马铃薯绿原酸调控因子的筛选与功能鉴定

doi:10.3724/SP.J.1006.2024.34185

作物学报 ›› 2024, Vol. 50 ›› Issue (7): 1740-1749.doi: 10.3724/SP.J.1006.2024.34185

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

马铃薯绿原酸调控因子的筛选与功能鉴定

周洪源(), 杨慧芹, 罗威, 石振明, 马玲^*()

云南师范大学云南省生物质能与环境生物技术重点实验室, 云南昆明 650000

收稿日期:2023-11-07 接受日期:2024-01-31 出版日期:2024-07-12 网络出版日期:2024-06-15
通讯作者: *马玲, E-mail: may_ynnu@ynnu.edu.cn
作者简介:E-mail: 2153211602@qq.com
基金资助:
广东省基础与应用基础研究重大项目(2021B0301030004);云南省William John Lucas院士工作站(202105AF150028);国家自然科学基金项目“二倍体栽培种马铃薯RH中自交亲和基因的图位克隆”(32002032);云南省基础研究专项面上项目“薯肉龙葵素含量负调控因子StERF9基因的克隆和功能解析”(202201AT070037);昭通市千和农业科技开发有限公司“尚轶专家工作站”项目

Screening and functional identification of chlorogenic acid regulatory factors in potato

ZHOU Hong-Yuan(), YANG Hui-Qin, LUO Wei, SHI Zhen-Ming, MA Ling^*()

Key Laboratory of Yunnan Province for Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming 650000, Yunnan, China

Received:2023-11-07 Accepted:2024-01-31 Published:2024-07-12 Published online:2024-06-15
Contact: *E-mail: may_ynnu@ynnu.edu.cn
Supported by:
Basic and Applied Basic Research Project of Guangdong Province(2021B0301030004);Academician William John Lucas Workstation, Yunnan Province(202105AF150028);National Natural Science Foundation of China “Mapping Cloning of Self-compatibility Gene in Potato RH from Diploid Culture”(32002032);Yunnan Province Basic Research Special Surface Project “Cloning and Functional Analysis of StERF9 Gene, a Negative Regulator of Solanin Content in Potato Meat”(202201AT070037);Zhaotong Qianhe Agricultural Science and Technology Development Co., Ltd. “Shangyi Expert Workstation”

摘要/Abstract

摘要：

马铃薯是世界第三大粮食作物。绿原酸是马铃薯中最主要的酚类化合物, 也是马铃薯抗虫抗病的物质基础之一, 但过量绿原酸会影响薯块的口感。因此, 培育地上部分高绿原酸含量而薯肉中低绿原酸含量的品种能很好的兼顾马铃薯抗病性及品质口感的需求。为了明确马铃薯中绿原酸的分子调控机制, 本研究以绿原酸合成关键酶基因StHQT的启动子序列为诱饵, 进行酵母单杂交文库筛选, 鉴定出一个AT-hook基因家族的转录因子StAHL。进一步对StAHL的生物信息学特征、表达模式、蛋白质产物的亚细胞定位等信息进行了系统分析, 并利用酵母单杂交和双荧光素酶报告试验等方法验证了StAHL与StHQT启动子之间的转录活性。结果表明StAHL基因的表达没有组织特异性, 但根与花中的表达量相对较高, 其蛋白产物包含AT-Hook、DUF296两个保守结构域, 且定位于细胞核中。StAHL蛋白能直接作用于StHQT的启动子序列, 并抑制其转录活性, 表明StAHL可能通过抑制StHQT的表达来抑制马铃薯中绿原酸的积累。研究结果为全面揭示马铃薯绿原酸生物合成的分子机制奠定了基础, 为马铃薯精准育种提供了分子靶标。

关键词: 马铃薯, 绿原酸, 调控因子, StAHL, AT-Hook

Abstract:

