马铃薯Stpatatin 05基因转录调控因子筛选及互作验证

doi:10.13560/j.cnki.biotech.bull.1985.2024-0991

摘要/Abstract

摘要：

目的筛选对patatin启动子有调控作用的转录因子，为patatin蛋白积累提供新的基因资源。方法选取Stpatatin 05作为候选基因，克隆其启动子序列，构建pAbAi-Stpatatin 05pro诱饵载体，将其转化至酵母细胞中，获得诱饵酵母菌株。利用马铃薯幼嫩块茎组织的cDNA文库进行酵母单杂交筛库实验，对候选蛋白进行互作验证和互作位点分析，并利用双荧光素酶报告实验分析其对下游靶标基因表达的影响。结果经酵母单杂交筛选获得一个候选蛋白StMYB86-like。StMYB86-like属于MYB类转录因子家族，该蛋白编码320个氨基酸，定位在细胞核。克隆StMYB86-like基因全长序列，进行酵母单杂交验证，结果显示在30 mmol/L 3-氨基-1，2，4-三氮唑的条件下，对照组酵母不能正常生长，而实验组酵母能正常生长，表明转录因子与基因启动子存在互作。酵母单杂交结果表明StMYB86-like转录因子能够结合启动子中的CAACTG结构域，双荧光素酶报告实验证明StMYB86-like负调控下游靶标基因的表达。结论 StMYB86-like与Stpatatin 05启动子互作，可能对马铃薯块茎中patatin蛋白的积累发挥作用。

关键词: 马铃薯, patatin蛋白, 酵母单杂交, Stpatatin 05, StMYB86-like, 转录调控

Abstract:

Objective The aim of this study is to identify the transcription factors binding to patatin promoters and provide new gene resources for patatin protein accumulation. Method We selected Stpatatin 05 gene as a candidate gene. Its promoter was cloned and integrated into the pAbAi vector to obtain the pAbAi-Stpatatin 05 bait vector. The resultant vector was then transformed into Y1H yeast cells to yield the yeast bait strain. Yeast one-hybrid (Y1H) screening was conducted based on the cDNA library of young potato tuber tissues. The interaction of candidate proteins was verified and the interaction motif was analyzed. The effect on the expression of the target gene was investigated using dual-luciferase reporter system. Result A candidate protein StMYB86-like was obtatined by Y1H. StMYB86-like belongs to the MYB-like transcription factors family and encodes a protein of 320 amino acids. It is located in the nucleus. The full length sequence of StMYB86-like was cloned to verify the interaction. The Y1H results showed that the yeast cells in the control group did not grow normally under the condition of 30 mmol/L 3-amino-1, 2, 4-triazole (3-AT), while the yeast cells in the experimental group grew normally, supporting the interaction between StMYB86-like and the promoter of Stpatatin 05 gene. Y1H results indicated that StMYB86-like bound to the CAACTG motif in the promoter, and the dual-luciferase reporter system showed that StMYB86-like negatively regulates the expression of its downstream target gene. Conclusion StMYB86-like interacts with Stpatatin 05 promoter; which may play a role in the accumulation of patatin protein in potato tubers.

Key words: potato, patatin protein, yeast one-hybrid, Stpatatin 05, StMYB86-like, transcriptional regulation

俞婷, 黄丹丹, 朱炎辉, 杨梅宏, 艾菊, 高冬丽. 马铃薯Stpatatin 05基因转录调控因子筛选及互作验证[J]. 生物技术通报, 2025, 41(3): 137-145.

YU Ting, HUANG Dan-dan, ZHU Yan-hui, YANG Mei-hong, AI Ju, GAO Dong-li. Screening and Interaction Verification of Transcription Factors Stpatatin 05 Gene in Potato[J]. Biotechnology Bulletin, 2025, 41(3): 137-145.

