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Article(id=1276616348486734586, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276616263778562546, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2025.11.001, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1750262400000, receivedDateStr=2025-06-19, revisedDate=null, revisedDateStr=null, acceptedDate=1754323200000, acceptedDateStr=2025-08-05, onlineDate=1782298655864, onlineDateStr=2026-06-24, pubDate=1764000000000, pubDateStr=2025-11-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782298655864, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782298655864, creator=13701087609, updateTime=1782298655864, updator=13701087609, issue=Issue{id=1276616263778562546, tenantId=1146029695717560320, journalId=1235980609244409860, year='2025', volume='46', issue='11', pageStart='2549', pageEnd='2815', issueExtLink='null', onlineDate='null', pubDate='1764000000000', pubDateStr='2025-11-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782298635668, creator='13701087609', updateTime=1782299117657, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276618285483426694, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276616263778562546, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276618285487620999, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276616263778562546, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2549, endPage=2558, ext={EN=ArticleExt(id=1276616348734198524, articleId=1276616348486734586, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Bioinformatics, Subcellular Localization and Expression Analysis of Gene Tai6.33665 in Sweet Potato, columnId=1236256430337085821, journalTitle=Chinese Journal of Tropical Crops, columnName=Omics & Biotechnology, runingTitle=null, highlight=null, articleAbstract=

Sweet potato (Ipomoea batatas) is a globally significant food and functional crop, with its purple-fleshed varieties drawing particular attention due to the high anthocyanin content. Anthocyanins not only give the tubers distinct color but also possess antioxidant, anti-inflammatory, and disease-preventive physiological functions, making them highly valuable in the development of health foods. However, the biosynthesis of anthocyanins is regulated by a multi-level network, and the role of bHLH transcription factors in sweet potatoes has not been fully elucidated. This study focused on the sweet potato gene Tai6.33665, aiming to clarify its molecular characteristics, subcellular localization pattern, and regulatory role in anthocyanin accumulation, providing a theoretical basis for molecular breeding of high-anthocyanin sweet potato varieties. Using purple-fleshed sweet potatoe (PFSP) and white-fleshed sweet potatoe (WFSP) as materials, the study analyzed the protein structure and evolutionary relationship of Tai6.33665 through bioinformatics, detected the gene expression pattern using qRT-PCR, constructed a Tai6.33665-GFP fusion vector for transient transformation in tobacco, determined the subcellular localization using laser confocal microscopy, and analyzed the correlation between gene expression and anthocyanin content and the miRNA interaction mechanism. The results showed that the protein encoded by Tai6.33665 had a typical bHLH domain and was highly homologous to anthocyanin synthesis regulatory factors. Its expression level was significantly negatively correlated with anthocyanin accumulation (P<0.05), and it was targeted and inhibited by ib-miR52. Subcellular localization indicated that the protein was located in the endoplasmic reticulum, suggesting that it might indirectly regulate metabolism through post-translational modification or the endoplasmic reticulum pathway. This study confirmed that Tai6.33665 acted as a negative regulator of anthocyanin biosynthesis in sweet potatoes, and its function depended on subcellular localization specificity and miRNA-mediated post-transcriptional regulatory networks. This discovery not only enriches the theoretical framework of plant secondary metabolism regulation but also provides a dual-track strategy for molecular breeding by targeting knockout of Tai6.33665 or overexpression of ib-miR52 using CRISPR/Cas9, the genetic limitations of anthocyanin accumulation could be overcome, and new high-anthocyanin sweet potato varieties could be developed. Future work would focus on elucidating the interaction mechanism between the gene and MYB/WD40 proteins, as well as the regulatory pathway of its transcriptional activity by endoplasmic reticulum localization.

, authors=null, authorsList=Jun XIONG, Xiuhua TANG, Minzheng WEI, authorCompany=null, correspAuthors=Xiuhua TANG, Minzheng WEI, 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=1276616350516777742, articleId=1276616348486734586, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=甘薯Tai6.33665基因生物信息学、亚细胞定位及表达分析, columnId=1236256430517440904, journalTitle=热带作物学报, columnName=组学与生物技术, runingTitle=null, highlight=null, articleAbstract=

