Article(id=1276530133552992319, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276530095770693736, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.07.004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1742227200000, receivedDateStr=2025-03-18, revisedDate=null, revisedDateStr=null, acceptedDate=1743091200000, acceptedDateStr=2025-03-28, onlineDate=1782278100621, onlineDateStr=2026-06-24, pubDate=1753372800000, pubDateStr=2025-07-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782278100621, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782278100621, creator=13701087609, updateTime=1782278100621, updator=13701087609, issue=Issue{id=1276530095770693736, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='7', pageStart='1533', pageEnd='1784', issueExtLink='null', onlineDate='null', pubDate='1753372800000', pubDateStr='2025-07-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782278091614, creator='13701087609', updateTime=1782299002258, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276617801443971243, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276530095770693736, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276617801448165548, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276530095770693736, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1562, endPage=1570, ext={EN=ArticleExt(id=1276530134156972098, articleId=1276530133552992319, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Cloning and Functional Analysis of the SGR Gene in Capsicum annum Fruits, columnId=1236256430337085821, journalTitle=Chinese Journal of Tropical Crops, columnName=Omics & Biotechnology, runingTitle=null, highlight=null, articleAbstract=

STAY-GREEN (SGR) is a key gene that regulates chlorophyll degradation in plants and plays an important role in controlling plant senescence. To investigate the regulatory role of the SGR gene in the fruit coloration process of pepper, the homologous gene cloning method was employed to amplify the CaSGR gene from pepper fruit cDNA using PCR technology in this study. Bioinformatics analysis was conducted on its protein sequence, and a virus-induced gene silencing (VIGS) vector was constructed to preliminarily verify the gene function. The results showed that ORF of CaSGR was 792 bp, encoding 263 amino acids. Bioinformatics analysis revealed that SGR was relatively conserved among Solanaceae crops, and SGR had a closer evolutionary relationship with SGR from tomato and potato. CaSGR VIGS vector was constructed and injected into pepper leaves, successfully obtaining silenced plants. The silenced plants exhibited leaf whitening, fruit color changed, and CaSGR expression levels reduced significantly. Additionally, the chlorophyll and β-carotene content in the fruits of the silenced plants increased markedly. This study would provide a foundation for further elucidating the molecular regulatory mechanisms of CaSGR in pepper fruit coloration and offer theoretical support for breeding new pepper varieties with high carotenoid content.

, authors=null, authorsList=Yuling QIN, Weixia LIU, Zhenmu CAO, Lin LIU, Dan ZHU, Xiaomin YIN, Ziji LIU, authorCompany=null, correspAuthors=Ziji LIU, 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=1276530138271584339, articleId=1276530133552992319, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=辣椒果实SGR基因的克隆与功能分析, columnId=1236256430517440904, journalTitle=热带作物学报, columnName=组学与生物技术, runingTitle=null, highlight=null, articleAbstract=

滞绿基因(STAY-GREENSGR)是一个调控植物叶绿素降解的关键基因,在调控植物衰老方面发挥着重要作用。为研究SGR基因在辣椒果实着色过程中的调控作用,采用同源基因克隆法,利用PCR技术从辣椒果实cDNA中扩增出CaSGR基因,对其蛋白序列进行生物信息学分析,并构建病毒诱导基因沉默(VIGS)载体,初步验证基因功能。结果表明:CaSGR基因的ORF为792 bp,编码263个氨基酸;生物信息学分析发现,茄科作物SGR基因相对保守,克隆的CaSGR蛋白与番茄、马铃薯的SGR蛋白进化关系较近;构建CaSGR VIGS载体并注射辣椒叶片,成功获得沉默植株,沉默植株叶片白化,果实颜色改变,CaSGR基因的表达量显著降低,果实中叶绿素与β-胡萝卜素含量明显增高。本研究为深入解析CaSGR基因在辣椒果实着色中的分子调控机制提供基础,并为培育高类胡萝卜素含量的辣椒新品种提供理论依据。

, authors=

秦于玲(1982—),女,博士,副研究员,研究方向:蔬菜遗传育种。

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* 刘子记(LIU Ziji),E-mail:
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秦于玲(1982—),女,博士,副研究员,研究方向:蔬菜遗传育种。

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秦于玲(1982—),女,博士,副研究员,研究方向:蔬菜遗传育种。

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M: DL2000 DNA marker.