图/表 8

表1 分子实验所用引物

Table 1 Primers used for molecular experiments

引物名称 Primer name	引物序列 Primer sequence（5′-3′）	引物用途 Primer usage
pAbAi-Stpatatin 05pro-F	TTGAATTCGAGCTCGGTACCAAGATAAAAATGTAAGAATTATTAT	靶基因启动子
pAbAi-Stpatatin 05pro-R	TCGACAGATCCCCGGGTACCTTCTCCCAACGTAGCAAATG	Target gene promoter
pAbAi-F	CTTCAACACTACATATGCGT	阳性克隆鉴定
pAbAi-R	ATGCTACAAAGGACCTAATG	Identification of positive clones
pGADT7-F	TATAACTATCTATTCGATGA	阳性克隆鉴定
pGADT7-R	CACGATGCACAGTTGAAGTG	Identification of positive clones
pGADT7-StMYB86-like-F	GGAGGCCAGTGAATTCATGGGTCATCACTGCTGCAG	候选蛋白
pGADT7-StMYB86-like-R	CACCCGGGTGGAATTCTTAACAATTCCATGCAAGCTGATC	Candidate protein
pHis2-Stpatatin 05pro-F	GGCGAATTCCCGGGGAGCTCTTCTCTCTTTCTCGTTTTATACAAC	酵母单杂互作验证
pHis2-Stpatatin 05pro-R	ATTCGCGAACGCGTGAGCTCTTCTCCCAACGTAGCAAATG	Verification of interaction by yeast one-hybrid
pHis2-3CAACTG*-F	AATTATGACAGTTGCGGTATGACAGTTGCGGTATGACAGTTGCGGTAGCT	结合位点分析
pHis2-3CAACTG*-R	ACCGCAACTGTCATACCGCAACTGTCATACCGCAACTGTCAT	Analysis of binding sites
pHis2-3TAACCA*-F	AATTTTATTGGTTATGTCTTATTGGTTATGTCTTATTGGTTATGTCAGCT	结合位点分析
pHis2-3TAACCA*-R	GACATAACCAATAAGACATAACCAATAAGACATAACCAATAA	Analysis of binding sites
pRI101-StMYB86-like-F	CACTGTTGATACATATGATGGGTCATCACTGCTGCAG	亚细胞定位；双荧光素酶报告实验
pRI101-StMYB86-like-R	TTGCTCACCATCATATGACAATTCCATGCAAGCTGATC	Subcellular localization. Dual-luciferase reporter system
pGreen 0800-Stpatatin 05pro-F	TATAGGGCGAATTGGGTACCTTAATTTAAAATCACAAAGTTCAAA	双荧光素酶报告实验
pGreen 0800-Stpatatin 05pro-R	TAGAACTAGTGGATCCTTCTCCCAACGTAGCAAA	Dual-luciferase reporter system

图1 Stpatatin 05的表达模式分析A： Stpatatin 05在马铃薯DM各组织的FPKM值；B： Stpatatin 05在块茎发育早期的表达模式，Y-1到Y-7表示块茎从0.2 cm膨大到4 cm的7个发育时期

Fig. 1 Analysis of expression pattern of Stpatatin 05A: FPKM values of Stpatatin 05 in various tissues of potato DM. B: Expression pattern of Stpatatin 05 in early tuber development. Y-1 to Y-7 indicate the seven stages of tuber development when the tuber expands from 0.2 to 4 cm

图2 pAbAi-Stpatatin 05pro诱饵表达载体的构建A：Stpatatin 05基因启动子（Stpatatin 05pro）扩增；B：将pAbAi-Stpatatin 05pro重组载体线性化（1：未酶切的质粒，2：酶切后的质粒）；C：线性化重组载体pAbAi-Stpatatin 05pro的酵母阳性克隆鉴定

Fig. 2 Construction of the bait expressing vector of pAbAi-Stpatatin 05proA: Amplification of Stpatatin 05 gene promoter (Stpatatin 05pro). B: Linearization of the pAbAi-Stpatatin 05pro recombinant vector (lane 1 is the undigested plasmid, lane 2 is the digested plasmid). C: Identification of the linearized pAbAi-Stpatatin 05pro clone from yeast cells

图3 酵母单杂交筛库A：pGADT7空载转入pAbAi-Stpatatin 05pro酵母菌株后的AbA浓度筛选；B：酵母单杂交筛库部分菌落PCR鉴定；C：其他物种中StMYB86-like同源蛋白的多重比对

Fig. 3 Yeast one-hybrid screening libraryA: AbA concentration screening when pGADT7 was transformed into pAbAi-Stpatatin 05pro yeast strain. B: PCR identification of positive colonies from yeast one-hybrid screening. C: Multiple alignments of homologous proteins for the StMYB86-like

表2 Stpatatin 05启动子结合蛋白预测

Table 2 Predicted proteins binding to Stpatatin 05 promoter

酵母编号 Yeast number	基因号 Gene No.	描述 Description	结构域 Domains
3	PGSC0003DMT400026285	Cysteine protease inhibitor 1	Kunitz family trypsin and protease inhibitor protein
6	PGSC0003DMT400010908	Serine/arginine-rich protein	SC35-like splicing factor
7	PGSC0003DMT400011562	Proteinase inhibitor type-2 P303.51	Potato type II proteinase inhibitor family domain containing protein
15	PGSC0003DMT400012898	Glucanendo-1，3-beta-glucosidase	Glycosyl hydrolase superfamily protein
16	PGSC0003DMT400009304	Ubiquitin carrier protein	Ubiquitin-conjugating enzyme
21	PGSC0003DMT400018897 （StMYB86-like）	Transcription factor MYB	MYB domain protein
25	PGSC0003DMT400035380	ADP，ATP carrier protein	ADP/ATP carrier

图4 酵母单杂交验证StMYB86-like与Stpatatin 05pro的互作A：StMYB86-like与Stpatatin 05pro的酵母单杂验证；B：StMYB86-like结合基序的确定

Fig. 4 Yeast one-hybrid assays of interactions between StMYB86-like and Stpatatin 05proA: Yeast one-hybrid assays of StMYB86-like with Stpatatin 05pro. B: Determination of StMYB86-like binding motifs

图5 马铃薯StMYB86-like的亚细胞定位

Fig. 5 Subcellular localization of StMYB86-like in potato

图6 双荧光素酶报告实验*0.05>P≥0.01

Fig. 6 Dual-luciferase reporter experiment

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