Article(id=1276531660757799528, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.04.019, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1727798400000, receivedDateStr=2024-10-02, revisedDate=null, revisedDateStr=null, acceptedDate=1734278400000, acceptedDateStr=2024-12-16, onlineDate=1782278464736, onlineDateStr=2026-06-24, pubDate=1745510400000, pubDateStr=2025-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782278464736, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782278464736, creator=13701087609, updateTime=1782278464736, updator=13701087609, issue=Issue{id=1276531538535781212, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='4', pageStart='777', pageEnd='1024', issueExtLink='null', onlineDate='null', pubDate='1745510400000', pubDateStr='2025-04-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782278435595, creator='13701087609', updateTime=1782278607615, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276532260098675208, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276532260098675209, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276531538535781212, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=968, endPage=977, ext={EN=ArticleExt(id=1276531661026234986, articleId=1276531660757799528, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Effects of DNA Methylation on Physiological Indexes Related to Drought Hardening in Watermelon with Different Ploidy, columnId=1236256434120348225, journalTitle=Chinese Journal of Tropical Crops, columnName=Plant Cultivation, Physiology & Biochemistry, runingTitle=null, highlight=null, articleAbstract=

In order to explore the effect of DNA methylation on drought hardening of different ploidy watermelon, mannitol was used to mimic drought stress in suspension cultured cells of different ploidy watermelon. The cell growth and resistance related physiological indexes were measured after stress, recovery and re-stress treatments. Meanwhile, the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) was used to explore whether methylation is involved in resistance regulation. The results showed that 5-AzaC could retard the growth, inhibit the extracellular alkalization, increase the content of malondialdehyde, bring down the content proline and the activity of protective enzymes such as SOD in all types of cells. Moreover, it caused the membrane permeability raising in diploid and triploid cells. Above results suggest that 5-AzaC can aggravate the osmotic stress injury and reverse the effects of drought hardening partially. Principal component analysis revealed that malondialdehyde content, proline content, PAL, CAT and APX activity could be used as the main evaluation indexes for evaluation the effect of drought hardening. This study would provide clues for mining the physiological indexes for evaluate the effects of stress hardening and the mechanism of DNA methylation involved in watermelon resistance regulation.

, authors=null, authorsList=Xiaoqing WANG, Yu GAO, Xuefei JIANG, Fei QIAO, authorCompany=null, correspAuthors=Xuefei JIANG, Fei QIAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1276531664004190840, articleId=1276531660757799528, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=DNA甲基化影响不同倍性西瓜抗旱锻炼相关生理指标的分析, columnId=1236256434313286224, journalTitle=热带作物学报, columnName=作物栽培与生理生化, runingTitle=null, highlight=null, articleAbstract=

为了探索DNA甲基化对不同倍性西瓜抗旱锻炼的影响,本研究以不同倍性西瓜悬浮培养细胞为材料,使用甘露醇(Mannitol)模拟干旱胁迫,经过胁迫、恢复、再胁迫的方法测定细胞生长情况和抗性相关生理指标。同时,利用DNA甲基转移酶抑制剂5-氮杂胞苷(5-AzaC)处理,探索甲基化对这些生理指标的影响。结果表明,在干旱条件下,抗逆锻炼对细胞的生长有显著的保护作用。但是,5-AzaC抑制了细胞的质外体碱化,提升了丙二醛(MDA)含量,使脯氨酸(Pro)含量和超氧化物歧化酶(SOD)等保护酶活性降低,加剧了渗透胁迫的损伤,部分逆转了细胞抗旱锻炼的效果,且在西瓜二倍体和三倍体细胞中尤其明显。通过主成分分析发现,MDA、Pro含量和苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性指标可以作为西瓜细胞干旱胁迫抗逆锻炼效果的主要评定指标。本研究可为DNA甲基化参与西瓜抗性锻炼效果的评价指标筛选和形成机制提供参考。

, authors=

王潇晴(1998—),男,硕士研究生,研究方向:园艺植物逆境生理与分子生物学。

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* 江雪飞(JIANG Xuefei),E-mail:
乔飞(QIAO Fei),E-mail:
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2.Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
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王潇晴(1998—),男,硕士研究生,研究方向:园艺植物逆境生理与分子生物学。