, figureFileSmall=/48BIQJ4akQTAAJs3D8SZQ==, figureFileBig=VGDmDMljAnyHfvAF+XfEcQ==, tableContent=null), ArticleFig(id=1276530150963548301, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 2, caption=Homology alignment of SGR sequences, figureFileSmall=FG7q5kM0nhsXD/PDj50Kzw==, figureFileBig=cQ2bejmPCUTIXqYpQdE/DA==, tableContent=null), ArticleFig(id=1276530151366201486, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图2, caption=SGR蛋白序列同源比对, figureFileSmall=FG7q5kM0nhsXD/PDj50Kzw==, figureFileBig=cQ2bejmPCUTIXqYpQdE/DA==, tableContent=null), ArticleFig(id=1276530151718523023, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 3, caption=Analysis of genetic evolutionary relationships of SGR in Arabidopsis thaliana and Solanaceae crops, figureFileSmall=A6INApwnjjtHHcrEJljmpQ==, figureFileBig=wAISBJGi/jmOWdpwTiUAjw==, tableContent=null), ArticleFig(id=1276530151806603408, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图3, caption=拟南芥、茄科作物SGR蛋白遗传进化关系分析, figureFileSmall=A6INApwnjjtHHcrEJljmpQ==, figureFileBig=wAISBJGi/jmOWdpwTiUAjw==, tableContent=null), ArticleFig(id=1276530152171507857, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 4, caption=PCR amplification of CaSGR target sequence, figureFileSmall=SG8EvmDKyYJ+tVAcet+kRg==, figureFileBig=LOuOT3mTBaLjfFtZymPkDg==, tableContent=null), ArticleFig(id=1276530152548995218, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图4, caption=CaSGR靶序列的PCR扩增

M: DL2000 DNA marker.

, figureFileSmall=SG8EvmDKyYJ+tVAcet+kRg==, figureFileBig=LOuOT3mTBaLjfFtZymPkDg==, tableContent=null), ArticleFig(id=1276530152624492691, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 5, caption=Restriction enzyme digestion of TRV2-CaSGR vector, figureFileSmall=D/h1WYuO89k3zQGOXjPH0g==, figureFileBig=Y7gm9oQKpsrwg96c5bunlA==, tableContent=null), ArticleFig(id=1276530153052311700, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图5, caption=TRV2-CaSGR载体的酶切鉴定

M:DL12000 DNA marker;1:TRV2-CaSGR载体未酶切;2:TRV2- CaSGR载体酶切。

, figureFileSmall=D/h1WYuO89k3zQGOXjPH0g==, figureFileBig=Y7gm9oQKpsrwg96c5bunlA==, tableContent=null), ArticleFig(id=1276530153379467413, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 6, caption=Phenotype of pepper plants after VIGS injection, figureFileSmall=xd62XIbEfIdZgmoIBNLgUA==, figureFileBig=mHi1DpnOfNZUd4nW2lST7g==, tableContent=null), ArticleFig(id=1276530153459159191, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图6, caption=VIGS注射后的辣椒植株表型, figureFileSmall=xd62XIbEfIdZgmoIBNLgUA==, figureFileBig=mHi1DpnOfNZUd4nW2lST7g==, tableContent=null), ArticleFig(id=1276530153832452248, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Fig. 7, caption=Detection of CaSGR gene silencing efficiency, figureFileSmall=hWETOtn2EI+7hMvgHSuThA==, figureFileBig=r8dP8esMKcw0VlIbm3Q+9w==, tableContent=null), ArticleFig(id=1276530154209939609, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=图7, caption=CaSGR基因沉默效率检测

*表示显著差异(P<0.05)。

, figureFileSmall=hWETOtn2EI+7hMvgHSuThA==, figureFileBig=r8dP8esMKcw0VlIbm3Q+9w==, tableContent=null), ArticleFig(id=1276530154335768730, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Tab. 1, caption=

Primer sequences used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列(5′-3′)Primer sequences (5′-3′)
CaSGR-FCGGGATCCCGATGGGGACTTTGACTGCTTCT
CaSGR-RGGGGTACCCCTCAGCTTTGCTGCTCTTGC
VIGS-FCTCTAGAG TCTAGAACGTTCATAGTTGTCAAGT
VIGS-RCGGATCCG GGATCCCATCCTTATTATACCACCC
qRT-PCR-FCCTGCTGTTCTCCTTTCT
qRT-PCR-RCAACTTCTTCCCCTTTTA
Actin-FCCACCTCTTCACTCTCTGCTCT
Actin-RACTAGGAAAAACAGCCCTTGGT
), ArticleFig(id=1276530154658730139, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=表1, caption=