甘薯（Ipomoea batatas）作为全球重要的粮食与功能作物，其紫肉品种因富含花青素而备受关注。花青素不仅赋予块根鲜明的色泽，更具有抗氧化、抗炎及预防慢性疾病的生理功能，因此在健康食品开发中具有重要价值。然而，花青素的生物合成受多层级调控网络影响，其中bHLH转录因子的功能尚未在甘薯中被充分解析。本研究聚焦甘薯基因Tai6.33665，旨在阐明其分子特性、亚细胞定位模式及其在花青素积累中的调控作用，为高花青素甘薯品种的分子育种提供理论依据。本研究以紫肉甘薯（PFSP）和白肉甘薯（WFSP）为材料，通过生物信息学分析Tai6.33665蛋白的结构与进化关系，利用qRT-PCR检测基因表达模式，构建Tai6.33665-GFP融合载体进行烟草瞬时转化，结合激光共聚焦显微镜明确亚细胞定位，分析基因表达与花青素含量的相关性及miRNA互作机制。结果表明：Tai6.33665编码的蛋白具有典型bHLH结构域，与花青素合成调控因子高度同源，其表达量与花青素积累呈显著负相关（P<0.05），且受ib-miR52靶向抑制；亚细胞定位显示该蛋白定位于内质网，推测其可能通过翻译后修饰或内质网通路间接调控代谢。本研究证实了Tai6.33665作为负调控因子抑制甘薯花青素生物合成，其功能实现依赖于亚细胞定位特异性及miRNA介导的转录后调控网络。这一发现不仅丰富了植物次生代谢调控的理论框架，更为分子育种提供了双轨策略：通过CRISPR/Cas9靶向敲除Tai6.33665或过表达ib-miR52，可突破花青素积累的遗传限制，培育高花青素含量甘薯新品种。未来工作将聚焦于解析该基因与MYB/WD40蛋白的互作机制，以及内质网定位对其转录活性的调控路径。

, authors=

熊军（1978—），男，博士，副研究员，研究方向：作物育种和栽培技术。

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* 韦民政（WEI Minzheng），E-mail：
唐秀桦（TANG Xiuhua），E-mail：。
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熊军（1978—），男，博士，副研究员，研究方向：作物育种和栽培技术。

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熊军（1978—），男，博士，副研究员，研究方向：作物育种和栽培技术。

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Molecular Plant, 2010, 3(3): 509-523., articleTitle=The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine, refAbstract=null)], funds=[Fund(id=1276616362504098626, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, awardId=桂农科AB241484015, language=CN, fundingSource=广西重点研发计划项目(桂农科AB241484015), fundOrder=null, country=null), Fund(id=1276616362567013187, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, awardId=桂农科2022JM37; 桂农科2021YT060, language=CN, fundingSource=广西农业科学院基本科研业务专项(桂农科2022JM37; 桂农科2021YT060), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1276616350722298640, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, xref=null, ext=[AuthorCompanyExt(id=1276616350726492945, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, companyId=1276616350722298640, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China), AuthorCompanyExt(id=1276616350734881554, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, companyId=1276616350722298640, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=广西壮族自治区农业科学院经济作物研究所，广西南宁　530007)])], figs=[ArticleFig(id=1276616357252829996, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 1, caption=Amino acid sequence alignment of Tai6.33665 and its homologous proteins, figureFileSmall=xwFBu+lsmxpQ4XEp4gCVWQ==, figureFileBig=wQZWnB8vc+bME233VCwSZQ==, tableContent=null), ArticleFig(id=1276616357311550253, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图1, caption=Tai6.33665及其同源蛋白氨基酸序列比对, figureFileSmall=xwFBu+lsmxpQ4XEp4gCVWQ==, figureFileBig=wQZWnB8vc+bME233VCwSZQ==, tableContent=null), ArticleFig(id=1276616357483516718, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 2, caption=Phylogenetic tree of amino acids of Tai6.33665 and its homologous proteins, figureFileSmall=7r1xlb2kQ0UJnGjyGVbCmg==, figureFileBig=Qb/xebsUpFiZkZDbfehjsg==, tableContent=null), ArticleFig(id=1276616357647094575, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图2, caption=Tai6.33665及其同源蛋白氨基酸的系统发育进化树, figureFileSmall=7r1xlb2kQ0UJnGjyGVbCmg==, figureFileBig=Qb/xebsUpFiZkZDbfehjsg==, tableContent=null), ArticleFig(id=1276616357705814832, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 3, caption=Amino acid hydrophilic analysis of Tai6.33665, figureFileSmall=slAfDaaAXViGVNY3IC09nA==, figureFileBig=iIi1rvwyBjigFUTubXi4Lw==, tableContent=null), ArticleFig(id=1276616357777118001, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图3, caption=Tai6.33665氨基酸亲疏水性分析

