马铃薯G6PDH基因家族鉴定及其在损伤块茎的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0243

摘要/Abstract

摘要：

【目的】葡萄糖-6-磷酸脱氢酶（G6PDH）在植物响应非生物胁迫中发挥重要作用，鉴定马铃薯中G6PDH基因家族，并分析其在损伤块茎的表达模式，为深入研究马铃薯G6PDH基因在损伤胁迫中的作用提供参考。【方法】利用生物信息学对马铃薯G6PDH基因家族进行鉴定，并对该基因家族成员编码蛋白的染色体分布、蛋白理化性质和二级结构、进化关系、基因结构、保守基序和启动子顺式作用元件，以及在不同器官和损伤块茎中的表达模式进行分析。【结果】在马铃薯基因组中共鉴定到4个StG6PDHs家族成员，分别分布在4条染色体上，命名为StG6PDH1-StG6PDH4。根据亚细胞定位和系统进化分析，StG6PDH1、StG6PDH3和StG6PDH4位于叶绿体，属于质体型；StG6PDH2位于细胞质，属于胞质型。马铃薯G6PDH蛋白的氨基酸个数介于511-596 aa，分子量为58.48-66.65 kD，等电点为5.83-8.57，不稳定系数为39.79-47.53。蛋白二级结构以α-螺旋和无规则卷曲占比最多，β-转角最少。此外，StG6PDHs启动子含大量植物激素、光和胁迫响应元件。4个StG6PDHs在马铃薯根、茎、叶和块茎均有表达，且在叶片中的表达高于其他组织。StG6PDHs各成员共同参与马铃薯块茎对损伤胁迫的响应，其中，StG6PDH1、StG6PDH2和StG6PDH3在块茎损伤后36 h内上调表达，StG6PDH4在损伤后下调表达。【结论】在马铃薯中共鉴定出4个StG6PDHs基因家族成员，不均匀地分布于4条染色体上，其中，1个为胞质型，3个为质体型。StG6PDHs启动子区有光、激素和胁迫响应元件。损伤块茎中StG6PDHs的表达具有差异性，各成员协同调控了马铃薯块茎对损伤胁迫的应答。

关键词: 马铃薯, G6PDH基因家族, 生物信息学分析, 损伤胁迫, 表达分析

Abstract:

【Objective】Glucose-6-phosphate dehydrogenase（G6PDH）plays an important role in plant response to abiotic stress. Identification of the G6PDH gene family in potato and analysis of its expression pattern in damaged tubers would provide a reference for the subsequent in-depth study of the role of potato G6PDH genes in damage stress. 【Method】We used bioinformatics methods to identify the G6PDH gene family members of the potato w, and to analyze the chromosomal distribution, protein physicochemical properties, secondary structure, evolutionary relationships, gene structure, conserved motifs, and promoter cis-acting elements of proteins encoded by members of the gene family, as well as the expression pattern in different organs and damaged tubers of potato.【Result】A total of 4 G6PDH family members in potato genome were identified and distributed on 4 chromosomes, named StG6PDH1-StG6PDH4. Based on the subcellular localization and phylogenetic analysis, StG6PDH1, StG6PDH3 and StG6PDH4 were located in the chloroplasts and belonged to the plastidic type, StG6PDH2 was located in the cytoplasm and belonged to the cytoplasmic type. The number of amino acids of potato G6PDH proteins ranged from 511 to 596 aa, molecular weights from 58.48 to 66.65 kD, isoelectric points from 5.83 to 8.57, and instabilities coefficient from 39.79 to 47.53. The secondary structure of protein showed the highest percentage of α-helices and irregular coils and the least of β-turns. In addition, the promoters of StG6PDHs contained a large number of phytohormone, light, and stress response elements. The four StG6PDHs were expressed in the roots, stems, leaves and tubers of potato, and the expression in the leaves was higher than in other organs. The members of StG6PDHs were involved in the responses of potato tubers to injury stress. The expressions of StG6PDH1, StG6PDH2 and StG6PDH3 were upregulated within 36 h after tubers injured, and StG6PDH4 was down-regulated in injured potato tubers. 【Conclusion】A total of four potato StG6PDHs gene family members are identified, unevenly distributed on four chromosomes, of which one is cytoplasmic and three are plastidic. The promoter region of StG6PDHs includes light, hormone, and stress response elements. The expression of StG6PDHs in wounded tubers is differential, and the members synergistically regulate the responses of potato tubers to damage stress.

Key words: potato, G6PDH gene family, bioinformatics analysis, damage stress, expression analysis

宋兵芳, 柳宁, 程新艳, 徐晓斌, 田文茂, 高悦, 毕阳, 王毅. 马铃薯G6PDH基因家族鉴定及其在损伤块茎的表达分析[J]. 生物技术通报, 2024, 40(9): 104-112.

SONG Bing-fang, LIU Ning, CHENG Xin-yan, XU Xiao-bin, TIAN Wen-mao, GAO Yue, BI Yang, WANG Yi. Identification of Potato G6PDH Gene Family and Its Expression Analysis in Damaged Tubers[J]. Biotechnology Bulletin, 2024, 40(9): 104-112.

