kg, which is 2.4 times that of the control. Subcellular localization results indicates that ScDHNS is localized in the peroxisome. 【Conclusion】 The ScDHNS gene in potato may regulate the expression of the key gene SGT1, which is involved in glycoalkaloid synthesis. It influences glycoalkaloid synthesis through the β-oxidation and mevalonate pathways. This gene is significantly associated with the subcellular compartmentalization of glycoalkaloids and has considerable application value in the cultivation of potato varieties with reduced glycoalkaloid content.

Key words: wild potato, Solanum chacoense, ScDHNS, gene cloning, subcellular localization

QIAO Yan, YANG Fang, REN Pan-rong, QI Wei-liang, AN Pei-pei, LI Qian, LI Dan, XIAO Jun-fei. Cloning and Function Analysis of the ScDHNS Gene of Crotonase/Enoyl-CoA Superfamily from a Wild Potato Species[J]. Biotechnology Bulletin, 2024, 40(9): 92-103.

Figures/Tables 15

Table 1 Primers for the 5'- and 3'- RACE analysis

引物Primer	序列Sequence（5'-3'）
P1（g21236-5GSP1）	ACATTAAGGCGACCAAAACTTTCA
P2（g21236-5GSP2）	AACTTTCAAAATCAGCATAACCATCC
P3（outer primer）	GCTGTCAACGATACGCTACGTAAC
P4（inner primer）	GCTACGTAACGGCATGACAGTG
P5（g21236-3GSP1）	CCATTAGATAAGTTGGAGGCAG
P6（g21236-3GSP2）	GTCCTACAGCGATACGAGTGC
P7（outer primer）	TACCGTCGTTCCACTAGTGATTT
P8（inner primer）	CGCGGATCCTCCACTAGTGATTTCACTATAGG

Table 2 Primers’ sequences for RT-qPCR

引物名称Primer name	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	长度 Length/bp
SGT1-F	AAGCCACAATCCTCACTACCC	57.7	132
SGT1-R	AGGCAACCCAACTTCAGCAG	59.2	132
ScDHNS-F	TTGATGATGGACATGCTGGACTTC	58.0	84
ScDHNS-R	TGCCTTCATTGCCTTCTTCAGTTC	59.0	84
ef1-α-F	ATTCAAGTATGCCTGGGTGCT	58.9	144
ef1-α-R	TTCTTGATAAAGTCTCTGTGTCCG	58.4	144

Fig. 1 The core fragment, 5'- and 3'- end fragments of gene ScDHNS A: The core fragment of ScDHNS gene（Lane M: 2000 DNA marker; lane 1: CDS cloning of ScDHNS gene）. B: 5'-RACE end fragments of ScDHNS gene（Lane M: 500 DNA marker. Lane 1: 5'- RACE fragments of ScDHNS gene）. C: 3'-RACE fragments of ScDHNS gene（Lane M: 2000 DNA marker. Lane 1: 5'- RACE fragments of ScDHNS gene）

Fig. 2 Nucleotide sequences and deduced amino acid sequences of gene ScDHNS The black boxes indicate the initiation and termination codons, respectively. Asterisk indicates the stop codon

Fig. 3 Multiple comparisons of amino acid sequences between ScDHNS and other plants The ECH domain is displayed as line. β-strands are rendered as arrows, strict β-turns as TT letters. The helix line indicates the α helix or 310- helix（η）area. DHNS gene sources: ScDHNS（Solanum chacoense）, SlDHNS（Solanum lycopersicum）, StDHNS（Solanum tuberosum）, NaDHNS（Nicotiana attenuata）, BdDHNS（Brachypodium distachyon）, AtDHNS（Arabidopsis thaliana）and MtDHNS（Medicago truncatula）

Fig. 4 Functional domain analysis of ScDHNS protein SMART domain indicates the predicted structural domain of ScDHNS. The scale indicates the order of the codon starting from the initial codon

Fig. 5 Phylogenetic tree analysis of ScDHNS

Fig. 6 Profile of pBWA-ScDHNS-Glosgfp subcellular localization vector and its digestion verification A: Profile of pBWA-ScDHNS-Glosgfp subcellular localization vector. B: Digestion result of pBWA-ScDHNS-Glosgfp vector（Lane M: DNA marker. Lane 1: Digestion result of vector）

Fig. 7 Subcellular localization of ScDHNS in Arabidopsis protoplasts A: Empty vector pBWA-Glosgfp was transformed into Arabidopsis protoplasts. B: Recombinant vector pBWA（V）HS-ScDHNS-Glosgfp was transformed into Arabidopsis protoplasts

Fig. 8 Profile of pBWA（V）HS-ScDHNS expressing vector and its enzymatic digestion verification A: pBWA（V）HS-ScDHNS express vector. B: Digestion result of pBWA（V）HS-ScDHNS vector（Lane M: DNA marker. Lane 1: Digestion result of vector）

Fig. 9 pBWA（V）HS-ScDHNS vector transformed into Agrobacterium tumefaciens GV3101 strain to tissue-cultivated seedlings of potato

Fig. 10 PCR identification of potato tissue-cultivated seedlings transformed with pBWA（V）HS-ScDHNS M: DNA marker 1000. 1-18: PCR product

Fig. 11 Phenotypes of wild-type and ScDHNS- transgenic potato lines

Fig. 12 RT-qPCR analysis of ScDHNS and SGT1 genes in transgenic potato lines ** P<0.01，* P<0.05, the same below

Fig. 13 Total SGA content in potato tissue-cultivated seedlings transformed with pBWA（V）HS-ScDHNS

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