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作物学报 ›› 2025, Vol. 51 ›› Issue (8): 2220-2227.doi: 10.3724/SP.J.1006.2025.44203

• 研究简报 • 上一篇    下一篇

LsSAT2与LsAAE3互作调控山黧豆ODAP水平

刘晓宁1(), 张颖2, 蔡曼蕾2, 马昊2, 苗智博2, 曹宁3, 连荣芳3,*(), 徐全乐2,*()   

  1. 1黄河科技学院医学院, 河南郑州 450006
    2西北农林科技大学生命科学学院, 陕西杨凌 712100
    3定西市农业科学研究院, 甘肃定西 743000
  • 收稿日期:2024-12-04 接受日期:2025-04-25 出版日期:2025-08-12 网络出版日期:2025-05-07
  • 通讯作者: *连荣芳, E-mail: gsdxlianrongfang@163.com;徐全乐, E-mail: xuql03@163.com
  • 作者简介:E-mail: Xiaoningliu2016@126.com
  • 基金资助:
    河南省教育厅高等学校重点科研项目(25B180013);陕西省自然科学基金项目(2023-JC-YB-152);财政部和农业农村部国家现代农业产业技术体系建设专项(食用豆, CARS-08-Z21);河南省科技攻关项目(252102110271);国家级大学生创新创业训练计划项目(202401360B1)

Regulation of ODAP levels in Lathyrus sativus L. via interaction between LsSAT2 and LsAAE3

LIU Xiao-Ning1(), ZHANG Ying2, CAI Man-Lei2, MA Hao2, MIAO Zhi-Bo2, CAO Ning3, LIAN Rong-Fang3,*(), XU Quan-Le2,*()   

  1. 1School of Medicine, Huanghe S&T University, Zhengzhou 450006, Henan, China
    2College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China
    3Dingxi Academy of Agricultural Sciences, Dingxi 743000, Gansu, China
  • Received:2024-12-04 Accepted:2025-04-25 Published:2025-08-12 Published online:2025-05-07
  • Contact: *E-mail: gsdxlianrongfang@163.com;E-mail: xuql03@163.com
  • Supported by:
    Key Scientific Research Projects of Henan Provincial Department of Education for Institutions of Higher Learning(25B180013);Natural Science Foundation of Shaanxi Province(2023-JC-YB-152);China Agriculture Research System of MOF and MARA(Food Legumes, CARS-08-Z21);Tackling-Plan Project of Henan Department of Science and Technology(252102110271);National Undergraduate Training Programs fo Innovation and Entrepreneurship(202401360B1)

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摘要：

丝氨酸乙酰基转移酶LsSAT2 (serine acetyltransferase, SAT)是山黧豆中神经活性成分β-N-草酰-L-α,β-二氨基丙酸(β-N-oxalyl-L-α,β-diaminopropionic acid, β-ODAP)生物合成的关键酶。为进一步分析LsSAT2的功能及其活性调控, 本研究构建了LsSAT2过表达山黧豆毛状根株系, 利用免疫沉淀和质谱联用技术(immunoprecipitation mass spectrum, IP-MS)筛选到了LsSAT2的潜在互作蛋白草酰辅酶A合成酶LsAAE3, 通过蛋白对接、酵母双杂交(yeast two-hybrid, Y2H)、蛋白质下拉试验(pull down assay)等进行蛋白互作验证及功能分析。结果表明, 利用IP-MS从LsSAT2过表达山黧豆毛状根株系中筛选到其潜在的互作蛋白LsAAE3; Y2H和Pull down试验证实了LsAAE3与LsSAT2存在蛋白互作关系; 蛋白对接分析表明, LsSAT2通过羧基末端与LsAAE3发生蛋白互作。LsSAT2在山黧豆毛状根株系中的过表达使得β-ODAP水平相比对照降低52.4%, LsAAE3在山黧豆毛状根株系中的过表达则使得β-ODAP水平升高55.9%。上述结果加深了对山黧豆β-ODAP生物合成调控的理解。

关键词: 山黧豆, 三七素, 丝氨酸乙酰基转移酶, 草酰辅酶A合成酶, 蛋白互作

Abstract:

Serine acetyltransferase 2 (LsSAT2) in Lathyrus sativus functions as the rate-limiting enzyme in the biosynthesis of the neuroactive compound β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid). To explore the regulatory mechanisms underlying LsSAT2 activity, we overexpressed LsSAT2 in L. sativus hairy roots. Proteins interacting with LsSAT2 were subsequently identified via immunoprecipitation followed by mass spectrometry (IP-MS), and their interactions were confirmed using protein-protein docking, yeast two-hybrid (Y2H) analysis, and pull-down assays. The results revealed that LsSAT2 interacts with an acyl-CoA synthetase, LsAAE3, through its C-terminal region. Overexpression of LsSAT2 or LsAAE3 in hairy roots led to a 52.4% reduction or a 55.9% increase in β-ODAP content, respectively, indicating a functional interplay between these two proteins. These findings provide important insights into the genetic regulation of β-ODAP biosynthesis and establish a foundation for future metabolic engineering in L. sativus.