本研究中的引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列(5′-3′)Primer sequences (5′-3′)
CaSGR-FCGGGATCCCGATGGGGACTTTGACTGCTTCT
CaSGR-RGGGGTACCCCTCAGCTTTGCTGCTCTTGC
VIGS-FCTCTAGAG TCTAGAACGTTCATAGTTGTCAAGT
VIGS-RCGGATCCG GGATCCCATCCTTATTATACCACCC
qRT-PCR-FCCTGCTGTTCTCCTTTCT
qRT-PCR-RCAACTTCTTCCCCTTTTA
Actin-FCCACCTCTTCACTCTCTGCTCT
Actin-RACTAGGAAAAACAGCCCTTGGT
), ArticleFig(id=1276530155048800412, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Tab. 2, caption=

Relative analysis of members of SGR family in Solanaceae crops

, figureFileSmall=null, figureFileBig=null, tableContent=
作物种类Crop species基因ID Gene ID核苷酸长度Gene length/bp氨基酸长度Amino acid length等电点pI分子量Molecular weight/kDa亚细胞定位Predicted subcellular localization
辣椒(Capsicum annuumEU196733.112812669.2629.94叶绿体
AM746208.28012669.2629.94叶绿体
NM_001324918.19952669.2629.94叶绿体
CaSGR9182639.1929.60叶绿体;细胞核
野生番茄(Solanum pennelliiXM_015229006.212712728.8430.55叶绿体;细胞核
樱桃番茄(Lycopersicon esculentumDQ100158.112062728.8530.53叶绿体
番茄(Solanum lycopersicumNM_001247794.111692728.8530.53叶绿体
XM_004252594.410392478.5627.90细胞核
野生马铃薯(Solanum verrucosumXM_049506185.112102728.9930.46叶绿体;细胞核
窄叶马铃薯(Solanum stenotomum)XM_049521326.112212728.8830.46叶绿体;细胞核
XM_049543335.19482478.9028.15细胞核
马铃薯(Solanum tuberosumXM_015303798.18782488.3228.13细胞膜;细胞核
XM_015303797.19102488.3228.13细胞膜;细胞核
XM_006364069.211772468.3227.90细胞核
XM_006358786.212832728.8730.48叶绿体;细胞核
XM_006364068.211822478.7928.18细胞核
绒毛状烟草(Nicotiana tomentosiformis)XM_009608848.414682658.8929.77叶绿体;细胞核
XM_009628189.412912598.8529.10细胞膜;细胞核
烟草(Nicotiana tabacumXM_016644658.114402679.1029.99叶绿体;细胞核
EU294209.18052679.1929.95叶绿体;细胞核
XM_016651072.114602678.7529.86叶绿体;细胞核
XM_016642907.112892598.8529.10细胞膜;细胞核
XM_016645914.114522598.7229.08细胞核
南美烟草(Nicotiana sylvestris)XM_009800664.215142678.7529.86叶绿体;细胞核
XM_009804691.214712598.7229.08细胞核
美洲烟草(Nicotiana attenuate)XM_019401856.115232678.8829.86叶绿体;细胞核
XM_019404849.114232548.7928.68细胞核
XM_019406621.110122609.0029.49叶绿体;细胞核
拟南芥(Arabidopsis thalianaAY699948.111162718.8830.75叶绿体
AY850161.113012688.8330.05叶绿体
), ArticleFig(id=1276530155480813725, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=表2, caption=

