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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (7): 1740-1749.doi: 10.3724/SP.J.1006.2024.34185

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-07-12 Published: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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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

Table 1

Primers and sequences in this study"

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

Table 2

StHQT promoter element analysis"

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

Table 3

Result of yeast single hybrid screening pool"

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

Fig. 1

Analysis of StAHL expression patterns a: expression patterns of StAHL in 7 tissues of potato; b: expression of StHQT in the same tissue of the same potato."

Fig. 2

StAHL subcellular localization"

Fig. 3

Interaction between StAHL and StHQT a: StAHL and StHQT promoter full-length yeast single hybrid results. b: StHQT promoter segment diagram, the yellow part is segment 1, the green part is segment 2, the red part is segment 3, and the blue part is segment 4. c: StAHL and StHQT promoter segmental yeast monohybrid results. d: StAHL and StHQT promoter full-length double luciferase test results. e: StAHL and StHQT promoter segmenting double luciferase assay results. * and *** indicate significant difference at the 0.05 and 0.001 probability levels, respectively. ns indicates no significant difference."

Fig. 4

StAHL phylogenetic tree (a) and protein domain conservation analysis (b)"

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