负数值表示亲水性质，正数值表示疏水性。

, figureFileSmall=slAfDaaAXViGVNY3IC09nA==, figureFileBig=iIi1rvwyBjigFUTubXi4Lw==, tableContent=null), ArticleFig(id=1276616357835838258, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 4, caption=Prediction of transmembrane structure of Tai6.33665, figureFileSmall=3OuUW5co2j5BsXvkqJZVAw==, figureFileBig=oVltBvID8Yefn/VD5RgFgw==, tableContent=null), ArticleFig(id=1276616357898752819, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图4, caption=Tai6.33665蛋白的跨膜结构预测, figureFileSmall=3OuUW5co2j5BsXvkqJZVAw==, figureFileBig=oVltBvID8Yefn/VD5RgFgw==, tableContent=null), ArticleFig(id=1276616357982638900, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 5, caption=Prediction of phosphorylation sites of Tai6.33665, figureFileSmall=h9200+spJKEgTycFHSOnrg==, figureFileBig=CLUavcZ9WQpSCm6x4j6oNA==, tableContent=null), ArticleFig(id=1276616358053942069, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图5, caption=Tai6.33665蛋白的磷酸化位点预测, figureFileSmall=h9200+spJKEgTycFHSOnrg==, figureFileBig=CLUavcZ9WQpSCm6x4j6oNA==, tableContent=null), ArticleFig(id=1276616358112662326, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 6, caption=Prediction of tertiary structure of Tai6.33665 protein, figureFileSmall=TG2bbqrFLG3juOTjrZQDTQ==, figureFileBig=WtWydfLYedvFvIco9h7r2w==, tableContent=null), ArticleFig(id=1276616358171382583, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图6, caption=Tai6.33665蛋白的三级结构预测, figureFileSmall=TG2bbqrFLG3juOTjrZQDTQ==, figureFileBig=WtWydfLYedvFvIco9h7r2w==, tableContent=null), ArticleFig(id=1276616358234297144, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 7, caption=Subcellular localization of Tai6.33665 protein, figureFileSmall=inZSpj6k6rUykXU16Uak5A==, figureFileBig=w7eVHgsp+6nrqK9lPWwLgQ==, tableContent=null), ArticleFig(id=1276616358301406009, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图7, caption=Tai6.33665蛋白亚细胞定位, figureFileSmall=inZSpj6k6rUykXU16Uak5A==, figureFileBig=w7eVHgsp+6nrqK9lPWwLgQ==, tableContent=null), ArticleFig(id=1276616358364320570, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Fig. 8, caption=Expression analysis of ib-miR52 and Tai6.33665 gene, figureFileSmall=1G3GhZm0nEz3cJqtHH6+iw==, figureFileBig=fliaUrhtMCsap88OhhMgUQ==, tableContent=null), ArticleFig(id=1276616358439818043, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=图8, caption=ib-miR52和Tai6.33665基因的表达分析

*表示差异显著（P<0.05），**表示差异极显著（P<0.01）。

, figureFileSmall=1G3GhZm0nEz3cJqtHH6+iw==, figureFileBig=fliaUrhtMCsap88OhhMgUQ==, tableContent=null), ArticleFig(id=1276616358511121212, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Tab. 1, caption=

Gene function annotation of Tai6.33665

, figureFileSmall=null, figureFileBig=null, tableContent=
功能描述Function description学名Scientific name最大得分Max score总得分Total score查询覆盖率Query cover/%期望比对Evalue匹配百分比Percent identity访问号长度Accession length访问号Accession
Anthocyanin bHLH transcriptional regulatorIpomoea batatas1169116980099.85667QMP81308.1
Putative transcription factor bHLH2Ipomoea batatas1129112980097.46667AEA34965.1
bHLH2 transcription factorIpomoea batatas1110111080093.93674AFV33952.1
Transcriptional regulator bHLH2Ipomoea batatas1095109580092.9672AHH29256.1
Putative anthocyanin transcriptional regulatorIpomoea lacunosa1070107080093.31669ABY26934.1
Basic helix-loop-helix protein AIpomoea triloba1067106780093.73665XP_031100393.1
Putative anthocyanin transcriptional regulatorIpomoea trifida1059105980092.61676ABY26933.1
Putative anthocyanin transcriptional regulatorIpomoea hochstetteri1011101180083.70683ABY26937.1
Putative anthocyanin transcriptional regulatorIpomoea violacea98098080082.82684ABY26935.1
Putative anthocyanin transcriptional regulatorIpomoea quamoclit97197180081.33659ABY26931.1
), ArticleFig(id=1276616362063696701, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=表1, caption=

