马铃薯StPTST2a基因的克隆及互作分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0043

摘要/Abstract

摘要：

目的 PROTEIN TARGETING TO STARCH（PTST）在淀粉合成中发挥重要功能，本研究旨在探究马铃薯（Solanum tuberosum L.）StPTST2a基因的作用。方法利用反转录技术克隆StPTST2a基因，并对其氨基酸序列进行生物信息分析；利用荧光定量PCR技术分析StPTST2a在不同组织的表达量；利用原核表达系统在体外表达融合MBP标签的StPTST2a蛋白，并对融合蛋白进行体外淀粉结合实验；克隆7个淀粉合成相关基因，利用酵母双杂交及荧光素酶互补实验揭示StPTST2a与淀粉合成相关基因之间的互作关系。结果 StPTST2a基因编码了一个具有526个氨基酸的蛋白，在C端含有一个CBM48结构域。该基因在各个组织均有表达，其中在叶片中的表达较高。体外淀粉结合实验表明，StPTST2a是一个淀粉结合蛋白。分析StPTST2a与7个淀粉合成相关基因的相互作用关系，发现StPTST2a与StSS4、StSS6和StISA1.1在酵母和植物体内均有互作。结论 StPTST2a能够与多个淀粉合成基因形成复合体，在马铃薯淀粉合成和淀粉颗粒形成中发挥潜在作用。

关键词: 马铃薯, 淀粉, StPTST2a, 蛋白互作

Abstract:

Objective PROTEIN TARGETING TO STARCH (PTST) plays an important role in starch biosynthesis. This study aims to investigate the functional role of StPTST2a in potato (Solanum tuberosum L.). Method RT-PCR was used to clone StPTST2a gene and its amino acid sequence was analyzed by the bioinformatic method. RT-qPCR was performed to examine the expressions of StPTST2a in various organs. Prokaryotic expression system was used to express MBP-tagged StPTST2a in vitro and subsequently the recombinant protein was tested for its binding to starch. Seven starch biosynthesis-related genes were cloned, and the protein-protein interaction analysis was performed with yeast two-hybrid (Y2H) assays and luciferase complementation imaging (LCI) assays. Result StPTST2a encoded a protein of 532 amino acids, and it harboured a CBM48 domain at the C-terminus. StPTST2a was expressed at various tissues, and the transcript level was higher in the leaves than that in other organs. In vitro starch binding assays showed that StPTST2a bound to the starch. Y2H and LCI assays demonstrated that StPTST2a interacted with StSS4, StSS6 and StISA1.1 in yeast cells and in plants. Conclusion StPTST2a form a complex with starch biosynthesis-related genes, which may collaboratively control starch biosynthesis and formation of starch granules.

Key words: Solanum tuberosum L., starch, StPTST2a, protein-protein interaction

黄丹丹, 吴云翼, 邹建华, 俞婷, 朱炎辉, 杨梅宏, 董文丽, 高冬丽. 马铃薯StPTST2a基因的克隆及互作分析[J]. 生物技术通报, 2025, 41(7): 172-180.

HUANG Dan-dan, WU Yun-yi, ZOU Jian-hua, YU Ting, ZHU Yan-hui, YANG Mei-hong, DONG Wen-li, GAO Dong-li. Cloning and Interaction Analysis of StPTST2a Gene in Potato[J]. Biotechnology Bulletin, 2025, 41(7): 172-180.