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王潇晴(1998—),男,硕士研究生,研究方向:园艺植物逆境生理与分子生物学。

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不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=nUQt7MrcGWf5fOKBRNaymA==, figureFileBig=c02iZG31P/UCzuSjaZpsCA==, tableContent=null), ArticleFig(id=1276531678847832752, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Fig. 3, caption=Effects of osmotic stress and methylation on extracellular alkalization, figureFileSmall=+vIQI3hN8Savsvo11YAPGQ==, figureFileBig=OW79QBOPmuuyG+YZNeVQlQ==, tableContent=null), ArticleFig(id=1276531678935913137, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=图3, caption=渗透胁迫和甲基化对质外体碱化的影响

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=+vIQI3hN8Savsvo11YAPGQ==, figureFileBig=OW79QBOPmuuyG+YZNeVQlQ==, tableContent=null), ArticleFig(id=1276531679003022002, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Fig. 4, caption=Effects of osmotic stress and methylation on membrane permeability, figureFileSmall=4sSS5YkaJgvc2XO7wK0jwA==, figureFileBig=IJuOuNPGYnHCUnyqwVoczA==, tableContent=null), ArticleFig(id=1276531679275651763, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=图4, caption=渗透胁迫和甲基化对细胞膜透性的影响

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=4sSS5YkaJgvc2XO7wK0jwA==, figureFileBig=IJuOuNPGYnHCUnyqwVoczA==, tableContent=null), ArticleFig(id=1276531679346954932, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Fig. 5, caption=Influence of DNA methylation on MDA content and proline content, figureFileSmall=jjeZU4Zdqj2pvRS1tTkNiQ==, figureFileBig=p751uqbr5QJNBAdrR+uFkg==, tableContent=null), ArticleFig(id=1276531679418258101, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=图5, caption=DNA甲基化对丙二醛含量和脯氨酸含量的影响

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=jjeZU4Zdqj2pvRS1tTkNiQ==, figureFileBig=p751uqbr5QJNBAdrR+uFkg==, tableContent=null), ArticleFig(id=1276531679481172662, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Fig. 6, caption=Influence of DNA methylation on activities of antioxidase, figureFileSmall=wMJl4PjGxBViJBJN4kUyuw==, figureFileBig=+IlDFpULT4gcQZdpK+/fzQ==, tableContent=null), ArticleFig(id=1276531679728636599, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=图6, caption=DNA甲基化对抗氧化酶活性的影响

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=wMJl4PjGxBViJBJN4kUyuw==, figureFileBig=+IlDFpULT4gcQZdpK+/fzQ==, tableContent=null), ArticleFig(id=1276531679808328376, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Tab. 1, caption=

Correlation analysis of physiological and biochemical indexes of watermelon suspension cells with different ploidy under drought hardening and DNA methylation

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index鲜质量Fresh weight∆PCVpH电导率ConductivityMDAProPODSODAPXGPXCAT
∆PCV0.953**
pH0.0030.020
电导率0.1190.1060.263**
MDA0.0440.201–0.1950.435**
Pro–0.433**–0.566**0.254–0.337*–0.664**
POD–0.698**–0.742**–0.037–0.103–0.0770.180
SOD–0.651**–0.590**0.126–0.626**–0.2850.570**0.330*
APX–0.561**–0.612**0.154–0.661**–0.583**0.654**0.538**0.811**
GSH-Px–0.152–0.1580.187–0.552**–0.629**0.475**0.2550.416*0.670**
CAT–0.714**–0.725**0.138–0.244–0.340*0.350*0.800**0.449**0.645**0.496**
PAL–0.473**–0.292–0.1420.2580.569**–0.2740.2260.090–0.254–0.2930.071
), ArticleFig(id=1276531679892214457, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=表1, caption=