Tai6.33665的基因功能注释

, figureFileSmall=null, figureFileBig=null, tableContent=
功能描述Function description学名Scientific name最大得分Max score总得分Total score查询覆盖率Query cover/%期望比对Evalue匹配百分比Percent identity访问号长度Accession length访问号Accession
Anthocyanin bHLH transcriptional regulatorIpomoea batatas1169116980099.85667QMP81308.1
Putative transcription factor bHLH2Ipomoea batatas1129112980097.46667AEA34965.1
bHLH2 transcription factorIpomoea batatas1110111080093.93674AFV33952.1
Transcriptional regulator bHLH2Ipomoea batatas1095109580092.9672AHH29256.1
Putative anthocyanin transcriptional regulatorIpomoea lacunosa1070107080093.31669ABY26934.1
Basic helix-loop-helix protein AIpomoea triloba1067106780093.73665XP_031100393.1
Putative anthocyanin transcriptional regulatorIpomoea trifida1059105980092.61676ABY26933.1
Putative anthocyanin transcriptional regulatorIpomoea hochstetteri1011101180083.70683ABY26937.1
Putative anthocyanin transcriptional regulatorIpomoea violacea98098080082.82684ABY26935.1
Putative anthocyanin transcriptional regulatorIpomoea quamoclit97197180081.33659ABY26931.1
), ArticleFig(id=1276616362160165694, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Tab. 2, caption=

Prediction of signal peptide of Tai6.33665 protein

, figureFileSmall=null, figureFileBig=null, tableContent=
项目Item位置Position分值Value剪切位点Cut off信号肽Signal peptide
最大原始剪切（C）290.129
最大综合剪切（Y）120.133
信号肽最大值（S）380.197
信号肽平均值1-110.140
加权平均值1-110.1370.450NO
), ArticleFig(id=1276616362231468863, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=表2, caption=

Tai6.33665蛋白的信号肽预测

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项目Item位置Position分值Value剪切位点Cut off信号肽Signal peptide
最大原始剪切（C）290.129
最大综合剪切（Y）120.133
信号肽最大值（S）380.197
信号肽平均值1-110.140
加权平均值1-110.1370.450NO
), ArticleFig(id=1276616362311160640, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=EN, label=Tab. 3, caption=

Secondary structure prediction of Tai6.33665

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结构Structure数量Number比例Percentage/%
Alpha helix（Hh）28642.88
310 helix（Gg）00
Pi helix（Ii）00
Beta bridge（Bb）00
Extended strand（Ee）7411.09
Beta turn（Tt）304.50
Bend region（Ss）00
Random coil（Cc）27741.53
Other states00
), ArticleFig(id=1276616362403435329, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276616348486734586, language=CN, label=表3, caption=

Tai6.33665蛋白的二级结构预测

, figureFileSmall=null, figureFileBig=null, tableContent=
结构Structure数量Number比例Percentage/%
Alpha helix（Hh）28642.88
310 helix（Gg）00
Pi helix（Ii）00
Beta bridge（Bb）00
Extended strand（Ee）7411.09
Beta turn（Tt）304.50
Bend region（Ss）00
Random coil（Cc）27741.53
Other states00
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甘薯Tai6.33665基因生物信息学、亚细胞定位及表达分析
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熊军 , 唐秀桦 * , 韦民政 *
热带作物学报 | 组学与生物技术 2025,46(11): 2549-2558
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热带作物学报 |组学与生物技术 2025 , 46 (11) : 2549 -2558
甘薯Tai6.33665基因生物信息学、亚细胞定位及表达分析
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熊军（1978—），男，博士，副研究员，研究方向：作物育种和栽培技术。

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熊军（1978—），男，博士，副研究员，研究方向：作物育种和栽培技术。

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熊军, 唐秀桦* , 韦民政*
作者信息
  • 广西壮族自治区农业科学院经济作物研究所，广西南宁　530007
通讯作者:
* 韦民政（WEI Minzheng），E-mail：
唐秀桦（TANG Xiuhua），E-mail：。
Bioinformatics, Subcellular Localization and Expression Analysis of Gene Tai6.33665 in Sweet Potato
Jun XIONG, Xiuhua TANG* , Minzheng WEI*
Affiliations
  • Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530007, China
出版时间: 2025-11-25 doi: 10.3969/j.issn.1000-2561.2025.11.001
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摘要
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甘薯（Ipomoea batatas）作为全球重要的粮食与功能作物，其紫肉品种因富含花青素而备受关注。花青素不仅赋予块根鲜明的色泽，更具有抗氧化、抗炎及预防慢性疾病的生理功能，因此在健康食品开发中具有重要价值。然而，花青素的生物合成受多层级调控网络影响，其中bHLH转录因子的功能尚未在甘薯中被充分解析。本研究聚焦甘薯基因Tai6.33665，旨在阐明其分子特性、亚细胞定位模式及其在花青素积累中的调控作用，为高花青素甘薯品种的分子育种提供理论依据。本研究以紫肉甘薯（PFSP）和白肉甘薯（WFSP）为材料，通过生物信息学分析Tai6.33665蛋白的结构与进化关系，利用qRT-PCR检测基因表达模式，构建Tai6.33665-GFP融合载体进行烟草瞬时转化，结合激光共聚焦显微镜明确亚细胞定位，分析基因表达与花青素含量的相关性及miRNA互作机制。结果表明：Tai6.33665编码的蛋白具有典型bHLH结构域，与花青素合成调控因子高度同源，其表达量与花青素积累呈显著负相关（P<0.05），且受ib-miR52靶向抑制；亚细胞定位显示该蛋白定位于内质网，推测其可能通过翻译后修饰或内质网通路间接调控代谢。本研究证实了Tai6.33665作为负调控因子抑制甘薯花青素生物合成，其功能实现依赖于亚细胞定位特异性及miRNA介导的转录后调控网络。这一发现不仅丰富了植物次生代谢调控的理论框架，更为分子育种提供了双轨策略：通过CRISPR/Cas9靶向敲除Tai6.33665或过表达ib-miR52，可突破花青素积累的遗传限制，培育高花青素含量甘薯新品种。未来工作将聚焦于解析该基因与MYB/WD40蛋白的互作机制，以及内质网定位对其转录活性的调控路径。