茄科作物SGR基因家族成员的相关分析

, figureFileSmall=null, figureFileBig=null, tableContent=
作物种类Crop species基因ID Gene ID核苷酸长度Gene length/bp氨基酸长度Amino acid length等电点pI分子量Molecular weight/kDa亚细胞定位Predicted subcellular localization
辣椒(Capsicum annuumEU196733.112812669.2629.94叶绿体
AM746208.28012669.2629.94叶绿体
NM_001324918.19952669.2629.94叶绿体
CaSGR9182639.1929.60叶绿体;细胞核
野生番茄(Solanum pennelliiXM_015229006.212712728.8430.55叶绿体;细胞核
樱桃番茄(Lycopersicon esculentumDQ100158.112062728.8530.53叶绿体
番茄(Solanum lycopersicumNM_001247794.111692728.8530.53叶绿体
XM_004252594.410392478.5627.90细胞核
野生马铃薯(Solanum verrucosumXM_049506185.112102728.9930.46叶绿体;细胞核
窄叶马铃薯(Solanum stenotomum)XM_049521326.112212728.8830.46叶绿体;细胞核
XM_049543335.19482478.9028.15细胞核
马铃薯(Solanum tuberosumXM_015303798.18782488.3228.13细胞膜;细胞核
XM_015303797.19102488.3228.13细胞膜;细胞核
XM_006364069.211772468.3227.90细胞核
XM_006358786.212832728.8730.48叶绿体;细胞核
XM_006364068.211822478.7928.18细胞核
绒毛状烟草(Nicotiana tomentosiformis)XM_009608848.414682658.8929.77叶绿体;细胞核
XM_009628189.412912598.8529.10细胞膜;细胞核
烟草(Nicotiana tabacumXM_016644658.114402679.1029.99叶绿体;细胞核
EU294209.18052679.1929.95叶绿体;细胞核
XM_016651072.114602678.7529.86叶绿体;细胞核
XM_016642907.112892598.8529.10细胞膜;细胞核
XM_016645914.114522598.7229.08细胞核
南美烟草(Nicotiana sylvestris)XM_009800664.215142678.7529.86叶绿体;细胞核
XM_009804691.214712598.7229.08细胞核
美洲烟草(Nicotiana attenuate)XM_019401856.115232678.8829.86叶绿体;细胞核
XM_019404849.114232548.7928.68细胞核
XM_019406621.110122609.0029.49叶绿体;细胞核
拟南芥(Arabidopsis thalianaAY699948.111162718.8830.75叶绿体
AY850161.113012688.8330.05叶绿体
), ArticleFig(id=1276530155921215646, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=EN, label=Tab. 3, caption=

Determination of chlorophyll and β-carotene in pepper CaSGR gene-silenced lines

, figureFileSmall=null, figureFileBig=null, tableContent=
植株Plant叶绿素含量Chlorophyll content/(mg·g-1)β-胡萝卜素含量β-carotene content/(μg·g-1)
CK0.014±0.003c8.24±1.37b
TRV2-CaSGR-10.033±0.002b21.43±1.98a
TRV2-CaSGR-20.069±0.005a26.47±4.33a
TRV2-CaSGR-30.029±0.002b26.61±2.85a
), ArticleFig(id=1276530156307091615, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276530133552992319, language=CN, label=表3, caption=

辣椒CaSGR基因沉默株系的叶绿素与β-胡萝卜素的测定

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植株Plant叶绿素含量Chlorophyll content/(mg·g-1)β-胡萝卜素含量β-carotene content/(μg·g-1)
CK0.014±0.003c8.24±1.37b
TRV2-CaSGR-10.033±0.002b21.43±1.98a
TRV2-CaSGR-20.069±0.005a26.47±4.33a
TRV2-CaSGR-30.029±0.002b26.61±2.85a
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辣椒果实SGR基因的克隆与功能分析
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秦于玲 , 刘维侠 , 曹振木 , 刘琳 , 朱丹 , 殷晓敏 , 刘子记 *
热带作物学报 | 组学与生物技术 2025,46(7): 1562-1570
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热带作物学报 |组学与生物技术 2025 , 46 (7) : 1562 -1570
辣椒果实SGR基因的克隆与功能分析
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秦于玲, 刘维侠, 曹振木, 刘琳, 朱丹, 殷晓敏, 刘子记*
作者信息
  • 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
通讯作者:
* 刘子记(LIU Ziji),E-mail:
Cloning and Functional Analysis of the SGR Gene in Capsicum annum Fruits
Yuling QIN, Weixia LIU, Zhenmu CAO, Lin LIU, Dan ZHU, Xiaomin YIN, Ziji LIU*
Affiliations
  • Institute of Tropical Crops Genetic Resources, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
出版时间: 2025-07-25 doi: 10.3969/j.issn.1000-2561.2025.07.004
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滞绿基因(STAY-GREENSGR)是一个调控植物叶绿素降解的关键基因,在调控植物衰老方面发挥着重要作用。为研究SGR基因在辣椒果实着色过程中的调控作用,采用同源基因克隆法,利用PCR技术从辣椒果实cDNA中扩增出CaSGR基因,对其蛋白序列进行生物信息学分析,并构建病毒诱导基因沉默(VIGS)载体,初步验证基因功能。结果表明:CaSGR基因的ORF为792 bp,编码263个氨基酸;生物信息学分析发现,茄科作物SGR基因相对保守,克隆的CaSGR蛋白与番茄、马铃薯的SGR蛋白进化关系较近;构建CaSGR VIGS载体并注射辣椒叶片,成功获得沉默植株,沉默植株叶片白化,果实颜色改变,CaSGR基因的表达量显著降低,果实中叶绿素与β-胡萝卜素含量明显增高。本研究为深入解析CaSGR基因在辣椒果实着色中的分子调控机制提供基础,并为培育高类胡萝卜素含量的辣椒新品种提供理论依据。

