马铃薯野生种烯酰水合酶超家族基因ScDHNS的克隆与功能分析

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

摘要/Abstract

摘要：

【目的】1，4-二氢氧-2-石脑-CoA合成酶（1,4-dihydroxy-2-naphthoyl-CoA synthase，DHNS）基因是茄科植物糖苷生物碱合成代谢的潜在重要基因，开展马铃薯DHNS基因功能研究与验证，为低糖苷生物碱马铃薯品种（系）的选育提供基因和材料来源。【方法】利用RACE方法克隆得到马铃薯野生种恰柯薯（Solanum chacoense）ScDHNS基因，对其进行生物信息学分析和亚细胞定位，通过构建过表达载体pBWA（V）HS-DHNS转化马铃薯栽培种进行功能验证。【结果】ScDHNS cDNA序列开放阅读框1 023 bp，编码340个氨基酸，分子量为37.34 kD，等电点pI为8.592，具有典型的ECH保守结构域，属于烯酰水合酶超家族成员，在二穗短柄草（Brachypodium distachyon）、蒺藜苜蓿（Medicago truncatula）等植物基因组中都有其同源基因，且存在基因扩张和收缩事件。过表达ScDHNS基因后发现转化株ScDHNS和SGT1基因表达量显著上调，且表达量显著高于马铃薯WT植株。且对应转化植株的总糖苷生物碱含量显著高于马铃薯WT植株，最高可达到364.3 mg/kg，是对照的2.4倍。亚细胞定位结果显示ScDHNS定位于过氧化物酶体。【结论】马铃薯ScDHNS基因可能参与调控糖苷生物碱合成关键基因SGT1的表达，通过β-氧化途径和甲羟戊酸通路协同影响糖苷生物碱的合成，该基因与糖苷生物碱在亚细胞水平上的区室化有重要关系，对于培育低糖苷生物碱的马铃薯品种（系）具有重要的应用价值。

关键词: 马铃薯野生种, 恰柯薯, ScDHNS, 基因克隆, 亚细胞定位

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

乔岩, 杨芳, 任盼荣, 祁伟亮, 安沛沛, 李茜, 李丹, 肖俊飞. 马铃薯野生种烯酰水合酶超家族基因ScDHNS的克隆与功能分析[J]. 生物技术通报, 2024, 40(9): 92-103.

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.

图/表 15

表1 5'-和3'-RACE 所用引物

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

表2 荧光定量PCR引物序列

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

图1 ScDHNS 基因核心片段、5'-RACE 和3'-RACE 克隆 A：ScDHNS 基因核心片段克隆（泳道M：2000 DNA marker；泳道1：ScDHNS 基因CDS区域克隆片段）；B：5'-RACE克隆（泳道M：500 DNA marker；泳道1：ScDHNS 基因5'-RACE克隆片段）；C：3'-RACE克隆（泳道M：2000 DNA marker；泳道1：ScDHNS 基因3'-RACE克隆片段）

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）

图2 ScDHNS基因的核苷酸序列及其编码氨基酸序列图中黑色方框代表着起始密码子和终止密码子，星号代表终止子

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

图3 ScDHNS与其他植物氨基酸序列的多重比较直线表示ECH结构域，箭头为β折叠区，TT字母为β转角；螺旋线为α螺旋区和310-螺旋区（η）。各植物DHNS基因来源，恰柯薯（Solanum chacoense）ScDHNS、番茄（Solanum lycopersicum）SlDHNS、栽培马铃薯（Solanum tuberosum）StDHNS、渐狭叶烟草（Nicotiana attenuata）NaDHNS、二穗短柄草（Brachypodium distachyon）BdDHNS、拟南芥（Arabidopsis thaliana）AtDHNS、蒺藜苜蓿（Medicago truncatula）MtDHNS

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）

图4 ScDHNS蛋白质功能域分析 SMART domain 表示预测ScDHNS 蛋白结构域；刻度表示从起始密码子开始的密码子顺序

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

图5 ScDHNS的系统进化分析

Fig. 5 Phylogenetic tree analysis of ScDHNS

图6 pBWA-ScDHNS-Glosgfp亚细胞定位载体示意图及其酶切验证 A：pBWA-ScDHNS-Glosgfp亚细胞定位载体；B：pBWA-ScDHNS-Glosgfp载体酶切验证（M: DNA marker；泳道 1: 酶切后的载体）

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）

图7 ScDHNS在拟南芥原生质体中的亚细胞定位 A：拟南芥原生质体瞬时表达pBWA -Glosgfp空载体；B：拟南芥原生质体瞬时表达pBWA（V）HS-ScDHNS-Glosgfp重组载体

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

图8 pBWA（V）HS-ScDHNS表达载体及其酶切验证 A：pBWA（V）HS-ScDHNS表达载体；B：pBWA（V）HS-ScDHNS载体酶切验证（泳道M：DNA marker；泳道1：酶切后的载体）

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）

图9 农杆菌GV310（pBWA（V）HS-ScDHNS）转化马铃薯组培苗

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

图10 pBWA（V）HS-ScDHNS转化马铃薯组培苗的PCR鉴定 M: DNA marker 1000；1-18：PCR产物

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

图11 ScDHNS马铃薯遗传转化植株与野生型植株的表现型

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

图12 马铃薯遗传转化植株ScDHNS和SGT1基因的RT-qPCR分析

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

图13 pBWA（V）HS-ScDHNS转化马铃薯组培苗的总SGA含量分析

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

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