干旱锻炼和DNA甲基化处理不同倍性西瓜悬浮细胞生理生化指标相关性分析

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index鲜质量Fresh weight∆PCVpH电导率ConductivityMDAProPODSODAPXGPXCAT
∆PCV0.953**
pH0.0030.020
电导率0.1190.1060.263**
MDA0.0440.201–0.1950.435**
Pro–0.433**–0.566**0.254–0.337*–0.664**
POD–0.698**–0.742**–0.037–0.103–0.0770.180
SOD–0.651**–0.590**0.126–0.626**–0.2850.570**0.330*
APX–0.561**–0.612**0.154–0.661**–0.583**0.654**0.538**0.811**
GSH-Px–0.152–0.1580.187–0.552**–0.629**0.475**0.2550.416*0.670**
CAT–0.714**–0.725**0.138–0.244–0.340*0.350*0.800**0.449**0.645**0.496**
PAL–0.473**–0.292–0.1420.2580.569**–0.2740.2260.090–0.254–0.2930.071
), ArticleFig(id=1276531680169038522, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=EN, label=Tab. 2, caption=

Principal component analysis was used to assess contribution rate of suspended cells derived from watermelon with varying ploidy under drought hardening and DNA methylation

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index主成分1Principal component 1主成分2Principal component 2
MDA–0.9840.127
Pro0.973–0.198
PAL–0.9470.307
CAT0.928–0.220
APX0.8860.459
POD0.4370.892
SOD0.4520.880
pH–0.4810.823
GSH-Px0.5940.799
∆PCV–0.532–0.780
鲜质量0.637–0.755
电导率–0.5370.734
特征值6.4155.010
方差贡献率/%53.46241.754
累积方差贡献率/%53.46295.216
), ArticleFig(id=1276531681863537340, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, language=CN, label=表2, caption=

干旱锻炼和DNA甲基化处理不同倍性西瓜悬浮细胞主成分分析值及贡献率

, figureFileSmall=null, figureFileBig=null, tableContent=
指标Index主成分1Principal component 1主成分2Principal component 2
MDA–0.9840.127
Pro0.973–0.198
PAL–0.9470.307
CAT0.928–0.220
APX0.8860.459
POD0.4370.892
SOD0.4520.880
pH–0.4810.823
GSH-Px0.5940.799
∆PCV–0.532–0.780
鲜质量0.637–0.755
电导率–0.5370.734
特征值6.4155.010
方差贡献率/%53.46241.754
累积方差贡献率/%53.46295.216
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DNA甲基化影响不同倍性西瓜抗旱锻炼相关生理指标的分析
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王潇晴 1, 2, 3 , 高宇 1, 2, 3 , 江雪飞 1, 3, * , 乔飞 2, *
热带作物学报 | 作物栽培与生理生化 2025,46(4): 968-977
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热带作物学报 |作物栽培与生理生化 2025 , 46 (4) : 968 -977
DNA甲基化影响不同倍性西瓜抗旱锻炼相关生理指标的分析
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2.Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
3.Sanya Institute of Breeding and Multiplication, Hainan University, Sanya, Hainan 572025, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1276531665111487116, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276531660757799528, authorId=1276531664687862406, language=CN, stringName=王潇晴, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, 3, address=1.海南大学热带农林学院/海南省热带园艺作物品质调控重点实验室,海南儋州 571737
2.中国热带农业科学院热带作物品种资源研究所,海南海口 571101
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王潇晴(1998—),男,硕士研究生,研究方向:园艺植物逆境生理与分子生物学。

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王潇晴(1998—),男,硕士研究生,研究方向:园艺植物逆境生理与分子生物学。