辣椒  /  CaSGR基因  /  VIGS技术  /  叶绿素  /  β-胡萝卜素

STAY-GREEN (SGR) is a key gene that regulates chlorophyll degradation in plants and plays an important role in controlling plant senescence. To investigate the regulatory role of the SGR gene in the fruit coloration process of pepper, the homologous gene cloning method was employed to amplify the CaSGR gene from pepper fruit cDNA using PCR technology in this study. Bioinformatics analysis was conducted on its protein sequence, and a virus-induced gene silencing (VIGS) vector was constructed to preliminarily verify the gene function. The results showed that ORF of CaSGR was 792 bp, encoding 263 amino acids. Bioinformatics analysis revealed that SGR was relatively conserved among Solanaceae crops, and SGR had a closer evolutionary relationship with SGR from tomato and potato. CaSGR VIGS vector was constructed and injected into pepper leaves, successfully obtaining silenced plants. The silenced plants exhibited leaf whitening, fruit color changed, and CaSGR expression levels reduced significantly. Additionally, the chlorophyll and β-carotene content in the fruits of the silenced plants increased markedly. This study would provide a foundation for further elucidating the molecular regulatory mechanisms of CaSGR in pepper fruit coloration and offer theoretical support for breeding new pepper varieties with high carotenoid content.