Potato is the third largest food crop in the world. Chlorogenic acid is the most important phenolic compound in potato, and also one of the material basis of potato insect resistance and disease resistance. However, excessive chlorogenic acid can affect the taste of tuber, therefore, breeding varieties with high chlorogenic acid content in the ground part and low chlorogenic acid content in tuber flesh can well meet the needs of potato disease resistance and quality taste. In order to clarify the molecular regulation mechanism of chlorogenic acid in potato, we used the promoter sequence of StHQT, a key enzyme for chlorogenic acid synthesis, as bait to screen the yeast single hybridization library, and identified the transcription factor StAHL of an AT-hook gene family. Further, the bioinformatic characteristics, the relative expression patterns, and subcellular localization of protein products of StAHL were systematically analyzed, and the transcriptional activity between StAHL and StHQT promoters was verified by yeast single hybridization and double luciferase report experiments. The results showed that StAHL gene expression was not tissue specific, but relatively high in roots and flowers. StAHL protein products contained two conserved domains, AT-Hook and DUF296, and were localized in the nucleus. StAHL proteins acted directly on StHQT promoter sequences and inhibited transcriptional activity. This suggests that StAHL may inhibit the accumulation of chlorogenic acid in potato by inhibiting StHQT expression. The results laid a foundation for revealing the molecular mechanism of chlorogenic acid biosynthesis in potato, and provided a molecular target for the breeding practices in potato.

Key words: potato, chlorogenic acid, regulatory factor, StAHL, AT-Hook

周洪源, 杨慧芹, 罗威, 石振明, 马玲. 马铃薯绿原酸调控因子的筛选与功能鉴定[J]. 作物学报, 2024, 50(7): 1740-1749.

ZHOU Hong-Yuan, YANG Hui-Qin, LUO Wei, SHI Zhen-Ming, MA Ling. Screening and functional identification of chlorogenic acid regulatory factors in potato[J]. Acta Agronomica Sinica, 2024, 50(7): 1740-1749.