甘薯  /  Tai6.33665  /  生物信息学  /  亚细胞定位  /  表达分析

Sweet potato (Ipomoea batatas) is a globally significant food and functional crop, with its purple-fleshed varieties drawing particular attention due to the high anthocyanin content. Anthocyanins not only give the tubers distinct color but also possess antioxidant, anti-inflammatory, and disease-preventive physiological functions, making them highly valuable in the development of health foods. However, the biosynthesis of anthocyanins is regulated by a multi-level network, and the role of bHLH transcription factors in sweet potatoes has not been fully elucidated. This study focused on the sweet potato gene Tai6.33665, aiming to clarify its molecular characteristics, subcellular localization pattern, and regulatory role in anthocyanin accumulation, providing a theoretical basis for molecular breeding of high-anthocyanin sweet potato varieties. Using purple-fleshed sweet potatoe (PFSP) and white-fleshed sweet potatoe (WFSP) as materials, the study analyzed the protein structure and evolutionary relationship of Tai6.33665 through bioinformatics, detected the gene expression pattern using qRT-PCR, constructed a Tai6.33665-GFP fusion vector for transient transformation in tobacco, determined the subcellular localization using laser confocal microscopy, and analyzed the correlation between gene expression and anthocyanin content and the miRNA interaction mechanism. The results showed that the protein encoded by Tai6.33665 had a typical bHLH domain and was highly homologous to anthocyanin synthesis regulatory factors. Its expression level was significantly negatively correlated with anthocyanin accumulation (P<0.05), and it was targeted and inhibited by ib-miR52. Subcellular localization indicated that the protein was located in the endoplasmic reticulum, suggesting that it might indirectly regulate metabolism through post-translational modification or the endoplasmic reticulum pathway. This study confirmed that Tai6.33665 acted as a negative regulator of anthocyanin biosynthesis in sweet potatoes, and its function depended on subcellular localization specificity and miRNA-mediated post-transcriptional regulatory networks. This discovery not only enriches the theoretical framework of plant secondary metabolism regulation but also provides a dual-track strategy for molecular breeding by targeting knockout of Tai6.33665 or overexpression of ib-miR52 using CRISPR/Cas9, the genetic limitations of anthocyanin accumulation could be overcome, and new high-anthocyanin sweet potato varieties could be developed. Future work would focus on elucidating the interaction mechanism between the gene and MYB/WD40 proteins, as well as the regulatory pathway of its transcriptional activity by endoplasmic reticulum localization.