Capsicum annum  /  CaSGR gene  /  VIGS technology  /  chlorophyll  /  β-carotene
秦于玲, 刘维侠, 曹振木, 刘琳, 朱丹, 殷晓敏, 刘子记. 辣椒果实SGR基因的克隆与功能分析. 热带作物学报, 2025 , 46 (7) : 1562 -1570 . DOI: 10.3969/j.issn.1000-2561.2025.07.004
Yuling QIN, Weixia LIU, Zhenmu CAO, Lin LIU, Dan ZHU, Xiaomin YIN, Ziji LIU. Cloning and Functional Analysis of the SGR Gene in Capsicum annum Fruits[J]. Chinese Journal of Tropical Crops, 2025 , 46 (7) : 1562 -1570 . DOI: 10.3969/j.issn.1000-2561.2025.07.004
辣椒(Capsicum spp.)是全球重要的蔬菜和调味品,近几年我国辣椒种植面积均稳定在210万hm2左右,产业规模居蔬菜首位[1-3]。辣椒果色是最直观的外在品质性状和经济性状,辣椒果实因含有类胡萝卜素种类不同而呈现不同的颜色,同时辣椒果实着色过程伴随叶绿素的降解和类胡萝卜素的合成[4]
滞绿基因(Stay-green,SGR)可延迟植物衰老过程中的叶绿素降解,导致叶片保持绿色,对植物生物量、品质和保鲜有重要影响。SGR基因在高等植物中以基因家族形式存在,一般有2个或3个同源基因都参与植物叶绿素降解调控[5-6],目前SGR基因在拟南芥[7]、番茄[8]、辣椒[9]等作物中研究较多。SATO等[10]在拟南芥中过表达AtSGR1会加速叶片叶绿素的的降解,而呈现叶片黄化;SAKURABA等[11]研究发现,拟南芥中的STAY-GREEN2SGR2)基因是一个负调控因子,它在叶片衰老过程中抑制叶绿素降解,可见其与在拟南芥的同源基因发挥的功能完全不同;刘宇华[6]在研究辣椒果实着色中发现,CaSGR2CaSGR- LIKE在果实发育的整个过程中不表达或低表达,叶绿素降解主要由CaSGR1调控;番茄果实中RNA沉默SISGR1或CRISPR/Cas9编辑SISGR1,会使成熟番茄果实呈现棕色,番茄红素和β-胡萝卜素含量增加,研究发现是SISGR1SIPSY1相互作用,从而调控番茄果实番茄红素的积累[12-14]
对于尚未建立稳定的遗传转化体系的植物来说,利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)方法验证基因功能,是较高效、便捷的手段[15]。ZHOU等[16]建立了高效的辣椒VIGS体系,在多个品种实现高达100%的沉默效率;ZHANG等[17]通过构建与辣椒花青素合成相关的TRV2-MYB载体,发现MYB转录因子抑制辣椒花青素的合成;利用VIGS沉默辣椒素合成的基因(ComtpAmtKas),辣椒果实中辣椒素含量明显降低[18];申龙斌等[19]研究发现,在海南黄灯笼辣椒沉默凯氏带膜蛋白CASP,沉默植株中基因的表达量显著下降,可见VIGS技术在辣椒基因功能研究中具有广阔的应用前景。
本课题组前期在辣椒果实不同着色时期的转录组数据中发现SGR基因差异表达显著,本研究从辣椒果实中克隆CaSGR基因,利用生物信息学软件分析CaSGR基因亚细胞定位及进化关系等,构建VIGS载体,分析沉默植株的基因表达以及叶绿素、β-胡萝卜素的含量,以期为研究CaSGR在辣椒果实着色中的调控作用提供基础。
提取RNA的实验材料来自自主选育的辣椒高代自交系17SCa2m,种植于海南省儋州试验场五队(19°33.66′N,109°31.63′E),采取成熟辣椒果实,去除胎座和种子,立即放入液氮中,-80 ℃冰箱保存。
根据天根多糖多酚RNA提取试剂盒(DP441)说明书提取辣椒果实RNA,使用全式金反转录试剂盒(AE311)反转录cDNA,琼脂糖凝胶电泳检测RNA完整性,使用NanoDrop检测RNA浓度。
利用Primer 5.0软件设计CaSGR-F和CaSGR-R的引物(表1),以上述得到的cDNA为模板,PCR扩增目的片段。PCR反应体系为:10×PCR Buffer 5 μL、2 mmol/L dNTPs 5 μL、25 mmol/L MgSO4 3 μL、引物1.5 μL、TOYOBO KOD-Plus-Neo酶1 μL、cDNA 2 μL、ddH2O 32.5 μL。PCR扩增程序为:94 ℃ 2 min;98 ℃ 10 s,58 ℃ 30 s,72 ℃ 30 s,循环30次;72 ℃ 10 min;4 ℃保存。用1.0%琼脂糖凝胶电泳检测,将胶回收PCR产物、连接pEASY-T1 Cloning Kit载体和转化大肠杆菌感受态细胞DH5α,菌落PCR筛选阳性克隆送至生工生物工程(上海)股份有限公司测序。