图/表 7

表1

表2

表3

图1

图2

图3

图4

参考文献 24

[1]	Akyol H, Riciputi Y, Capanoglu E, Caboni M F, Verardo V. Phenolic compounds in the potato and its byproducts: an overview. Int J Mol Sci, 2016, 17: 835-853.
[2]	Leiss K A, Maltese F, Choi Y H, Verpoorte R, Klinkhamer P G. Identification of chlorogenic acid as a resistance factor for thrips in chrysanthemum. Plant Physiol, 2009, 150: 1567. doi: 10.1104/pp.109.138131 pmid: 19448039
[3]	Kumar P, Ortiz E V, Garrido E, Poveda K, Jander G. Potato tuber herbivory increases resistance to aboveground lepidopteran herbivores. Oecologia, 2016, 182: 177-187. doi: 10.1007/s00442-016-3633-2 pmid: 27147449
[4]	Lee G, Joo Y, Kim S G, Baldwin I T. What happens in the pith stays in the pith: tissue-localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores. Plant J, 2017, 92: 414-425.
[5]	Guo X Z, Ning Z X. Natural phenolic compounds and their health effects. Food Ind, 2002, 23: 28-29.
[6]	Hua X Y, Tao S, Sun S N, Guo N, Yan X F, Lin J X. Research progress of plant secondary metabolites-phenolic compounds. Bull Biotechnol, 2017, 33: 22-29.
[7]	Sinden S L, Deahl K L, Aulenbach B B. Effect of glycoalkaloids and phenolics on potato flavor. J Food Sci, 1976, 41: 520-523.
[8]	Ulbrich B, Zenk M H. Partial purification and properties of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase from higher plants. Phytochemistry, 1979, 18: 929-933.
[9]	Niggeweg R, Michael A J, Martin C. Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol, 2004, 22: 746-754. doi: 10.1038/nbt966 pmid: 15107863
[10]	Sonnante G D, Amore R, Blanco E, Pierri C L, Palma M, Luo J, Tucci M, Martin C. Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid. Plant Physiol, 2010, 153: 1224-1238. doi: 10.1104/pp.109.150144 pmid: 20431089
[11]	Payyavula R S, Shakya R, Sengoda V G, Munyaneza J E, Swamy P, Navarre D A. Synthesis and regulation of chlorogenic acid in potato: rerouting phenylpropanoid flux in HQT-silenced lines. Plant Biotechnol J, 2015, 13: 551-564. doi: 10.1111/pbi.12280 pmid: 25421386
[12]	Chen Z X, Liu G H, Liu Y Q, Xian Z Q, Tang N. Overexpression of the LmHQT1 gene increases chlorogenic acid production in Lonicera macranthoides Hand-Mazz. Acta Physiol Plant, 2017, 39: 27.
[13]	Yun J, Kim Y S, Jung J H, Seo P J, Park C M. The AT-hook motif-containing protein AHL22 regulates flowering initiation by modifying FLOWERING LOCUS T chromatin in Arabidopsis. J Biol Chem, 2012, 287: 15307-15316. doi: 10.1074/jbc.M111.318477 pmid: 22442143
[14]	Aravind L, Landsman D. AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res, 1998, 26: 4413-4421. doi: 10.1093/nar/26.19.4413 pmid: 9742243
[15]	Zhao J, Favero D S, Peng H, Neff M M. Arabidopsis thaliana AHL family modulates hypocotyl growth redundantly by interacting with each other via the PPC/DUF296 domain. Proc Natl Acad Sci USA, 2013, 110: E4688-E4697.
[16]	Huth J R, Bewley C A, Nissen M S, Evans J N, Reeves R, Gronenborn A M, Clore G M. The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif. Nat Struct Biol, 1997, 4: 657-665. doi: 10.1038/nsb0897-657 pmid: 9253416
[17]	Xiao C, Chen F, Yu X, Lin C, Fu Y F. Over-expression of an AT-hook gene AHL22 delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana. Plant Mol Biol, 2009, 71: 39-50.
[18]	Zhang W M, Cheng X Z, Fang D, Cao J. AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) proteins of ancient origin radiate new functions. Int J Biol Macromol, 2022, 214: 290-300.
[19]	Lim P O, Kim Y, Breeze E, Koo J C, Woo H R, Ryu J S, Park D H, Beynon J, Tabrett A, Buchanan-Wollaston V, Nam H G. Overexpression of a chromatin architecture-controlling AT-hook protein extends leaf longevity and increases the post-harvest storage life of plants. Plant J, 2007, 52: 1140-1153. pmid: 17971039
[20]	Wong M M, Bhaskara G B, Wen T N, Lin W D, Nguyen T T, Chong G L, Verslues P E. Phosphoproteomics of Arabidopsis highly ABA-induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation. Proc Natl Acad Sci USA, 2019, 116: 2354-2363. doi: 10.1073/pnas.1819971116 pmid: 30670655
[21]	Yadeta K A, Hanemian M, Smit P, Hiemstra J A, Pereira A, Marco Y, Thomma B P. The Arabidopsis thaliana DNA-binding protein AHL19 mediates Verticillium wilt resistance. Mol Plant-Microbe Interact, 2011, 24: 1582-1591.
[22]	Wang L, Li T, Liu N, Liu X. Identification of tomato AHL gene families and functional analysis their roles in fruit development and abiotic stress response. Plant Physiol Biochem, 2023, 202: 107931.
[23]	Kumar A, Singh S, Mishra A. Genome-wide identification and analyses of the AHL gene family in rice (Oryza sativa). 3 Biotech, 2023, 13: 248. doi: 10.1007/s13205-023-03666-0 pmid: 37366497
[24]	Zhou L, Liu Z, Liu Y, Kong D, Li T, Yu S, Mei H, Xu X, Liu H, Chen L, Luo L. A novel gene OsAHL1 improves both drought avoidance and drought tolerance in rice. Sci Rep, 2016, 6: 30264.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

