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作物学报 ›› 2025, Vol. 51 ›› Issue (11): 3096-3104.doi: 10.3724/SP.J.1006.2025.54028

• 研究简报 • 上一篇    下一篇

甘薯β淀粉酶基因IbBAM48829的功能解析

张顺杰1,2,3,4(), 吴维泰1, 冉禧玥1,2,3,4, 赵梓含1,2,3,4, 韩永辉1,2,3,4, 吴正丹1,5,*(), 张凯1,*()   

  1. 1 西南大学农学与生物科技学院, 重庆 400715
    2 薯类生物学与遗传育种重庆市重点实验室, 重庆 400715
    3 南方山地农业教育部工程研究中心, 重庆 400715
    4 重庆市作物种质北碚薯类资源库, 重庆 400715
    5 广西大学农学院 / 植物科学国家级实验教学示范中心, 广西南宁 530004
  • 收稿日期:2025-02-27 接受日期:2025-08-14 出版日期:2025-11-12 网络出版日期:2025-08-26
  • 通讯作者: *吴正丹, E-mail: wudandan0905@163.com; 张凯, E-mail: zhangkai2010s@163.com
  • 作者简介:E-mail: zhangshunjie0113@163.com
  • 基金资助:
    重庆市技术创新与应用发展专项重点项目(cstc2021jscx-gksbX0022);重庆市自然科学基金创新发展基金联合基金(市教委)项目(CSTB2025NSCQ-LZX0022);广西自然科学基金项目(2023GXNSFBA026297);重庆市作物种质北碚薯类资源库专项资金项目(ZWZZ2020005);重庆市薯类产业技术体系创新团队专项资金项目资助

Functional analysis of the sweetpotato β-Amylase IbBAM48829

ZHANG Shun-Jie1,2,3,4(), WU Wei-Tai1, RAN Xi-Yue1,2,3,4, ZHAO Zi-Han1,2,3,4, HAN Yong-Hui1,2,3,4, WU Zheng-Dan1,5,*(), ZHANG Kai1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2 Chongqing Key Laboratory of Biology and Genetic Breeding for Tuber and Root Crops, Chongqing 400715, China
    3 Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    4 Chongqing Beibei Germplasm Bank of Tuber and Root Crops, Chongqing 400715, China
    5 College of Agriculture, Guangxi University / National Demonstration Center for Experimental Plant Science Education, Nanning 530004, Guangxi, China
  • Received:2025-02-27 Accepted:2025-08-14 Published:2025-11-12 Published online:2025-08-26
  • Contact: *E-mail: wudandan0905@163.com; E-mail: zhangkai2010s@163.com
  • Supported by:
    Technology Innovation and Application Development Key Project of Chongqing(cstc2021jscx-gksbX0022);Key Project of Chongqing Natural Science Foundation Innovation and Development Joint Fund (Chongqing Municipal Education Commission)(CSTB2025NSCQ-LZX0022);Guangxi Natural Science Foundation Project(2023GXNSFBA026297);Special Fund for Chongqing Beibei Germplasm Bank of Tuber and Root Crops(ZWZZ2020005);and the Special Fund for the Innovation Team of Tuber and Root Crops Industry Technology System in Chongqing

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摘要：

甘薯(Ipomoea batatas (L.) Lam.)块根中的β-淀粉酶可以分解淀粉产生糖组分, 影响甘薯的干率、甜度、口感, 进而影响其经济价值。鉴定甘薯中关键β-淀粉酶基因, 解析其功能, 可为甘薯品质和适口性改良奠定重要基础。本研究针对前期甘薯转录组数据筛选到的块根发育过程中差异表达的β-淀粉酶候选基因IbBAM48829, 通过RT-qPCR分析其在甘薯不同部位的表达模式; 构建pCAMBIA1300-IbBAM48829-GFP表达载体, 瞬时转化本氏烟草进行亚细胞定位分析; 采用Gateway技术构建pEarleyGate101-IbBAM48829超量表达载体, 通过蘸花法转入拟南芥野生型Col-0中进行异源表达和功能鉴定。结果表明, IbBAM48829在甘薯块根中的表达量较低, 而在叶柄和叶片中的表达量较高。亚细胞定位结果显示, IbBAM48829可能定位于细胞质和叶绿体中。异源表达IbBAM48829基因的拟南芥植株与野生型相比生长无明显差异, 转基因株系营养生长和开花表现正常。异源表达IbBAM48829基因的拟南芥叶片中的淀粉含量、可溶性糖含量和种子千粒重均显著提高, 但根尖淀粉含量无明显差别。推测IbBAM48829在叶片淀粉代谢过程发挥重要作用, 可能通过加速淀粉代谢产生促进气孔开放的渗透活性代谢物, 进而促进气孔开放, 提高光合产物的积累。