通过NCBI数据库的ORF finder(https://www.ncbi.nlm.nih.gov/orffinder/)工具找出基因的开放阅读框ORF和翻译出的蛋白序列;在NCBI数据库Blastp并下载拟南芥和茄科作物的SGR蛋白序列,使用MEGA 7软件采用neighbour joining(NJ)系统发育方法分析进化关系[20],并用ITOL(https://itol.embl.de/)在线软件美化;使用ProtParam(https://web.expasy.org/protparam/)和Cell-PLoc 2.0(http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/[21]在线软件预测分子量、等电点和亚细胞定位;使用DNAMAN软件对SGR蛋白序列进行多重比对分析。
利用SGN VIGS(https://vigs.solgenomics.net/)在线软件设计CaSGR VIGS片段,根据片段设计引物(表1),以上述1.2.2中的阳性质粒为模板PCR扩增VIGS片段,经测序验证后,与酶切后的TRV2连接,转化到DH5α感受态细胞。经双酶切验证后,将重组质粒转化到GV3101感受态细胞,PCR检测成功的菌液用于VIGS注射实验。
待辣椒幼苗生长至3~4片真叶期,分别挑取TRV2-CaPDS、TRV2-CaSGR、TRV2和TRV1已转化农杆菌GV3101的单克隆,在含有卡那霉素的液体LB培养基中28 ℃震荡培养24 h,4000 r/min离心20 min,收集菌体。用添加乙酰丁香酮的MES缓冲液重悬浮2次,调整TRV1的OD600为0.4,TRV2-CaPDS、TRV2-CaSGR和TRV2的OD600为0.2,将TRV2-CaPDS、TRV2-CaSGR和TRV2分别与TRV1等量混合,28 ℃黑暗放置3~4 h。用2 mL的注射器将菌液注入到辣椒子叶和真叶中,于16 ℃,16 h光照/8 h黑暗培养3 d。移到23 ℃ 16 h光照、20 ℃ 8 h黑暗的环境下培养,直至出现白化表型和目标表型。
注射120 d后,采取红熟期的辣椒,去除果柄、胎座和种子,利用试剂盒提取辣椒果实RNA,通过Primer 5.0软件设计CaSGR基因的qRT-PCR特异性引物(表1)。qRT-PCR反应体系(20 μL)为:SYBR 10 μL,cDNA模板1 μL,正反向引物各0.8 μL,ddH2O 7.4 μL。qRT-PCR反应程序:95 ℃ 1 min;95 ℃ 10 s,55 ℃ 30 s,72 ℃ 15 s,40个循环。每个样品3次重复,以actin作为内参基因,采用2–∆∆Ct的方法分析目标基因的表达情况,利用Excel软件作图,利用SPSS软件作显著性分析。
以未注射辣椒为对照(CK),对照植株和沉默植株在23 ℃16 h光照、20 ℃ 8 h黑暗的培养箱中培养约120 d,采取辣椒果实,取样方法同1.2.6,采用分光光度法测定叶绿素含量[22];β-胡萝卜素含量测定参照QIN等[23]的方法。
利用特异引物CaSGR-F/R和高保真酶,以辣椒果实的cDNA为模板进行PCR扩增,经琼脂糖电泳检测得到1条约800 bp的条带(图1)。测序后经过NCBI的ORF finder工具分析发现,该基因的ORF为792 bp,编码263个氨基酸。将该基因在NCBI数据库Blast,发现与辣椒(EU196733.1、AM746208.2、NM_001324918.1)的SGR基因一致,将其命名为CaSGR
利用克隆的基因核苷酸序列在NCBI数据库中搜索分析,获得茄科作物中的27个SGR基因,其中辣椒SGR基因3个,番茄SGR基因4个,马铃薯SGR基因8个,烟草SGR基因12个,另外在数据库中搜索到拟南芥SGR基因2个。辣椒SGR同源基因的序列长度差异明显,最短的为801 bp,最长的为1281 bp。茄科作物之间的SGR蛋白质序列长度和分子量差异不明显,说明不同种属间SGR基因相对保守。理论等电点均大于7,表明SGR均是碱性蛋白。亚细胞定位预测显示,有5个SGR蛋白定位在叶绿体,12个同时定位在叶绿体、细胞核,4个同时定位在细胞膜、细胞核,7个定位在细胞核(表2)。
利用DANMAN软件将克隆的CaSGR基因编码的蛋白序列与辣椒、番茄的SGR蛋白序列进行多序列比对结果显示,蛋白序列相似度为88.94%,与数据库中辣椒SGR蛋白的序列相比,CaSGR在155~157处有3个氨基酸(VLK)缺失(图2)。
利用MEGA 7软件将CaSGR蛋白序列与拟南芥、茄科作物的SGR蛋白序列进行系统进化树分析发现,CaSGR蛋白与番茄、马铃薯的SGR蛋白进化关系较近,与烟草和拟南芥的SGR蛋白进化关系较远(图3)。
使用带有酶切位点的特异性引物扩增CaSGR基因的病毒诱导基因沉默(VIGS)靶片段。扩增产物经琼脂糖凝胶电泳检测,条带大小一致(图4)。回收靶片段,同时双酶切回收产物和TRV2载体,并将酶切后的片段与TRV2载体连接,转化到DH5α感受态细胞中,对重组质粒进行酶切鉴定(图5),并送公司测序,经比对和靶片段一致,表明成功构建CaSGR VIGS载体。