引物名称 Primer name	正向引物 Forward sequences (5'-3')	反向引物 Reverse sequences (5'-3')
pProStHQT-AbAi	agcttgaattcgaATTTGTAAAGTTTTAAAAAATTCAAAGATAA	cctcgaggtcgacGTAATTGTTGTGGTACAAGAAATATAGTGTGTTT
pBI121s-StAHL	ctctcgagctttcgcgagctcATGAGTATGAGTGGAGTAACAGTGG	caggtcgactctagaggatccCTATGCAGGCAACGTTATGTTGA
Luc-ProStHQT	ctatagggcgaattgggtaccTTGAGATAGCTTGAGTTTTGAGAA	aagcttatcgataccgtcgacGTAATTGTTGTGGTACAAGAAATATAGT
Luc-ProStHQT-1	ctatagggcgaattgggtaccTTGAGATAGCTTGAGTTTTGAGAA	aagcttatcgataccgtcgacTATATATTTTTGGGGCCATATAAAACC
Luc-ProStHQT-2	ctatagggcgaattgAAAGACATATAAAAGAAAAGTACTAGAC	aagcttatcgataccgtcgacAGTATTTTCTTTGGAGTTCAATTTGG
Luc-ProStHQT-3	ctatagggcgaattgggtaccAAAGGGCATCTTTCTCAAATTTACT	aagcttatcgataccgtcgacAGTTACACTGATATATTCAGTAGTGGTC
Luc-ProStHQT-4	ctatagggcgaattgggtaccCAAAGAAGCTTCACAGAATTTGGC	aagcttatcgataccgtcgacGTAATTGTTGTGGTACAAGAAATATAGT
pR101-StAHL-GFP	gagaacacgggggactctagaATGAGTATGAGTGGAGTAACAGT	gcccttgctcaccatcatatgTGCAGGCAACGTTATGTTGATG
pJG4-5-StAHL	gattatgcctctcccgaattcATGAGTATGAGTGGAGTAACAGTG	agaagtccaaagcttctcgagCTATGCAGGCAACGTTATGTTGA
pLacZ-ProStHQT	atcgaattcgagctcggtaTTGAGATAGCTTGAGTTTTGAGAAGTT	agcacatgcctcgaggtcgaGTAATTGTTGTGGTACAAGAAATATAGT
pLacZ-ProStHQT-1	atcgaattcgagctcggtaTTGAGATAGCTTGAGTTTTGAGAAG	agcacatgcctcgaggtcgacTATATATTTTTGGGGCCATATAAAACC
pLacZ-ProStHQT-2	atcgaattcgagctcgAAAGACATATAAAAGAAAAGTACTAGAC	agcacatgcctcgaggtcgacAGTATTTTCTTTGGAGTTCAATTTGG
pLacZ-ProStHQT-3	atcgaattcgagctcggtaccAAAGGGCATCTTTCTCAAATTTACT	agcacatgcctcgaggtcgAGTTACACTGATATATTCAGTAGTGGTCG
pLacZ-ProStHQT-4	atcgaattcgagctcggtaccCAAAGAAGCTTCACAGAATTTGGC	agcacatgcctcgaggtcgacGTAATTGTTGTGGTACAAGAAATATAGT
StAHL-qPCR	TCCAGATTGTTGTGGGGAGC	AATCGAGGAAGCGGGCATAG
StActin-qPCR	GGATCTTGCTGGTCGTGATTT	CATAGGCAAGCTTTTCCTTCAT