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王潇晴1, 2, 3, 高宇1, 2, 3, 江雪飞1, 3, * , 乔飞2, *
作者信息
  • 1.海南大学热带农林学院/海南省热带园艺作物品质调控重点实验室,海南儋州 571737
  • 2.中国热带农业科学院热带作物品种资源研究所,海南海口 571101
  • 3.海南大学三亚南繁研究院,海南三亚 572025
通讯作者:
* 江雪飞(JIANG Xuefei),E-mail:
乔飞(QIAO Fei),E-mail:
Effects of DNA Methylation on Physiological Indexes Related to Drought Hardening in Watermelon with Different Ploidy
Xiaoqing WANG1, 2, 3, Yu GAO1, 2, 3, Xuefei JIANG1, 3, * , Fei QIAO2, *
Affiliations
  • 1.School of Tropical Agriculture and Forestry, Hainan University / Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Danzhou, Hainan 571737, China
  • 2.Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
  • 3.Sanya Institute of Breeding and Multiplication, Hainan University, Sanya, Hainan 572025, China
出版时间: 2025-04-25 doi: 10.3969/j.issn.1000-2561.2025.04.019
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为了探索DNA甲基化对不同倍性西瓜抗旱锻炼的影响,本研究以不同倍性西瓜悬浮培养细胞为材料,使用甘露醇(Mannitol)模拟干旱胁迫,经过胁迫、恢复、再胁迫的方法测定细胞生长情况和抗性相关生理指标。同时,利用DNA甲基转移酶抑制剂5-氮杂胞苷(5-AzaC)处理,探索甲基化对这些生理指标的影响。结果表明,在干旱条件下,抗逆锻炼对细胞的生长有显著的保护作用。但是,5-AzaC抑制了细胞的质外体碱化,提升了丙二醛(MDA)含量,使脯氨酸(Pro)含量和超氧化物歧化酶(SOD)等保护酶活性降低,加剧了渗透胁迫的损伤,部分逆转了细胞抗旱锻炼的效果,且在西瓜二倍体和三倍体细胞中尤其明显。通过主成分分析发现,MDA、Pro含量和苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性指标可以作为西瓜细胞干旱胁迫抗逆锻炼效果的主要评定指标。本研究可为DNA甲基化参与西瓜抗性锻炼效果的评价指标筛选和形成机制提供参考。

西瓜  /  倍性  /  DNA甲基化  /  干旱胁迫  /  生理指标  /  抗旱锻炼

In order to explore the effect of DNA methylation on drought hardening of different ploidy watermelon, mannitol was used to mimic drought stress in suspension cultured cells of different ploidy watermelon. The cell growth and resistance related physiological indexes were measured after stress, recovery and re-stress treatments. Meanwhile, the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) was used to explore whether methylation is involved in resistance regulation. The results showed that 5-AzaC could retard the growth, inhibit the extracellular alkalization, increase the content of malondialdehyde, bring down the content proline and the activity of protective enzymes such as SOD in all types of cells. Moreover, it caused the membrane permeability raising in diploid and triploid cells. Above results suggest that 5-AzaC can aggravate the osmotic stress injury and reverse the effects of drought hardening partially. Principal component analysis revealed that malondialdehyde content, proline content, PAL, CAT and APX activity could be used as the main evaluation indexes for evaluation the effect of drought hardening. This study would provide clues for mining the physiological indexes for evaluate the effects of stress hardening and the mechanism of DNA methylation involved in watermelon resistance regulation.