sweet potato  /  Tai6.33665  /  bioinformatics  /  subcellular localization  /  expression analysis
熊军, 唐秀桦, 韦民政. 甘薯Tai6.33665基因生物信息学、亚细胞定位及表达分析. 热带作物学报, 2025 , 46 (11) : 2549 -2558 . DOI: 10.3969/j.issn.1000-2561.2025.11.001
Jun XIONG, Xiuhua TANG, Minzheng WEI. Bioinformatics, Subcellular Localization and Expression Analysis of Gene Tai6.33665 in Sweet Potato[J]. Chinese Journal of Tropical Crops, 2025 , 46 (11) : 2549 -2558 . DOI: 10.3969/j.issn.1000-2561.2025.11.001
正文
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花青素是一类广泛存在于植物中的水溶性类黄酮化合物，具有抗氧化、抗炎及抗癌等多种生理活性，在食品工业和人类健康领域具有重要价值[1]。甘薯（Ipomoea batatas）作为全球重要的粮食作物，其紫肉品种因富含花青素而备受关注。然而，花青素的生物合成受多基因调控网络控制，其中bHLH（basic Helix-Loop-Helix）转录因子在调控花青素合成途径中起关键作用[2]。明确甘薯中bHLH家族基因的功能及其调控机制，不仅有助于解析花青素合成的分子基础，还可为高花青素甘薯品种的分子育种提供理论依据。
前人在植物花青素合成途径及其调控机制的研究方面取得了显著进展。在模式植物中，花青素生物合成的调控机制研究较为深入，该过程主要由编码相关酶的结构基因以及关键的转录调控因子（MYB、bHLH和WD40蛋白）协同控制[3]，bHLH转录因子通过与MYB和WD40蛋白形成MBW复合体，激活结构基因（如DFR、ANS等）的表达，进而调控花青素的积累[4–6]。在甘薯中，多个bHLH家族基因已被鉴定，如IbMYC1和IbMYC2被证实参与花青素合成的正调控[7]；在紫心甘薯块根中，IbbHLH2的表达量与花青素合成酶基因（CHS、CHI、F3H、DFR、ANS、3GT）呈正相关，且与块根着色程度一致，表明其直接调控花青素途径，而IbbHLH1无此关联[8]。此外，亚细胞定位分析表明，部分bHLH蛋白定位于细胞核或内质网，其定位特性可能影响其转录活性[9]。然而，甘薯中多数bHLH的基因功能尚未明确，尤其是不同品种间基因表达差异与花青素含量变化的关联机制仍有待探索。
尽管已有研究揭示了bHLH家族基因在花青素合成中的重要性，但甘薯Tai6.33665基因的具体功能及其调控机制尚未明确。前期生物信息学分析显示，Tai6.33665编码的蛋白具有典型的bHLH结构域，并与花青素合成相关转录因子高度同源（NCBI数据库注释号QMP81308.1）。然而，该基因是否直接参与花青素合成调控、其亚细胞定位特性以及与miRNA的相互作用仍缺乏实验证据。
本研究以紫肉甘薯和白肉甘薯为材料，结合生物信息学、亚细胞定位及表达分析，初步解析Tai6.33665基因的功能及其在花青素积累中的调控作用。通过生物信息学分析明确Tai6.33665基因编码蛋白的结构、理化性质及进化关系；探究Tai6.33665蛋白的亚细胞定位，揭示其潜在作用位点；分析Tai6.33665在不同品种和发育阶段的表达差异，阐明其与花青素积累的负调控关系；验证Tai6.33665是否受ib-miR52靶向调控；以期为后续解析其在花青素合成通路中的分子机制提供理论依据。
1　材料与方法
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1.1　材料
供试材料为广西农业科学院经济作物研究所选育的紫肉甘薯（purple-fleshed sweet potato，PFSP，桂经薯8号）和白肉甘薯（white-fleshed sweet potato，WFSP，桂经薯8号的自然突变品系118）。2个品种（系）种植于广西农业科学院院部基地（22°51′11″N，108°14′45″E，海拔：55.59 m）。主要试剂：难提取植物总RNA试剂盒（HiPure HP Plant RNA Mini Kit，美基生物），反转录试剂盒[HiScript II Q RT SuperMix for qPCR（+gDNA wiper），南京诺唯赞生物科技有限公司]，高保真DNA聚合酶（Vazyme公司），重组试剂盒（ONE STEP CLONG KIT，Vazyme公司）。
1.2　方法
本课题组前期通过对2个品种（PFSP和WFSP）花青素含量高、低2个时期进行全长转录组和miRNA测序，并进行联合分析，推测出novel_miR_52（命名为ib-miR52）与靶基因Tai6.33665存在互作关系，共同调控甘薯块根花青素的生物合成，因此，本研究针对靶基因Tai6.33665进行生物信息学分析、亚细胞定位和表达模式分析。
1.2.1　生物信息学分析