Key words: Lathyrus sativus, dencichine, serine acetyltransferase, oxalyl-CoA synthase, protein-protein interaction

表1

本研究所用引物"

引物名称 Name of primer 引物序列Sequence of primer (5'-3')
pCAMBIA1300UBQ10-LsSAT2-F GTCGACTCTAGAGGATCCGGTACCATGTTTGTTCTTGTTCTTGGTCGTT
pCAMBIA1300UBQ10-LsSAT2-R
 CGAGCTCACTAGTCTCGACTACTTATCGTCGTCATCCTTGTAATCGATAACATAATCAGACCAATCCGAA
pCAMBIA1300UBQ10-LsAAE3-F GTCGACTCTAGAGGATCCGATGGAAACCGCAACCACC
pCAMBIA1300UBQ10-LsAAE3-R GCTCCTCGCCCTTGCTCACAACTTTAGAAACAAAGTGTTCTGCT
pGBKT7-LsSAT2-F CCGCTGCAGGTCGACGGATCCCTAGATAACATAATCAGACCAATCCG
pGBKT7-LsSAT2-R CCGCTGCAGGTCGACGGATCCCTAGATAACATAATCAGACCAATCCG
pGADT7-LsAAE3-F GAGGCCAGTGAATTCATGGAAACCGCAACCACC
pGADT7-LsAAE3-R TTCATCTGCAGCTCGAGCTCAAACTTTAGAAACAAAGTGTTCTGC
pET28a-LsAAE3-F CTGGTGCCGCGCGGCAGCCATATG GAAACCGCAACCACCCTCAC
pET28a-LsAAE3-R GTGGTGGTGGTGGTGGTGCTCGAGTCAAACTTTAGAAACAAAGTGTTCTGC
pGEX2T-LsSAT2-F TCGGATCTGGTTCCGCGTATGTTTGTTCTTGTTCTTGGTC
pGEX2T-LsSAT2-R TCGTCAGTCAGTCACGAT CTAGATAACATAATCAGACCAATCC

图1

LsSAT2和LsAAE3过表达山黧豆毛状根系的Western blot验证 A: 转空载山黧豆毛状根系; B: LsSAT2过表达山黧豆毛状根系; C: LsAAE3过表达山黧豆毛状根系; D: LsSAT2过表达山黧豆毛状根系的Western blot分析; E: LsAAE3过表达山黧豆毛状根系的Western blot分析。"

图2

绿色荧光蛋白在LsAAE3过表达山黧豆毛状根中的表达 A和C为转空载山黧豆毛状根; B和D为LsAAE3过表达山黧豆毛状根。"

表2

LsSAT2和LsAAE3蛋白的质谱鉴定"

序号
Number		 登录号
Accession		 蛋白名称
Protein name		 覆盖率Coverage (%) 特异肽段数
Unique peptides		 蛋白匹配度得分
Score sequest HT
1 LATSA3860_EIv1.0_0145300.1 丝氨酸乙酰基转移酶
Serine O-acetyltransferase (SAT2)		 25 8 35.73
2 LATSA3860_EIv1.0_0424890.1 草酰辅酶A合成酶
Oxalate-CoA ligase (AAE3)		 20 4 22.52

图3

酵母双杂交试验分析LsAAE3与LsSAT2的蛋白互作关系"

图4

蛋白质下拉试验验证LsAAE3与LsSAT2的蛋白互作关系"

图5

基于Alphofold3.0进行LsAAE3与LsSAT2蛋白的分子对接 红色表示LsAAE3蛋白, 绿色表示LsSAT2蛋白。右下角黑框是LsSAT2蛋白的羧基末端深入到LsAAE3空腔内部的局部放大。"

表3

LsAAE3和LsSAT2蛋白相互作用面上形成的次级键"

次级键
Secondary bonds		 LsAAE3参与次级键原子
Atoms of LsAAE3 involved in bonds formation		 键长
Distance (Å)		 LsSAT2参与次级键原子
Atoms of LsSAT2 involved in bonds formation
氢键
Hydrogen bonds		 SER 250 OG 3.16 ASP 214 OD1
GLN 445 NE2 3.21 GLY 345 O
GLY 506 N 3.12 TYR 388 O
THR 322 OG1 2.39 ASP 394 OD1
ARG 416 NH2 3.57 TYR 395 O
LYS 507 NZ 3.45 VAL 396 O
ARG 510 NH1 3.34 ILE 397 OXT
ASN 280 O 3.00 THR 282 OG1
GLU 468 OE2 2.65 THR 303 OG1
GLN 445 OE1 3.10 ASN 361 N
ASP 498 O 3.08 ALA 363 N
LEU 500 O 3.45 LYS 364 N
THR 505 O 2.72 TRP 392 NE1
ALA 320 O 2.44 TYR 395 N
GLU 317 OE1 2.67 TYR 395 OH
SER 296 O 3.20 VAL 396 N
盐桥
Salt bridges		 ARG 246 NH1 3.34 ASP 384 OD2
ARG 510 NH1 3.69 ILE 397 O
ARG 510 NH1 3.34 ILE 397 OXT
ARG 510 NH2 3.90 ILE 397 OXT

表4

高效液相测定不同转基因毛状根ODAP含量"

毛状根材料
Hairy root material		 β-ODAP峰面积
β-ODAP peak area (mAU s)		 α-ODAP峰面积
α-ODAP peak area (mAU s)		 β-ODAP与α-ODAP比值
β-ODAP/α-ODAP ratio
对照
Control		 262,219.33±14,673.63 c 116,719.33±9543.39 a 2.25±0.08 b
LsSAT2过表达株系
LsSAT2 overexpression line		 124,752.33±17,058.04 b 67,523±23,737.76 b 1.92±0.40 b
LsAAE3过表达株系
LsAAE3 overexpression line		 408,908.67±50,329.23 a 80,460.67±10,518.05 b 5.11±0.56 a
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