关键词: 甘薯, β-淀粉酶, 淀粉, 异源表达, 功能分析

Abstract:

β-Amylase in the storage roots of sweetpotato (Ipomoea batatas (L.) Lam.) catalyzes the hydrolysis of starch into sugars, influencing dry matter content, sweetness, texture, and ultimately, commercial value. Identifying key β-amylase genes and elucidating their functions is essential for improving the quality and palatability of sweetpotato. In this study, we focused on the β-amylase candidate gene IbBAM48829, previously identified through transcriptome analysis based on its differential expression during storage root development. The expression pattern of IbBAM48829 across various sweetpotato tissues was analyzed by RT-qPCR. To investigate its subcellular localization, the pCAMBIA1300-IbBAM48829-GFP expression vector was constructed and transiently expressed in Nicotiana benthamiana leaves. In addition, the pEarleyGate101-IbBAM48829 overexpression vector was generated using Gateway cloning and introduced into Arabidopsis thaliana Col-0 plants via the floral dip method for heterologous expression and functional analysis. The results showed that IbBAM48829 was expressed at lower levels in storage roots but at higher levels in petioles and leaves. Subcellular localization analysis suggested that the IbBAM48829 protein may localize to both the cytoplasm and chloroplasts. Transgenic Arabidopsis lines overexpressing IbBAM48829 displayed no significant differences in growth or flowering phenotypes compared to wild-type plants. However, these lines exhibited significantly increased starch and soluble sugar contents in the leaves, along with a higher thousand-seed weight, while starch content in the root tips remained unchanged. These findings suggest that IbBAM48829 may play a critical role in leaf starch metabolism, potentially accelerating starch degradation and generating osmotically active metabolites that promote stomatal opening and enhance the accumulation of photosynthetic products.

Key words: sweetpotato, β-amylase, starch, heterologous expression, functional analysis

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 用途
Application
T48829-F CACCATGAGTTTGGCACACCAAAT PCR扩增
PCR amplification
T48829-R ATGCCTGAGAGCTTCTTGCAC
488-OE-F AGAACACGGGGGACGATGAGTTTGGCACACCAAATTGG
488-OE-R CGACTCTAGAGGATCATGCCTGAGAGCTTCTTGCAC
IbUBI-F CTTGCTGGAGATTCCGATGT 表达模式分析
Expression pattern analysis
IbUBI-R CTTGCTGGAGATTCCGATGT
AtActin-F ACACTGTGCCAATCTACGAGGGTT
AtActin-R ACAATTTCCCGCTCTGCTGTTGTG
48829Q-F GGAGGAGGAATTATGAGTATGTATC
48829Q-R CTTGAATTTCCACTATGGTTTCAC
Fbar CGACATCCGCCGTGCCACCGA 阳性鉴定
Positive detection
Rbar GTACCGGCAGGCTGAAGTCCAGC

图1

IbBAM48829基因在甘薯品种“徐薯22”中的表达模式"

图2

IbBAM48829亚细胞定位"

图3

IbBAM48829基因在拟南芥中异源表达 A: 异源表达IbBAM48829拟南芥转基因植株表型; 1: Col-0; 2~4: 异源表达IbBAM48829拟南芥植株。B: IbBAM48829在拟南芥转基因植株叶片中表达量。"

图4

IbBAM48829异源表达影响拟南芥淀粉和可溶性糖含量 A: 转基因拟南芥种子千粒重。B: 转基因拟南芥叶片可溶性糖含量。C: 转基因拟南芥叶片淀粉含量。D: 转基因拟南芥叶片碘染; 1: Col-0; 2~4: 异源表达IbBAM48829基因的拟南芥株系。E: Col-0拟南芥根尖碘染。F: 异源表达IbBAM48829基因的拟南芥根尖碘染。标尺: 50 μm。*、**、***和****分别表示在0.05、0.01、0.001和0.0001水平差异显著。"

图5

异源表达IbBAM48829影响拟南芥叶片保卫细胞形态 A: Col-0。 B: 异源表达IbBAM48829基因的拟南芥。标尺: 50.0 μm。"

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