从NCBI数据库中检索得到目标基因Tai6.33665的功能注释和蛋白质序列；利用Expasy Protparma（https://web.expasy.org/protparam/）在线软件分析蛋白质的一级结构；利用SOPMA（https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html）在线软件预测蛋白质的二级结构；利用SWISS-MODE（https://swissmodel.expasy.org/interactive）在线软件预测蛋白质的三级结构；通过SignalP 4.1软件分析信号肽特征；通过TMHMM 2.0软件预测蛋白质的跨膜特性；利用ProtScale软件分析其亲水性和疏水性；通过InerPro（https://www.ebi.ac.uk/interpro/search/sequence/）在线软件分析蛋白质功能性结构域。为了解靶基因家族在甘薯和其他物种之间的进化关系，本研究从NCBI数据库中提取该家族其他成员的蛋白序列，并使用MEGA 5.0软件进行全长蛋白序列的详尽比较，采用邻接法构建系统进化树。利用DNAMAN软件进行多序列比对。
1.2.2　Tai6.33665蛋白亚细胞定位
从甘薯块根中提取RNA，反转录成cDNA。根据Tai6.33665开放阅读框（ORF）基因序列设计克隆引物（CZ-Tai6.33665-101SalI-F：5′–CTTCACTGTTGATACATATGGCGGCGGAAAACTCT–3′；CZ-Tai6.33665-101SalI-R：5′–ATCCGGTACCCCCGGGAAACTGAGGAATTATACTATGAATTG–3′，下划线区域为载体同源重组序列），引物退火温度为47 ℃。采用先前提取的cDNA作为模板，使用Vazyme公司的Phanta Max超高保真度DNA聚合酶进行PCR。PCR扩增的具体步骤如下：95 ℃预变性30 s；95 ℃变性15 s，47 ℃退火15 s，72 ℃延伸60 s，39个循环；72 ℃延伸5 min。将PCR产物通过1.0%的琼脂糖凝胶进行电泳分析，并将目标DNA条带（在预期的2 000 bp目标位置有一条单一条带）用Axygen割胶回收试剂盒切胶回收，并构建到载体上进行单克隆测序。
用Sal I（TaKaRa公司：No.1080S）内切酶酶切载体pRI101-GFP线性化。将纯化后的酶切片段与Tai6.33665基因的PCR扩增产物进行重组（所用重组试剂盒是Vazyme公司的ClonExpress II一步克隆试剂盒）。将重组反应所获得的产物转化大肠杆菌DH5α细胞，从PCR结果显示为阳性的转化菌株中提取质粒DNA，将获得的DNA扩增产物测序，以验证插入基因正确性。使用的扩增和序列分析引物为目标基因两侧的载体序列（35S-F：5′–GACGCACAATCCCACTATCC–3′；GFP-J-R：5′–GGGTGAGCTTGCCGTAGGTG–3′），因基因较长，另设计中间引物（Tai6.33665-Z-F：5′-CCCCATCCCCACCACCAT-3′）用于加测，以获得基因全长。
烟草种植约1个月后，将构建好的Tai6.33665定位载体质粒转入农杆菌GV3101，瞬时转化到烟草叶片，将烟草置于低光照条件下培养2 d，第3天之后，将烟草叶片中已接种的部分剪下，制作成显微镜玻片，在激光共聚焦显微镜下进行细致观察并记录图片。使用未携带有效载体的农杆菌转化样本，执行相同的步骤进行对照试验。
1.2.3　 Tai6.33665的表达模式分析
使用难提取植物总RNA试剂盒提取紫肉和白肉甘薯花青素含量高、低2个时期的甘薯块根（8H、118H和8L、118L）总RNA。按照HiScript® II Q RT SuperMix for qPCR（+gDNA wiper）试剂盒操作流程对Tai6.33665进行反转录。以反转录合成的cDNA为模板进行qRT-PCR分析。以IbActin为内参基因，通过Primer Premier 5设计引物（IbActin：5′–GACTACCATGTTCCCCGGTA–3ʹ/5ʹ–TTGTATGCCACGAGCATCTT–3′；Tai6.33665：5′–CCACCACCATCATCATCATCCA–3′/5′–CCTCCTCCTCGTCTTCCTCTT–3′）。qRT-PCR反应体系（20.0 μL）：2×Universal SYBR qPCR Master Mix 10.0 μL，正反向引物（10 μmol/L）各0.4 μL，Template cDNA 1.0 μL，ddH2O 8.2 μL。PCR反应程序：95 ℃预变性3 min；95 ℃ 10 s，56 ℃/59 ℃ 15 s，72 ℃延伸20 s，40个循环。完成上述步骤后，把加好样品的96孔板置于德国Analytik Jena qTOWERE 2.2荧光定量PCR仪中进行qRT-PCR。采用2-ΔΔCt方法计算基因的相对表达量。
1.3　数据处理
所有试验数据均通过3次重复试验。使用Excel、GraphPad Prism 9.5软件进行数据整理、分析及图表的制作，使用DPS 7.05软件进行方差分析和差异显著性检验，使用PowerPoint软件对图表进行合并和整理。
2　结果与分析
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2.1　甘薯Tai6.33665基因的生物信息学分析
2.1.1　 Tai6.33665基因结构预测和功能注释