图/表 6

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引物名称Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
pMAL-C2X-StPTST2a-F	GGATCGAGGGAAGGATTTCAATGGTTTTGACCCAGTTCGC	体外淀粉结合实验
pMAL-C2X-StPTST2a-R	CAGGTCGACTCTAGAGGATCCTTATGTGACAATGAGAAGATT	In vitro starch-binding assays
pGBKT7-StPTST2a-F	CATATGGCCATGGAGGCCATGGTTTTGACCCAGTTCG	酵母双杂交实验
pGBKT7-StPTST2a-R	CTAGTTATGCGGCCGCTGCAGTTATGTGACAATGAGAAG	Yeast two-hybrid assays
pGADT7-StSS1-F	CATATGGCCATGGAGGCCAGTATGGGGTCTCTGCAAACAC	酵母双杂交实验
pGADT7-StSS1-R	CGATGCCCACCCGGGTGTCATCTGACATATGGAGGATC	Yeast two-hybrid assays
pGADT7-StSS2-F	CATATGGCCATGGAGGCCAGTATGGAGAATTCCATTCTTC	酵母双杂交实验
pGADT7-StSS2-R	CGATGCCCACCCGGGTGTCACCACTGATACTTAGCAG	Yeast two-hybrid assays
pGADT7-StSS3-F	CATATGGCCATGGAGGCCAGTATGGACTCACTTACAGTTCT	酵母双杂交实验
pGADT7-StSS3-R	CGATGCCCACCCGGGTGCTATTCTAACTTTCTAGCAGC	Yeast two-hybrid assays
pGADT7-StSS4-F	CATATGGCCATGGAGGCCAGTATGGAGATGAAGATCTCCAA	酵母双杂交实验
pGADT7-StSS4-R	CGATGCCCACCCGGGTGTCAACTACGACTTGCAGCTCTT	Yeast two-hybrid assays
pGADT7-StSS5-F	ATGGCCATGGAGGCCAGTATGATCACGGAGATGCTTTC	酵母双杂交实验
pGADT7-StSS5-R	CGATGCCCACCCGGGTGTTACAAGTTTTTTATTGCTGTA	Yeast two-hybrid assays
pGADT7-StSS6-F	CATGGAGGCCAGTATGGATTTCACATCCGGCCTGTC	酵母双杂交实验
pGADT7-StSS6-R	CGATGCCCACCCGGGTGTCAAAGCAAGTGACGTCTAAG	Yeast two-hybrid assays
pGADT7-StISA1.1-F	ATGGCCATGGAGGCCAGTATGGTACGGCAGTTCATCAA	酵母双杂交实验
pGADT7-StISA1.1-R	CGATGCCCACCCGGGTGCTATGCATCATCAGCAGATG	Yeast two-hybrid assays
pCAMBIA-nLUC-StPTST2a-F	GAACAGGGGGACGAGCTCATGGTTTTGACCCAGTTCG	荧光素酶互补实验
pCAMBIA-nLUC-StPTST2a-R	CCCGGGACGCGTACGAGATCTGTGTGACAATGAGAAGATTA	Luciferase complementation assays
pCAMBIA-cLUC-StSS4-F	CTCGTACGCGTCCCGGGGCATGGAGATGAAGATCTCC	荧光素酶互补实验
pCAMBIA-cLUC-StSS4-R	CGTCCTTGTAGTCCATTTGTTTCAACTACGACTTGCAGCTCTTGC	Luciferase complementation assays
pCAMBIA-cLUC-StSS6-F	CTCGTACGCGTCCCGGGGCATGGATTTCACATCCGG	荧光素酶互补实验
pCAMBIA-cLUC-StSS6-R	GTCCTTGTAGTCCATTTGTTTCAAAGCAAGTGACGTCTAAGTAC	Luciferase complementation assays
pCAMBIA-cLUC-StISA1.1-F	GTACGCGTCCCGGGGCATGGTACGGCAGTTCAT	荧光素酶互补实验
pCAMBIA-cLUC-StISA1.1-R	CGTCCTTGTAGTCCATTTGTTCTATGCATCATCAGCAGATGATAG	Luciferase complementation assays
qPCR-StPTST2a-F	CCAGCTTCCTCATCTGGTAGATC	荧光定量PCR
qPCR-StPTST2a-R	CATGTCGTGAGGCTTCCATTTTT	Quantitative real-time PCR