watermelon  /  ploidy  /  DNA methylation  /  drought stress  /  physiological index  /  drought hardening
王潇晴, 高宇, 江雪飞, 乔飞. DNA甲基化影响不同倍性西瓜抗旱锻炼相关生理指标的分析. 热带作物学报, 2025 , 46 (4) : 968 -977 . DOI: 10.3969/j.issn.1000-2561.2025.04.019
Xiaoqing WANG, Yu GAO, Xuefei JIANG, Fei QIAO. Effects of DNA Methylation on Physiological Indexes Related to Drought Hardening in Watermelon with Different Ploidy[J]. Chinese Journal of Tropical Crops, 2025 , 46 (4) : 968 -977 . DOI: 10.3969/j.issn.1000-2561.2025.04.019
西瓜(Citrullus lanatus)是一种非常重要的园艺作物。在世界上,中国西瓜种植面积最大,其年产量占世界总产量的67%左右[1-2]。干旱不仅会影响西瓜的光合作用,抑制细胞有丝分裂,使细胞膜发生膜质过氧化,还会影响植物的电导率、渗透调节功能及活性氧代谢等[3-5]。因此,生产上经常在苗期利用适度干旱锻炼即“蹲苗”的措施来提高作物的抗旱能力[6]
抗逆锻炼是指植物经过短期逆境处理,植物再次遭遇逆境时表现出更强的抗性,这种响应也被称为“胁迫记忆”[4-5]。例如,干旱锻炼后的小麦遇到二次胁迫时,与未经过干旱锻炼的小麦相比可以缓解其减产[7];再次遭遇干旱时,其苗高和鲜质量都相对较高[8]。并且丙二醛(MDA)含量减少、脯氨酸(Pro)含量升高,过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等活性降低,说明干旱锻炼可以减少活性氧的产生并且提高植物抗旱性[9-11]。然而,植物胁迫记忆的产生机制目前仍不清楚。
研究发现,胁迫记忆的产生过程与DNA甲基化修饰紧密相关[12]。植物在多倍体形成及其后续稳定阶段,基因结构会经历重构,基因的表达模式也会发生改变;在此过程中,表观遗传修饰(甲基化等)对基因功能和结构的变化起到关键作用,促进了植物的适应性和多样性[13-15],而多倍体的产生经常会引起DNA甲基化变异[16]。西瓜多倍体材料丰富,不同倍性材料DNA甲基化研究方面已取得长足进展。例如,已明确西瓜基因组中DNA甲基化水平与倍性之间缺乏关联[17],并且观察到三倍体西瓜表现出一种独特的去甲基化模式[18];在逆境条件下,发现不同倍性材料之间甲基化差异不明显,但在相同NaCl浓度处理下二倍体甲基化率大于四倍体和三倍体[19]。然而,DNA甲基化是否参与了西瓜的抗旱锻炼目前尚未见报道。
悬浮培养细胞具有生长一致、周期稳定、受环境影响小等优点,在细胞水平研究抗性形成机制方面有着独特的优势[20]。因此,本研究以不同倍性西瓜悬浮细胞为材料,通过测定DNA甲基化对细胞生长量、电导率、质外体碱、MDA、Pro含量和SOD、POD活性等生理指标的变化,初步了解不同倍性西瓜抗旱锻炼抗性形成机制,为西瓜抗旱生理研究和栽培提供参考。
利用西瓜叶片诱导愈伤组织,培育并筛选出一致性、分散性较好的二倍体(2n)、三倍体(3n)和四倍体(4n)西瓜悬浮细胞作为试验材料。该材料目前保存于中国热带农业科学院热带作物品种资源研究所。悬浮细胞使用恒温摇床150 r/min,25 ℃,黑暗条件下进行培养,培养基配方与文献[21]相同,初始接种量为5 mL,继代周期为10 d。
使用甘露醇(Mannitol)模拟干旱胁迫,参考ZHU等[22]的方法设定浓度(100 mmol/L);使用DNA甲基化酶抑制剂5-氮杂胞苷(5-AzaC)调节基因组DNA甲基化水平,参考ZHU等[20]的方法设定浓度(100 μmol/L)。每个倍性细胞系设计4个试验处理:(1)CK(对照);(2)S(胁迫处理);(3)S-R(胁迫处理+恢复);(4)A(胁迫处理+甲基化)(图1)。
处理后的细胞于平台期(10 d)连同培养基倒入真空抽滤装置,抽干培养液,称取0.10 g细胞于2 mL离心管中液氮速冻,然后存放于–80 ℃超低温冰箱待用。