图/表 9

表1 RT-qPCR引物序列

Table 1 Primers’ sequences for RT-qPCR

基因名称 Gene name	基因ID Gene ID	引物序列 Primer sequence（5'-3'）
StG6PDH1	Soltu.DM.01G040700	F: GCTTTCACCAGTATCGCTATCA
StG6PDH1	Soltu.DM.01G040700	R: CGGACAACAACGGTAGGTAAT
StG6PDH2	Soltu.DM.02G033900	F: CAAGAAACCTGGGCTTGAAATG
StG6PDH2	Soltu.DM.02G033900	R: CGTTCATAAGCCTCTGGAATGA
StG6PDH3	Soltu.DM.05G012290	F: ATACCAGGAGTGCGGAAATAAG
StG6PDH3	Soltu.DM.05G012290	R: CGTCAGGTTGAACACGGATAA
StG6PDH4	Soltu.DM.07G015360	F: GCTGACCAGATCCCTAAAGAAG
StG6PDH4	Soltu.DM.07G015360	R: GACAAGATTCGAGAACCGAAGA
StEF1α	Soltu.DM.06G005580	F: ATTGATGCCCCTGGTCACAG
StEF1α	Soltu.DM.06G005580	R: CATGTTCACGGGTCTGACCA

图1 马铃薯G6PDH基因家族染色体分布

Fig. 1 Chromosome location of G6PDH gene family in potato

表2 马铃薯G6PDH蛋白亚细胞定位预测

Table 2 Prediction of subcellular localization of G6PDH protein in potato

基因名称 Gene name	基因ID Gene ID	染色体位置 Chromosome location	亚细胞定位 Subcellular location
StG6PDH1	Soltu.DM.01G040700	chr.01	叶绿体Chloroplast
StG6PDH2	Soltu.DM.02G033900	chr.02	细胞质Cytoplasmic
StG6PDH3	Soltu.DM.05G012290	chr.05	叶绿体Chloroplast
StG6PDH4	Soltu.DM.07G015360	chr.07	叶绿体Chloroplast

表3 马铃薯G6PDH基因家族蛋白理化性质和二级结构分析

Table 3 Protein physicochemical properties and secondary structures of potato G6PDH gene family

基因名称 Gene name	理化性质Physicochemical property				二级结构Secondary structure
基因名称 Gene name	氨基酸长度 Amino acid length/aa	相对分子量 Mw/kD	等电点 pI	不稳定系数Instability index	α-螺旋 α-helix/%	β-转角 β-turn/%	无规则卷曲 Random coil/%	延伸链 Extended strand/%
StG6PDH1	596	66.65	5.83	47.31	36.91	5.87	40.27	16.95
StG6PDH2	511	58.48	5.97	47.53	40.51	5.28	39.33	16.87
StG6PDH3	582	66.21	8.57	39.79	38.14	6.53	39.87	17.53
StG6PDH4	577	65.72	6.88	39.84	37.78	6.59	39.17	16.46

图2 马铃薯、拟南芥、番茄、玉米和水稻G6PDH蛋白的系统进化树

Fig. 2 Phylogenetic tree of G6PDH proteins in Solanum tuberosum, Arabidopsis thaliana, Solanum lycopersicum, Zea mays and Oryza sativa

图3 马铃薯G6PDH基因家族成员的进化关系（A）、基因结构（B）和保守基序（C）分析

Fig. 3 Evolutionary relationships（A）, gene structure（B）and conserved motifs（C）of the potato G6PDH gene family

图4 马铃薯G6PDH基因家族顺式作用元件分析光响应元件：GATA-motif、GT1、Box-4、MRE、G-Box、AT1-motif；低温响应元件：LTR；厌氧诱导：ARE；赤霉素响应元件：GARE、TATC-box；生长素响应元件：AuxRE-core、TGA-element；脱落酸响应元件：ABRE；茉莉酸响应元件：CGTCA-motif；水杨酸响应元件：TCA-element；干旱响应元件：MBS；玉米醇溶蛋白代谢调节元件：O2-site；胁迫和防御响应元件：TC-rich；其他：BoxIII、AT-rich

Fig. 4 Cis-acting elements of potato G6PDH gene family Light responsiveness: GATA-motif, GT1, Box-4, MRE, G-Box, AT1-motif. Low-temperature responsiveness: LTR. Anaerobic induction: ARE. Gibberellin responsiveness: GARE, TATC-box. Auxin responsiveness: AuxRE-core, TGA-element. Abscisic acid responsiveness: ABRE. Me-JA responsiveness: CGTCA-motif. Salicylic acid responsiveness: TCA-element. Drought-induced response element: MBS. Zein metabolism regulation: O2-site. Defense and stress responsiveness: TC-rich. Other: Box III, AT-rich

图5 马铃薯不同器官中G6PDH的相对表达量同一基因中的不同小写字母表示差异显著（P<0.05）

Fig. 5 Relative expressions of G6PDH in different organs of potato Different lowercase letters in the same gene indicate significant difference（P<0.05）

图6 损伤马铃薯块茎中StG6PDHs的相对表达量 *代表同一时间的对照组和处理组之间差异显著（P<0.05）

Fig. 6 Relative expression of StG6PDH1s in damaged potato tubers * indicates a significant difference between the control group and the treatment group（P<0.05）

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