引物名称Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
pMAL-C2X-StPTST2a-F	GGATCGAGGGAAGGATTTCAATGGTTTTGACCCAGTTCGC	体外淀粉结合实验
pMAL-C2X-StPTST2a-R	CAGGTCGACTCTAGAGGATCCTTATGTGACAATGAGAAGATT	In vitro starch-binding assays
pGBKT7-StPTST2a-F	CATATGGCCATGGAGGCCATGGTTTTGACCCAGTTCG	酵母双杂交实验
pGBKT7-StPTST2a-R	CTAGTTATGCGGCCGCTGCAGTTATGTGACAATGAGAAG	Yeast two-hybrid assays
pGADT7-StSS1-F	CATATGGCCATGGAGGCCAGTATGGGGTCTCTGCAAACAC	酵母双杂交实验
pGADT7-StSS1-R	CGATGCCCACCCGGGTGTCATCTGACATATGGAGGATC	Yeast two-hybrid assays
pGADT7-StSS2-F	CATATGGCCATGGAGGCCAGTATGGAGAATTCCATTCTTC	酵母双杂交实验
pGADT7-StSS2-R	CGATGCCCACCCGGGTGTCACCACTGATACTTAGCAG	Yeast two-hybrid assays
pGADT7-StSS3-F	CATATGGCCATGGAGGCCAGTATGGACTCACTTACAGTTCT	酵母双杂交实验
pGADT7-StSS3-R	CGATGCCCACCCGGGTGCTATTCTAACTTTCTAGCAGC	Yeast two-hybrid assays
pGADT7-StSS4-F	CATATGGCCATGGAGGCCAGTATGGAGATGAAGATCTCCAA	酵母双杂交实验
pGADT7-StSS4-R	CGATGCCCACCCGGGTGTCAACTACGACTTGCAGCTCTT	Yeast two-hybrid assays
pGADT7-StSS5-F	ATGGCCATGGAGGCCAGTATGATCACGGAGATGCTTTC	酵母双杂交实验
pGADT7-StSS5-R	CGATGCCCACCCGGGTGTTACAAGTTTTTTATTGCTGTA	Yeast two-hybrid assays
pGADT7-StSS6-F	CATGGAGGCCAGTATGGATTTCACATCCGGCCTGTC	酵母双杂交实验
pGADT7-StSS6-R	CGATGCCCACCCGGGTGTCAAAGCAAGTGACGTCTAAG	Yeast two-hybrid assays
pGADT7-StISA1.1-F	ATGGCCATGGAGGCCAGTATGGTACGGCAGTTCATCAA	酵母双杂交实验
pGADT7-StISA1.1-R	CGATGCCCACCCGGGTGCTATGCATCATCAGCAGATG	Yeast two-hybrid assays
pCAMBIA-nLUC-StPTST2a-F	GAACAGGGGGACGAGCTCATGGTTTTGACCCAGTTCG	荧光素酶互补实验
pCAMBIA-nLUC-StPTST2a-R	CCCGGGACGCGTACGAGATCTGTGTGACAATGAGAAGATTA	Luciferase complementation assays
pCAMBIA-cLUC-StSS4-F	CTCGTACGCGTCCCGGGGCATGGAGATGAAGATCTCC	荧光素酶互补实验
pCAMBIA-cLUC-StSS4-R	CGTCCTTGTAGTCCATTTGTTTCAACTACGACTTGCAGCTCTTGC	Luciferase complementation assays
pCAMBIA-cLUC-StSS6-F	CTCGTACGCGTCCCGGGGCATGGATTTCACATCCGG	荧光素酶互补实验
pCAMBIA-cLUC-StSS6-R	GTCCTTGTAGTCCATTTGTTTCAAAGCAAGTGACGTCTAAGTAC	Luciferase complementation assays
pCAMBIA-cLUC-StISA1.1-F	GTACGCGTCCCGGGGCATGGTACGGCAGTTCAT	荧光素酶互补实验
pCAMBIA-cLUC-StISA1.1-R	CGTCCTTGTAGTCCATTTGTTCTATGCATCATCAGCAGATGATAG	Luciferase complementation assays
qPCR-StPTST2a-F	CCAGCTTCCTCATCTGGTAGATC	荧光定量PCR
qPCR-StPTST2a-R	CATGTCGTGAGGCTTCCATTTTT	Quantitative real-time PCR

基因 Gene	基因号 Gene ID	花 Flower	叶 Leaf	叶柄 Petiole	茎 Stem	匍匐茎Stolon	发育块茎Young tuber	成熟块茎 Mature tuber
StSS1	PGSC0003DMG402018552	25.61	63.81	33.58	22.92	14.46	19.21	14.80
StSS2	PGSC0003DMG400001328	21.37	59.03	37.83	50.03	45.99	124.37	207.38
StSS3	PGSC0003DMG400016481	9.03	18.75	15.66	10.55	10.48	35.72	40.04
StSS4	PGSC0003DMG400008322	10.81	6.04	7.51	6.26	14.39	8.27	4.13
StSS5	PGSC0003DMG400030619	3.71	1.25	1.42	3.84	14.25	42.0	57.44
StSS6	PGSC0003DMG402013540	2.28	2.00	7.30	4.10	8.72	13.75	13.21
StISA1.1	PGSC0003DMG400020699	5.29	19.7	11.29	7.52	16.16	38.48	42.26

基因 Gene	基因号 Gene ID	花 Flower	叶 Leaf	叶柄 Petiole	茎 Stem	匍匐茎Stolon	发育块茎Young tuber	成熟块茎 Mature tuber
StSS1	PGSC0003DMG402018552	25.61	63.81	33.58	22.92	14.46	19.21	14.80
StSS2	PGSC0003DMG400001328	21.37	59.03	37.83	50.03	45.99	124.37	207.38
StSS3	PGSC0003DMG400016481	9.03	18.75	15.66	10.55	10.48	35.72	40.04
StSS4	PGSC0003DMG400008322	10.81	6.04	7.51	6.26	14.39	8.27	4.13
StSS5	PGSC0003DMG400030619	3.71	1.25	1.42	3.84	14.25	42.0	57.44
StSS6	PGSC0003DMG402013540	2.28	2.00	7.30	4.10	8.72	13.75	13.21
StISA1.1	PGSC0003DMG400020699	5.29	19.7	11.29	7.52	16.16	38.48	42.26