使用细胞压积法(packed cell volume,PCV)测定悬浮细胞的生长量[22]。在平台期将悬浮细胞连同培养基倒入50 mL量筒中密封,静置过夜,记录悬浮培养体系总体积(VT)以及细胞沉淀体积(VC),计算出细胞的总体积变化:DPCV=VC/VT。在平台期将细胞和培养基一同倒入真空抽滤装置,将悬浮细胞抽至微干,使用万分之一天平进行称重,记录细胞鲜质量。
在细胞快速生长期(7 d),无菌条件下吸取2 mL细胞于5 mL离心管中,放入摇床中150 r/min,25 ℃培养30~60 min后加入适量蒸馏水,再次稳定30 min后倒入小烧杯[22]。进行以下3个处理:(1)Mannitol处理(100 mmol/L Mannitol);(2)CK处理(等体积超纯水);(3)Mannitol+5-AzaC处理(100 mmol/L Mannitol+100 μmol/L 5-AzaC);以上3个处理30 min,分别使用pH计(Mettler toledo,pH 12)测定pH的相对变化量(DpH)。
在细胞指数生长期(7 d),无菌环境下吸取5 mL细胞于10 mL离心管中,将其放入150 r/min,25 ℃摇床培养30~60 min后加入适量蒸馏水,继续稳定30 min后,进行以下3个处理:(1)Mannitol处理(100 mmol/L Mannitol);(2)CK处理(等体积超纯水);(3)Mannitol+5-AzaC处理(100 mmol/L Mannitol+100 μmol/L 5-AzaC)。以上3个处理10 min,分别立即测其电导率。
参考郑子凡等[23]的方法,使用试剂盒(苏州科铭生物技术有限公司)分光光度法测定丙二醛(MDA)含量;磺基水杨酸法测定脯氨酸(Pro)含量。
参考郑子凡等[23]的方法,使用试剂盒(苏州科铭生物技术有限公司)愈创木酚法测定过氧化物酶(POD)活性;采用氮蓝四唑(NBT)法测定超氧化物歧化酶(SOD)活性;采用生化法测定抗坏血酸过氧化物酶(APX)活性;采用紫外分光光度计法测定谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)活性。
每个处理3个生物学重复。使用Excel软件整理数据,采用统计分析软件DPS 15.10的Duncan法进行差异显著性分析。用Graphpad Prism 8作图。使用隶属函数法[24]观察DNA甲基化对细胞影响并进行综合评价。当测定指标的变化率与抗旱性呈正相关,则Xu=(XXmin)/(XmaxXmin);当测定指标的变化率与抗旱性呈负相关,则Xu=1–(XXmin)/(XmaxXmin)。式中,Xu表示不同倍性西瓜悬浮细胞某一测定指标的变化率,Xmin表示该指标中的最小值,Xmax表示最大值。
图2可知,处理S可以显著降低细胞的鲜质量和总体积,其中,2n、3n、4n在胁迫处理后的鲜质量为对照的40%~50%。但经S-R处理,细胞的生长可以恢复至对照的70%。而2n、3n、4n处理A的鲜质量分别是处理S-R的71.31%、66.35%、41.79%,ΔPCV也呈现了相同的降低趋势,逆转了抗逆锻炼的效果。说明DNA的甲基化参与了抗逆锻炼对细胞生长的保护。
质外体碱化是植物细胞遭受逆境后的重要保护机制之一[25]。由图3可知,在2n、3n、4n中,Mannitol处理的ΔpH均比CK提高,而Mannitol+5-AzaC处理的ΔpH均比Mannitol处理降低。表明渗透胁迫可以使培养液pH迅速上升。在3n中,ΔpH变化最为明显,Mannitol处理比CK升高约0.32。同时,5-AzaC可以降低质外体碱化的程度,Mannitol+5-AzaC处理的ΔpH比Mannitol处理降低约0.06~0.10。表明DNA的甲基化也参与了质外体碱化。
当植物遭遇逆境时,细胞膜结构极易受损,这种损伤使细胞膜的通透性增加,细胞内部电解质等成分流失,细胞电导率上升[26-27]。因此,可以通过测量电导率评估细胞膜所遭受的损害程度。由图4可知,在2n、3n、4n中,Mannitol处理的电导率均比CK提高。而Mannitol+5-AzaC处理3n的电导率再次升高,且与CK差异显著,2n、4n电导率则基本保持不变。同时,Mannitol+5-AzaC处理2n、4n的电导率小幅度上升,但与CK差异不显著;而在3n中,电导率比CK约上升800 μS/cm,差异显著。表明在三倍体中,抑制DNA去甲基化可导致细胞膜受损程度加深。
