Screening and Interaction Verification of Transcription Factors Stpatatin 05 Gene in Potato

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

Abstract

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

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.

Figures/Tables 8

References 31

1	Pouvreau L, Gruppen H, Piersma SR, et al. Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana [J]. J Agric Food Chem, 2001, 49(6): 2864-2874.
2	杨炳南, 张小燕, 赵凤敏, 等. 常见马铃薯品种特性分析及加工适宜性分类 [J]. 食品科学技术学报, 2016, 34(1): 28-36.
	Yang BN, Zhang XY, Zhao FM, et al. Characteristic analysis and processing suitability clustering of common potato cultivars [J]. J Food Sci Technol, 2016, 34(1): 28-36.
3	刘垚彤, 孙伟, 董墨思, 等. 马铃薯糖蛋白Patatin的研究进展 [J]. 食品科学, 2019, 40(11): 331-337.
	Liu YT, Sun W, Dong MS, et al. Recent progress in research on patatin, the major potato tuber protein [J]. Food Sci, 2019, 40(11): 331-337.
4	Fu Y, Liu WN, Soladoye OP. Towards potato protein utilisation: insights into separation, functionality and bioactivity of patatin [J]. Int J Food Sci Tech, 2020, 55(6): 2314-2322.
5	Andrews DL, Beames B, Summers MD, et al. Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector [J]. Biochem J, 1988, 252(1): 199-206.
6	Gelley S, Lankry H, Glusac J, et al. Yeast-derived potato patatins: biochemical and biophysical characterization [J]. Food Chem, 2022, 370: 130984.
7	Bohac JR. A modified method to purify patatin from potato tubers [J]. J Agric Food Chem, 1991, 39(8): 1411-1415.
8	张笃芹, 木泰华, 孙红男. 马铃薯块茎特异蛋白Patatin的研究进展 [J]. 中国农业科学, 2016, 49(9): 1746-1756.
	Zhang DQ, Mu TH, Sun HN. Domestic and abroad research progress of potato tuber-specific storage protein patatin [J]. Sci Agric Sin, 2016, 49(9): 1746-1756.
9	宋东光, 王光清, 汪训明. Patatin启动子驱动的乙肝表面抗原基因在马铃薯中的表达 [J]. 高技术通讯, 2000, 10(2): 18-20.
	Song DG, Wang GQ, Wang XM. Expression of the HBsAg gene under the control of a patatin promoter in potato (Solanum tuberosum) [J]. High Technol Lett, 2000, 10(2): 18-20.
10	Abhary M, Siritunga D, Stevens G, et al. Transgenic biofortification of the starchy staple cassava (Manihot esculenta) generates a novel sink for protein [J]. PLoS One, 2011, 6(1): e16256.
11	Kolachevskaya OO, Alekseeva VV, Sergeeva LI, et al. Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro [J]. J Integr Plant Biol, 2015, 57(9): 734-744.
12	Zourelidou M, de Torres-Zabala M, Smith C, et al. Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression [J]. Plant J, 2002, 30(4): 489-497.
13	Choi HI, Baek SY, Kim SY. MYB class transcription factors bind to the tuber-specific and sucrose-response element of a class-I patatin promoter [J]. Plant Biotechnol Rep, 2017, 11(4): 239-245.
14	Chen JC, Zhao L, Li HR, et al. Nuclear factor-Y-polycomb repressive complex2 dynamically orchestrates starch and seed storage protein biosynthesis in wheat [J]. Plant Cell, 2024, 36(11): 4786-4803.
15	Guo DD, Hou QL, Zhang RQ, et al. Over-expressing TaSPA-B reduces prolamin and starch accumulation in wheat (Triticum aestivum L.) grains [J]. Int J Mol Sci, 2020, 21(9): 3257.
16	Zhu JT, Fang LL, Yu JQ, et al. 5-Azacytidine treatment and TaPBF-D over-expression increases glutenin accumulation within the wheat grain by hypomethylating the Glu-1 promoters [J]. Theor Appl Genet, 2018, 131(3): 735-746.
17	Wang J, Chen ZC, Zhang Q, et al. The NAC transcription factors OsNAC20 and OsNAC26 regulate starch and storage protein synthesis [J]. Plant Physiol, 2020, 184(4): 1775-1791.
18	Cao RJ, Zhao SL, Jiao GA, et al. OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice [J]. Plant Commun, 2022, 3(6): 100463.
19	王少阳. 马铃薯贮藏蛋白Patatin的鉴定及分析 [D]. 昆明: 云南大学, 2022.
	Wang SY. Identification and analysis of potato storage protein Patatin [D]. Kunming: Yunnan University, 2022.
20	Massa AN, Childs KL, Lin HN, et al. The transcriptome of the reference potato genome Solanum tuberosum Group Phureja clone DM1-3 516R44 [J]. PLoS One, 2011, 6(10): e26801.
21	郭凯, 侯留迪, 张莹莹, 等. 植物MYB基因家族研究进展 [J]. 长江大学学报: 自然科学版, 2020, 17(6): 93-98.
	Guo K, Hou LD, Zhang YY, et al. Research progress of MYB gene family in plants [J]. Journal of Yangtze University: Natural Science Edition, 2020, 17(6): 93-98.
22	Cao YP, Li K, Li YL, et al. MYB transcription factors as regulators of secondary metabolism in plants [J]. Biology, 2020, 9(3): 61.
23	Han JN, Li R, Zhang Z, et al. Genome-wide association and co-expression uncovered ZmMYB71 controls kernel starch content in maize [J]. J Integr Agric, 2024, (3).
24	Han Y, Yang RR, Zhang XJ, et al. MYB24, MYB144, and MYB168 positively regulate suberin biosynthesis at potato Tuber wounds during healing [J]. Plant J, 2024, 119(3): 1239-1257.
25	Li YM, Liang J, Zeng XZ, et al. Genome-wide analysis of MYB gene family in potato provides insights into tissue-specific regulation of anthocyanin biosynthesis [J]. Hortic Plant J, 2021, 7(2): 129-141.
26	Li XX, Guo C, Ahmad S, et al. Systematic analysis of MYB family genes in potato and their multiple roles in development and stress responses [J]. Biomolecules, 2019, 9(8): 317.
27	Chen BC, Wu XJ, Dong QJ, et al. Screening and functional analysis of StMYB transcription factors in pigmented potato under low-temperature treatment [J]. BMC Genomics, 2024, 25(1): 283.
28	Lin S, Singh RK, Moehninsi, et al. R2R3-MYB transcription factors, StmiR858 and sucrose mediate potato flavonol biosynthesis [J]. Hortic Res, 2021, 8(1): 25.
29	Chen C, Zhang KX, Khurshid M, et al. MYB transcription repressors regulate plant secondary metabolism [J]. Crit Rev Plant Sci, 2019, 38(3): 159-170.
30	Yang T, Wang HN, Guo LX, et al. Aba-induced phosphorylation of basic leucine zipper 29, abscisic acid insensitive 19, and Opaque2 by SnRK_2.2 enhances gene transactivation for endosperm filling in maize [J]. Plant Cell, 2022, 34(5): 1933-1956.
31	Wang XL, Liu YC, Hao CY, et al. Wheat NAC-A18 regulates grain starch and storage proteins synthesis and affects grain weight [J]. Theor Appl Genet, 2023, 136(6): 123.

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

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

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

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

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