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作物学报

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*()   

  1. 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*()   

  1. 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”

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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

表1

引物及序列表"

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

表2

StHQT启动子元件分析"

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

表3

酵母单杂筛库结果"

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

图1

StAHL的表达模式分析 a: StAHL在马铃薯7个组织中的表达; b: StHQT在同一马铃薯的相同组织中的表达。"

图2

StAHL亚细胞定位"

图3

StAHL与StHQT的相互作用 a: StAHL和StHQT启动子全长酵母单杂结果。b: StHQT启动子分段示意图, 黄色部分为片段1, 绿色部分为片段2, 红色部分为片段3, 蓝色部分为片段4。c: StAHL和StHQT启动子分段酵母单杂结果。d: StAHL和StHQT启动子全长双荧光素酶试验结果。e: StAHL和StHQT启动子分段双荧光素酶试验结果。*、***分别表示在0.05、0.001概率水平差异显著。ns表示无显著性差异。"

图4

StAHL系统进化树(a)和蛋白结构域保守性分析(b)"

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