采用叶片注射法将TRV2-CaSGR与TRV1等比例混合,注射到辣椒叶片中,22 d左右叶片出现较为明显的白化现象。注射120 d后观察植株表型,注射TRV2-CaPDS的植株白化依然明显,大部分叶片呈白化特征,辣椒果实也有变化,比对照和注射TRV2-CaSGR的颜色略浅(图6)。
为检测基因的沉默效率,取沉默植株的辣椒果实,利用qRT-PCR分析CaSGR基因的表达量。结果显示(图7),3组实验组辣椒果实中CaSGR基因的表达量显著低于对照组,说明成功沉默该基因。
测定对照和3个沉默辣椒植株的果实叶绿素与β-胡萝卜素含量,结果显示(表3),3个沉默辣椒植株的果实叶绿素与β-胡萝卜素含量显著高于对照,叶绿素含量分别是对照的2.35、4.92、2.07倍,β-胡萝卜素含量分别是对照的2.60、3.21、3.22倍。
本研究通过克隆辣椒CaSGR基因,构建VIGS载体,并测定沉默株系中与辣椒果实着色相关的叶绿素和β-胡萝卜素含量,为深入研究该基因在辣椒果实着色过程中的作用提供有力支撑。
本研究成功获得了长度为792 bp、编码263个氨基酸的基因片段,与数据库中辣椒SGR蛋白序列相比,CaSGR在第155~157位置存在3个氨基酸缺失,这一差异并未对辣椒果实的叶绿素降解产生影响。但在他人研究中指出编码区单碱基的突变可引起SGR基因功能的缺失[6],如LIU等[9]在辣椒滞绿突变体中发现CaSGR1编码区第342位核苷酸由G突变成A,导致第114位氨基酸转化成终止密码子,翻译提前终止;EFRATI等[24]研究叶绿素滞绿基因cl时发现,第340位核苷酸处碱基T突变成C,氨基酸由Trp变为Arg;在番茄[8]、豌豆[25]等滞绿突变体中也均发现有碱基突变,从而导致氨基酸的改变。
CaSGR VIGS载体的成功构建为研究该基因的功能提供了关键技术手段。本研究通过叶片注射法实现了CaSGR基因的沉默,沉默植株叶片出现明显白化现象,果实颜色也发生变化,同时基因表达量显著降低,证明了CaSGR基因在辣椒果实着色过程中发挥着重要作用。而沉默植株果实中叶绿素与β-胡萝卜素含量的显著增加,这与前人在研究SGR基因沉默后阳性植株的表型不太一致。如杨亮等[14]利用CRISPR/Cas9技术编辑番茄SlSGR1基因,阳性植株的番茄果实叶绿素、β-胡萝卜素和番茄红素含量显著提高;HU等[12]干扰沉默番茄的SlSGR1,沉默植株叶片和果实的叶绿素降解变低;ZHOU等[26]通过RNA干扰MsSGR获得沉默紫花苜蓿株系,沉默株系中苜蓿仍呈现绿色。但与SAKURABA等[11]研究拟南芥中的STAY-GREEN2SGR2)基因功能一致,该研究发现是一个负调控因子,在叶片衰老过程中抑制叶绿素降解,本研究中的CaSGR基因可能与拟南芥SGR2基因是同一类型,也有可能是3个氨基酸的缺失导致的沉默植株中叶绿素降解延缓。
本研究明确了CaSGR基因与辣椒果实叶绿素和β-胡萝卜素含量的关联,但具体的分子调控机制尚不清楚,有待进一步探索。未来可利用酵母双杂交、免疫共沉淀等技术筛选与CaSGR蛋白相互作用的蛋白,构建其调控网络。同时,还可以为分析该基因的启动子序列是否与叶绿素降解和类胡萝卜素合成过程中结构基因有相互作用奠定基础。摸清CaSGR在辣椒果实着色过程中的调控作用,将有助于培育高类胡萝卜素含量的辣椒新品种,提升辣椒的营养价值和商品价值。
  • 海南省科技计划三亚崖州湾科技城联合项目(320LH067)
  • 中央级公益性科研院所基本科研业务费专项(1630032022009)
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doi: 10.3969/j.issn.1000-2561.2025.07.004
  • 接收时间:2025-03-18
  • 首发时间:2026-06-24
  • 出版时间:2025-07-25
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  • 收稿日期:2025-03-18
  • 录用日期:2025-03-28
基金
海南省科技计划三亚崖州湾科技城联合项目(320LH067)
中央级公益性科研院所基本科研业务费专项(1630032022009)
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    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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* 刘子记(LIU Ziji),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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