MDA是细胞膜磷脂双分子层受损,引发细胞膜膜脂过氧化的重要产物,可以评估膜脂过氧化的变化[28]。由图5可知,S-R处理的MDA含量显著降低;而A处理2n、3n、4n的MDA含量均比S-R处理显著升高,MDA含量分别是S-R处理的1.13倍、1.12倍、1.31倍。说明抗逆锻炼可以降低干旱胁迫引起的MDA对质膜的伤害,并且DNA的甲基化可能参与了MDA的生成调控。但是,在三倍体和四倍体中,对照的MDA含量较高,倍性增加造成MDA水平升高的问题仍有待探索。
植物遭遇干旱胁迫时可以通过调节渗透调节物质增强植物的抗旱性[29]。S处理的Pro含量升高13~22 μg/g;2n的Pro含量升高约30 μg/g,3n、4n的Pro含量升高约11 μg/g;而复合使用5-AzaC后降低5~15 μg/g。在2n、3n、4n中,S处理的Pro含量均升高,而A、S-R处理的Pro含量的上升趋势均受到抑制。说明抑制DNA甲基化降低了抗旱锻炼的效果,表明DNA甲基化参与了细胞的渗透调节过程。
植物遭遇干旱胁迫时,可以通过调节抗氧化酶POD、SOD、APX、GSH-Px、CAT、PAL等的活性来减轻干旱胁迫带来的伤害[8-11]。由图6可知,在2n、3n、4n中,S处理的几种抗氧化酶活性均显著提高,而S-R处理均有所下降且差异显著。说明抗逆锻炼可以显著降低过氧化物的产生。而A处理的抗氧化酶活性在不同倍性细胞上的变化趋势不同。如SOD、APX、CAT、PAL活性在不同倍性细胞中,A处理的变化趋势均显著逆转。而POD、GSH-Px活性在不同倍性细胞中的变化趋势无明显规律。说明去甲基化/甲基化均有可能参与了细胞清除过氧化物的过程,但对不同保护酶的作用不尽相同。
对以上生理指标的相关性分析发现,很多指标间存在极显著相关性(表1),为了更有效地探讨众多生理指标间的相关性和重要性,使用主成分分析[24]进行进一步综合评价(表2)。在干旱胁迫抗逆锻炼下,MDA、Pro含量及PAL、CAT、APX活性可以作为评价DNA甲基化影响细胞生长的主要指标,SOD、POD活性和pH作为二级指标。通过计算细胞在5-AzaC处理后的隶属函数值发现,三倍体受甲基化的影响最大(平均隶属函数值0.700);二倍体次之(平均隶属函数值0.433),四倍体受甲基化的影响最小(平均隶属函数值0.365)。
不同植物在干旱胁迫下会表现出不同的内部适应性机制,即便是同一物种的不同品种,在遭遇相同或不同的胁迫时,也可能呈现出不同的应对策略与生理响应模式[30]。在拟南芥[31]和番茄[32]中均发现,遭遇干旱胁迫之后植物表现出更好的生长状况,盐和碱性胁迫诱导了水稻DNA甲基化的跨代改变[33],HERMAN等[34]研究也发现桃叶蓼存在抗性跨代遗传的现象。本研究通过干旱预处理细胞后,将其进行正常恢复培养,约经过2次细胞分裂之后再次干旱胁迫,发现细胞比直接胁迫生长更好,说明通过抗旱锻炼后细胞可能具有更强的抗旱性,由此推测西瓜细胞也可能存在跨代遗传的现象。复合使用5-AzaC处理后,抗旱锻炼的效果被抑制,说明DNA去甲基化过程和抗性调节相关,并且甲基化可能是胁迫记忆形成的重要机制之一。
在干旱胁迫下,还可以通过测定细胞外pH和电导率判断干旱对番茄细胞生长环境的影响[35]。铝胁迫复合使用5-AzaC处理后,还可引起质外体碱化现象,造成细胞生长环境改变,使生长受到抑制,并对细胞膜系统造成了伤害[26-27]。XU等[36]研究发现四倍体相比于二倍体和三倍体具有更大的基因组,基因表达水平保留更完整、维持相对较低的甲基化水平。此外,甲基化相关酶和调控因子的表达水平等差异也可能影响细胞对甲基化抑制剂的敏感性[37]。本研究发现,四倍体西瓜悬浮细胞使用甘露醇处理后略高于甘露醇和5-AzaC共处理的电导率。由此推测四倍体西瓜基因组较大,所以受到的影响较小,本研究通过对不同倍性细胞进行综合评价,结果也表明四倍体受到甲基化的影响较小。
