木薯采后生理变质相关基因MeGLYI-13上游转录因子的筛选

doi:10.13560/j.cnki.biotech.bull.1985.2025-1343

生物技术通报 ›› 2026, Vol. 42 ›› Issue (6): 98-106.doi: 10.13560/j.cnki.biotech.bull.1985.2025-1343

木薯采后生理变质相关基因MeGLYI-13上游转录因子的筛选

沈晨¹^,²(), 车延年¹^,³, 丁中平¹^,³, 王祥文¹^,³, 葛玉建¹, 符东青¹, 周亚星乔¹^,⁴, 李瑞梅¹()

^1.中国热带农业科学院热带生物技术研究所热带作物生物育种全国重点实验室（三亚研究院），海口 571101
^2.华中农业大学植物科学技术学院，武汉 430070
^3.海南大学生命健康学院热带农林学院，海口 570228
^4.佳木斯大学生物与农业学院，佳木斯 154007

收稿日期:2025-12-10 出版日期:2026-06-26 发布日期:2026-07-11
通讯作者: 李瑞梅，研究员，研究方向：木薯精准分子育种技术研发及新种质创制，E-mail: liruimei@catasitbb.cn
作者简介:沈晨，硕士研究生，研究方向：木薯抗逆基因功能解析，E-mail: 19850590632@163.com
第一联系人：车延年同为本文第一作者
基金资助:
海南省自然科学基金项目(323RC537);三亚崖州湾科技城“崖州湾”菁英人才项目(SCKJ-JYRC-2024-81);三亚市科技专项(2024KJFX014);中国热带农业科学院国家热带农业科学中心科技创新团队(CATASCXTD202301);中央公益性科研院所基本科研业务费(1630032025010);热带作物生物育种全国重点实验室科研项目(NKLTCBCXTD39)

Screening of Upstream Transcription Factors of MeGLYI-13 Gene Related to Postharvest Physiological Deterioration of Cassava

SHEN Chen¹^,²(), CHE Yan-nian¹^,³, DING Zhong-ping¹^,³, WANG Xiang-wen¹^,³, GE Yu-jian¹, FU Dong-qing¹, ZHOU Ya-xing-qiao¹^,⁴, LI Rui-mei¹()

^1.Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, National Key Laboratory of Tropical Crops Biobreeding (Sanya Research Institute), Haikou 571101
^2.College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070
^3.College of Life and Health College, College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228
^4.College of Biology and Agriculture, Jiamusi University, Jiamusi 154007

Received:2025-12-10 Published:2026-06-26 Online:2026-07-11

摘要/Abstract

摘要：

目的木薯（Manihot esculenta Crantz）作为全球范围内重要的经济作物与粮食作物之一，其采后生理变质（postharvest physiological deterioration, PPD）问题对贮藏期限及经济效益产生了严重影响。MeGLYI-13作为Ⅰ型乙二醛酶（glyoxalaseⅠ, GLYⅠ）家族成员，其表达水平与木薯PPD耐性呈正相关关系。深入解析MeGLYI-13参与木薯PPD耐性的上游调控分子机制，为探究木薯采后生理性变质分子机制、创制耐PPD木薯种质提供新思路。方法针对MeGLYI-13启动子区序列进行克隆分析，构建proMeGLYI-13-pAbAi诱饵载体并转化酵母细胞，获得诱饵菌株。利用木薯cDNA文库进行酵母单杂交筛库试验，对候选转录因子进行表达模式分析和互作验证。结果成功克隆MeGLYI-13上游2 000 bp启动子序列，该序列包含低温、干旱、生长素、赤霉素、光、防御和胁迫响应的顺式作用元件。通过酵母单杂交筛库试验，从木薯cDNA文库中筛选出4个候选转录因子，分别为乙烯响应转录因子ERF1以及AT-hook基序核定位蛋白（AT-hook motif nuclear localization proteins, AHL）AHL17、AHL29和AHL31。回转验证试验和双荧光素酶试验进一步证实，MeAHL17对MeGLYI-13的启动子活性起负调控作用。基因表达分析发现，MeAHL17的表达趋势与木薯PPD耐性呈负相关。结论 MeAHL17与MeGLYI-13的启动子结合调控基因的表达，可能在木薯采后生理性变质过程中发挥作用。

关键词: 木薯, 采后生理变质, 乙二醛酶, 转录因子, 启动子, AT-hook基序核定位蛋白

Abstract:

Objective Cassava (Manihot esculenta Crantz) is one of the most important economic and food crops in the world. The problem of postharvest physiological deterioration (PPD) causes a serious impact on the storage period and economic benefits. As a member of the glyoxalaseⅠ (GLYⅠ) family, the expressionof MeGLYI-13 was positively correlated with the PPD tolerance of cassava. In-depth analysis of the molecular mechanism of upstream regulation of MeGLYI-13 involved in cassava PPD tolerance may provide a new idea for exploring the molecular mechanism of cassava postharvest physiological deterioration process, and PPD-tolerant cassava germplasm innovation. Method The promoter region sequence of MeGLYI-13 was cloned and analyzed. The proMeGLYI-13-pAbAi bait vector was constructed and transformed into yeast cells to obtain the bait strain. The expression pattern analysis and interaction verification of candidate transcription factors were carried out by yeast one-hybrid screening library experiment using cassava cDNA library. Result The 2 000 bp upstream promoter sequence of MeGLYI-13 was successfully cloned. The sequence contained cis-acting elements responding to low temperature, drought, auxin, gibberellin, light, defense and stress. Through yeast one-hybrid screening, four candidate transcription factors were screened from cassava cDNA library, which were ethylene-responsive transcription factor ERF1 and AT-hook motif nuclear localization proteins AHL17, AHL29 and AHL31. The rotary verification test and dual luciferase test further confirmed that MeAHL17 played a negative regulatory role in the promoter activity of MeGLYI-13. Gene expression analysis showed that the expression trend of MeAHL17 was negatively correlated with PPD tolerance of cassava. Conclusion The combination of MeAHL17 and MeGLYI-13 promoter regulate gene expression, which may play a role in the physiological deterioration of cassava after harvest.

Key words: cassava, postharvest physiological deterioration, glyoxalase, transcription factors, promoter, AT-hook motif nuclear localization protein

沈晨, 车延年, 丁中平, 王祥文, 葛玉建, 符东青, 周亚星乔, 李瑞梅. 木薯采后生理变质相关基因MeGLYI-13上游转录因子的筛选[J]. 生物技术通报, 2026, 42(6): 98-106.

SHEN Chen, CHE Yan-nian, DING Zhong-ping, WANG Xiang-wen, GE Yu-jian, FU Dong-qing, ZHOU Ya-xing-qiao, LI Rui-mei. Screening of Upstream Transcription Factors of MeGLYI-13 Gene Related to Postharvest Physiological Deterioration of Cassava[J]. Biotechnology Bulletin, 2026, 42(6): 98-106.

图/表 9

表1 MeGLYI-13启动子截短片段的相关信息

Table 1 Related information of the truncated fragment of MeGLYI-13 promoter

名称

Name

在启动子序列中

起始位置

The start positions in the promoter sequence （bp）

在启动子序列中

终止位置

The end positions in the promoter sequence （bp）

片段长度

Fragment length （bp）

顺式作用元件

Cis-acting elements

ARE， TCCC-motif， MBS， LTR， P-box， Box 4， CAT-box

LTR， MRE， GA-motif， CAT-box， TGA-element

TCT-motif， GT1-motif， TC-rich repeats， Sp1， GATA-motif

图1 MeGLYI-13启动子的部分顺式作用元件

Fig. 1 Partial cis-acting elements of MeGLYI-13 gene promoter

图2 诱饵菌株AbA抑制浓度的筛选p53-pAbAi：阳性对照；proMeGLYI-13-pAbAi：诱饵菌株

Fig. 2 Screening of AbA inhibitory concentration of bait strainp53-pAbAi: Positive control. proMeGLYI-13-pAbAi: Bait strain

图3 诱饵酵母菌株AbA浓度的第2次筛选p53-pAbAi： Positive control. Seg1-pAbAi， Seg2-pAbAi， Seg3-pAbAi： Bait strain

Fig. 3 The second screening of AbA concentration of bait yeast strain

图4 候选阳性菌斑的筛选和菌斑PCR验证M：2 000 marker；A‒B：Seg1转录因子的筛选；C‒F：Seg2转录因子的筛选

Fig. 4 Candidate positive plaque screening and plaque PCR verificationM: 2 000 marker. A-B: Seg1 screening of transcription factors. C-F: Seg2 screening of transcription factors

表2 proMeGLYI-13启动子筛选的候选转录因子的信息

Table 2 Information of candidate transcription factors screened by proMeGLY13 promoter

序号 Number	登录号 Gene ID	名称 Name	功能注释 Functional annotation
1	Manes.02G145600	AHL17	AT-hook motif nuclear-localized protein 17
2	Manes.09G149500	AHL29	AT-hook motif nuclear-localized protein 29
3	Manes.15G039700	ERF1	Ethylene-responsive transcription factor 1
4	Manes.10G027000	AHL31	AT-hook motif nuclear-localized protein 31

图5 MeGLYI-13启动子与候选转录因子的回转验证

Fig. 5 Rotational verification of MeGLYI-13 promoter and candidate transcription factors

图6 块根采后MeGLYI-13、MeAHL17、MeAHL29、MeAHL31的表达分析与验证*： P0.05， **： P0.01. The same below

Fig. 6 Transcriptome expression analysis and verification of MeGLYI-13, MeAHL17, MeAHL29 and MeAHL31 in theroots after harvest

图7 双荧光素酶试验验证MeAHL17与MeGLYI-13启动子的关系

Fig. 7 Dual-luciferase assay to verify the relationship between MeAHL17and the promoter of MeGLYI-13

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木薯采后生理变质相关基因MeGLYI-13上游转录因子的筛选

Screening of Upstream Transcription Factors of MeGLYI-13 Gene Related to Postharvest Physiological Deterioration of Cassava

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