元件名称 Component name	核心序列 Core sequence	数量 Quantity	功能 Feature
AT-rich element	ATAGAAATCAA	1	富含AT碱基的DNA结合蛋白的结合位点 Binding site of AT-rich DNA binding protein (ATBP-1)
STRE	AGGGG	1	压力相关元件 Stress-related element
Myb	TAACTG	1	MYB识别序列 MYB recognition sequence
MYB-like sequence	TAACCA	3	MYB结合位点 MYB binding site
MYB	T/CAACCA	4	MYB结合位点 MYB binding site
Myc	TCTCTTA	1	MYC结合位点 MYC binding site
MYC	CATTTG	1	E-box, MYC结合位点 E-box, MYC binding site
TATA-box	TATA	72	在转录开始 -30 bp左右的核心启动子元件 Core promoter element around -30 bp of transcription start
TCA-element	CCATCTTTTT	1	参与水杨酸反应的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness
GT1-motif	GGTTAA	1	光响应元件 Light responsive element
chs-CMA1a	TTACTTAA	1	光响应元件的一部分 Part of a light responsive element
ABRE	ACGTG	1	参与脱落酸反应的顺式元件 Cis-acting element involved in the abscisic acid responsiveness
G-Box	CACGTT	1	参与光响应的顺式调节元件 Cis-acting regulatory element involved in light responsiveness
WUN-motif	AAATTACT	2	创伤反应元件 Wound response element
LTR	CCGAAA	1	参与低温反应的顺式元件 Cis-acting element involved in low-temperature responsiveness
CAAT-box	C(A)AAT	40	启动子和增强子区域中常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions
Box 4	ATTAAT	3	与光反应有关的保守DNA的一部分 Part of a conserved DNA module involved in light responsiveness
DRE core	GCCGAC	1	脱水反应元件 Dehydration response element
TC-rich repeats	ATTCTCTAAC	1	参与防御和应激反应的顺式作用因子 Cis-acting element involved in defense and stress responsiveness

马铃薯转录因子基因号 Potato transcription factor ID	比对率 Comparison rate (%)	马铃薯参考基因组注释 Potato reference genome notes
PGSC0003DMT400002870	100.00	组蛋白H3.2 Histone H3.2
PGSC0003DMT400009127	100.00	果糖二磷酸醛缩酶 Fructose-bisphosphate aldolase
PGSC0003DMT400014700	99.56	一种连接组蛋白的类似蛋白的连接子 A linker histone like protein
PGSC0003DMT400015124	100.00	40S核糖体蛋白S11 40S ribosomal protein S11
PGSC0003DMT400020708	100.00	26S蛋白酶体非ATP酶调控亚基 26S proteasome non-ATPase regulatory subunit
PGSC0003DMT400023621	98.21	核糖体生物发生蛋白 Ribosome biogenesis protein
PGSC0003DMT400023985	99.57	DCK/dGK-like脱氧核苷激酶 DCK/dGK-like deoxyribonucleoside kinase
PGSC0003DMT400027155	100.00	多泛素 Polyubiquitin
PGSC0003DMT400032575	97.67	DNA结合蛋白 DNA binding protein
PGSC0003DMT400047424	96.71	含有MTERF结构域的蛋白 MTERF domain containing protein
PGSC0003DMT400049672	97.36	富含F-box/亮氨酸的重复性蛋白 F-box/leucine rich repeat protein
PGSC0003DMT400052472	100.00	肌醇磷酸化神经酰胺合成酶2 Inositol phosphorylceramide synthase 2
PGSC0003DMT400052781	100.00	核RNA结合蛋白 Nuclear RNA binding protein
PGSC0003DMT400058778	100.00	组蛋白H3.2 Histone H3.2
PGSC0003DMT400061170	100.00	渗透应激活化蛋白激酶 Osmotic stress-activated protein kinase
PGSC0003DMT400078775	85.83	Armadillo/beta连环重复蛋白家族 Armadillo/beta-catenin repeat family protein
PGSC0003DMT400081297	100.00	组蛋白H3.2 Histone H3.2

马铃薯绿原酸调控因子的筛选与功能鉴定

Screening and functional identification of chlorogenic acid regulatory factors in potato