此外,渗透调节物质也会影响悬浮细胞的生长[29],且在不同的倍性中具有不同的表现。董民会[38]发现多倍体猕猴桃比二倍体染色体数量较多引起的基因剂量效应减缓了MDA的形成。使用秋水仙碱诱导猕猴桃发现,盐胁迫10 d时的MDA含量低于胁迫5 d的MDA含量[38]。本研究发现,三倍体和四倍体渗透胁迫10 d时MDA含量则低于对照,由此推测可能长时间胁迫导致细胞启动防御机制,增加了其适应能力,导致其MDA含量低于对照。而该现象并没有发生在在二倍体中,说明胁迫诱导产生的MDA可能和基因剂量负相关。复合使用5-AzaC处理细胞后与抗旱锻炼后相比,MDA含量显著上升,发生膜脂过氧化,细胞膜受损程度加深,表明5-AzaC抑制了抗旱锻炼的效果,影响细胞的生长。
脯氨酸具有亲水性,在干旱胁迫下其含量会增加,帮助细胞维持水分,脯氨酸含量越高其抗旱性越强[39-40]。水稻在干旱胁迫及恢复后其抗旱性会增强[40]。高鹏飞等[41]和陈翠红等[42]也发现脯氨酸含量随胁迫时间的增加而上升。本研究结果表明,在干旱胁迫下脯氨酸含量显著上升,抗旱锻炼后的脯氨酸含量最高,这表明经过预处理后的细胞抗旱性明显增加。复合使用5-AzaC后,抗旱锻炼诱导脯氨酸含量上升的效果被显著抑制,印证了DNA的去甲基化和抗性相关基因的启动紧密相关[43]。本研究还发现,利用5-AzaC抑制DNA甲基化可以逆转抗性锻炼的效果,说明DNA甲基化参与了抗逆锻炼抗性的形成。且不同倍性材料之间效果不同,说明不同倍性材料之间DNA甲基化水平存在差异。
植物细胞在逆境下会产生大量的活性氧,植物可以通过调节细胞内抗氧化防御系统,维持其正常的生长[3,44]。在西瓜[29]和黄瓜[44]的研究中发现干旱胁迫会导致抗氧化酶活性增加。此外,在玉米和番茄的研究结果表明低温锻炼可以提高其抗冷性[45]。莫言玲等[24]结果表明不同倍性的植物抗氧化酶变化不同。本研究结果表明,在渗透胁迫处理下不同倍性细胞抗氧化酶系统具有相同的变化趋势,POD、SOD、APX、GSH-Px、CAT、PAL活性与对照相比均显著增加;经过抗旱锻炼后这些酶的活性与胁迫处理相比均有所下降,这可能是经过抗旱锻炼细胞产生活性氧较少,其含量相应降低。本研究在细胞水平测定后发现,抗旱锻炼和DNA甲基化共同处理下这些生理指标变化趋势不尽相同,但经过相关性分析和主成分分析后发现部分指标具有显著相关性,并且在指标的重要性上尝试进行了排序,可为抗性锻炼效果的评价体系构建提供参考。
综上所述,本研究结果表明DNA甲基化在不同倍性西瓜抗旱锻炼中发挥着重要的调控作用。通过改变与抗旱相关基因的DNA甲基化水平,能够影响西瓜的生理生化特性,从而影响其抗旱能力。这为后期进一步探索DNA甲基化相关基因在抗逆锻炼中的具体机制提供科学依据。
  • 国家自然科学基金项目(32260801)
  • 海南省自然科学基金高层次人才项目(321RC473)
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2025年第46卷第4期
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doi: 10.3969/j.issn.1000-2561.2025.04.019
  • 接收时间:2024-10-02
  • 首发时间:2026-06-24
  • 出版时间:2025-04-25
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  • 收稿日期:2024-10-02
  • 录用日期:2024-12-16
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国家自然科学基金项目(32260801)
海南省自然科学基金高层次人才项目(321RC473)
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    1.海南大学热带农林学院/海南省热带园艺作物品质调控重点实验室,海南儋州 571737
    2.中国热带农业科学院热带作物品种资源研究所,海南海口 571101
    3.海南大学三亚南繁研究院,海南三亚 572025

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* 江雪飞(JIANG Xuefei),E-mail:
乔飞(QIAO Fei),E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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