RichHTML

PDF

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 24

相关文章 15

Metrics

本文评价

推荐阅读 10

关于本刊

在线期刊

作者中心

相关单位

联系我们

[1]	刘园园, 董建科, 应静文, 梅文祥, 程刚, 郭晶晶, 焦文标, 宋波涛. 利用野生种Solanum boliviense创制马铃薯抗寒种质[J]. 作物学报, 2024, 50(6): 1384-1393.
[2]	刘震, 陈丽敏, 李志涛, 朱金勇, 王玮璐, 齐喆颖, 姚攀锋, 毕真真, 孙超, 白江平, 刘玉汇. 马铃薯ARM基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2024, 50(6): 1451-1466.
[3]	赵娜, 刘宇曦, 张朝澍, 石瑛. 不同马铃薯淀粉含量差异的转录组学解析[J]. 作物学报, 2024, 50(6): 1503-1513.
[4]	李万, 李成, 程敏, 吴芳. 磷转运蛋白StPHO1.2提高马铃薯耐热性[J]. 作物学报, 2024, 50(2): 394-402.
[5]	刘洁, 蔡诚诚, 刘石锋, 邓孟胜, 王雪枫, 温和, 李罗品, 严奉君, 王西瑶. 马铃薯StCYP85A3促进萌芽及根系伸长的功能解析[J]. 作物学报, 2023, 49(9): 2462-2471.
[6]	索海翠, 刘计涛, 王丽, 李成晨, 单建伟, 李小波. 马铃薯锌转运蛋白基因StZIP12调控锌吸收功能[J]. 作物学报, 2023, 49(7): 1994-2001.
[7]	赵喜娟, 刘圣宣, 刘腾飞, 郑洁, 杜鹃, 胡新喜, 宋波涛, 何长征. 转录组分析揭示光诱导转录因子StMYB113调控马铃薯块茎表皮叶绿素合成[J]. 作物学报, 2023, 49(7): 1860-1870.
[8]	王硕, 鲍天旸, 刘建刚, 段绍光, 简银巧, 李广存, 金黎平, 徐建飞. 基于RGB颜色空间评价马铃薯块茎绿化程度[J]. 作物学报, 2023, 49(4): 1102-1110.
[9]	李红艳, 李洁雅, 李响, 叶广继, 周云, 王舰. 过表达LrAN2基因对马铃薯中花青素和糖苷生物碱含量的影响[J]. 作物学报, 2023, 49(4): 988-995.
[10]	张卫娜, 余慧芳, 安珍, 柳文凯, 康益晨, 石铭福, 杨昕宇, 张茹艳, 王勇, 秦舒浩. StEFR1正调控马铃薯对晚疫病的抗性[J]. 作物学报, 2023, 49(4): 996-1005.
[11]	赵朋, 陈广侠, 张宴萍, 杨晓慧, 刘芳, 董道峰. 马铃薯苗期耐碱性鉴定方法及86份种质资源耐碱性综合评价[J]. 作物学报, 2023, 49(11): 2923-2934.
[12]	朱金勇, 刘震, 曾钰婷, 李志涛, 陈丽敏, 李泓阳, 史田斌, 张俊莲, 白江平, 刘玉汇. 马铃薯PAL基因家族的全基因组鉴定及其在非生物胁迫下和块茎花色素苷合成中的表达分析[J]. 作物学报, 2023, 49(11): 2978-2990.
[13]	巩慧玲, 林红霞, 任小丽, 李彤, 王晨霞, 白江平. StvacINV1负调控马铃薯的耐旱性[J]. 作物学报, 2023, 49(11): 3007-3016.
[14]	赵富贵, 张龙, 李丹, 韩固, 王楠, 侯贤清. 不同气候年型下耕作覆盖对宁南旱区土壤水热及马铃薯产量的影响[J]. 作物学报, 2023, 49(10): 2806-2819.
[15]	杜鹃, 彭晓君, 侯娟, 刘腾飞, 刘增, 宋波涛. 马铃薯淀粉酶StBAM9互作蛋白的鉴定及其互作机制分析[J]. 作物学报, 2023, 49(10): 2643-2653.