利用softberry平台的FGENESH 2.6分析工具预测甘薯基因组DNA中Tai6.33665基因结构，其序列长度为2 491 nt，该基因来自正链。使用NCBI中的Blastx工具对Tai6.33665进行基因功能注释，共得到显著相关的注释100条，其中Max score得分前10的注释结果见表1。从表1中可知，Percent identity最高（99.85%）的注释结果为anthocyanin bHLH transcriptional regulator（Ipomoea batatas，QMP81308.1），推测其是调控甘薯花青素生物合成的bHLH转录因子。
2.1.2　Tai6.33665蛋白结构域预测
通过InerPro软件分析蛋白质结构域，对Tai6.33665所编码的氨基酸序列进行详细分析，发现该氨基酸在12~202位置有bHLH-MYC and R2R3-MYB transcription factors N-terminal结构域，在468~523位置有MYC-type，basic helix-loop-helix（bHLH）结构域。
2.1.3　Tai6.33665氨基酸的多重序列比对
利用Clustal Omega（https://www.ebi.ac.uk/Tools/msa/clustalo/）在线工具，结合Jalview软件，对Tai6.33665氨基酸序列（QMP81308.1）及其同源蛋白序列（percent identity>85%）进行多重序列比对分析。结果显示，这些序列存在较多的保守区间、比对质量较高及大量的共有序列（图1）。同时使用MEGA 11.0软件构建系统进化树（图2），结果表明：QMP81308.1[anthocyanin bHLH transcriptional regulator（Ipomoea batatas）]与AEA34965.1[putative transcription factor bHLH2（Ipomoea batatas）]聚在一起，均属于bHLH转录因子家族。
2.1.4　Tai6.33665蛋白的理化性质分析
理化性质分析结果显示，Tai6.33665蛋白由667个氨基酸组成，分子式为C3220H5058N925O1033S27，分子质量约为74 150。Tai6.33665蛋白含有全部20种氨基酸，其中谷氨酸（Glu）含量最高，占10.8%；丙氨酸（Ala）占9.1%；色氨酸（Trp）含量最低，仅占1.3%。该蛋白中带负电的氨基酸（Asp和Glu）共有98个，而带正电的氨基酸（Arg和Lys）有67个。该蛋白的理论等电点为5.36，表明其为酸性蛋白。其不稳定系数为58.39，高于50，推测该蛋白具有不稳定性。脂溶脂数为75.37，而总平均亲水性（GRAVY）为-0.511，表明该蛋白质具有亲水性。进一步通过ExPASy-ProtScale软件分析蛋白的亲疏水性，依据Hphob./Kyte & Doolittle标准打分，并使用线性加权法进行预测。如图3所示，亲水部分明显优于疏水部分，表明这是一种亲水性的可溶蛋白，与之前的预测结果相符。
2.1.5　Tai6.33665蛋白信号肽、跨膜结构、磷酸化位点预测
通过SignalP 4.1软件对Tai6.33665蛋白质的信号肽进行了预测分析，结果如表2所示，该蛋白的C-score值为0.129，Y-score值为0.133，S-score值为0.197，信号肽平均值为0.140，加权平均值为0.137，该蛋白不存在信号肽，说明其是非分泌蛋白，在细胞内起作用。
Tai6.33665蛋白的跨膜结构预测结果（图4）显示，在该蛋白中未检测到任何跨膜结构区域（TMHs数量为零），表明其不具备跨膜区域，因此不是跨膜蛋白。
通过NetPhos 3.1（https://services.healthtech.dtu.dk/services/NetPhos-3.1/）在线软件预测Tai6.33665蛋白的潜在磷酸化点，蛋白的潜在磷酸化点预测标准设定为当一个位点的纵轴预测值超过0.5时，该位点被认为是磷酸化位点。该蛋白在丝氨酸（Ser）上拥有42个磷酸化位点，在苏氨酸（Thr）上有23个，在酪氨酸（Tyr）上有5个（图5）。这表明Tai6.33665编码的蛋白质主要受到丝氨酸的磷酸化修饰，而苏氨酸和酪氨酸则起到辅助修饰的作用。
2.1.6　Tai6.33665蛋白二级、三级结构预测
通过SOPMA软件分析Tai6.33665蛋白的二级结构，结果如表3所示，该蛋白质主要存在4种类型的二级结构，其中数量最多的是α-螺旋（alpha helix），有286个，占比42.88%；无规则卷曲（random coil）277个，占比41.53%；延伸链（extended strand）74个，占比11.09%；β-折叠（beta turn）30个，占比4.5%。Tai6.33665蛋白的结构主要由α-螺旋和无规则卷曲构成。
利用SWISS-MODE软件预测Tai6.33665蛋白的三级结构，共搜索出50个模板（Templates），构建出4个模型（models）（图6A~图6D），构建的4个模型的蛋白未形成多聚体，均为单体（monomer）结构，且均无配体（ligands），其中model 01的GMQE值0.61最接近1，表明其一致性最好，可信度最高，其序列一致性（seq identity）达96.83%，覆盖度（coverage）为0.99，该模型描述为putative anthocyanin transcriptional regulator，其三维结构如图6E所示。
2.2　 Tai6.33665编码蛋白的亚细胞定位分析
为了确定Tai6.33665蛋白的亚细胞定位，将已构建好的Tai6.33665-GFP载体质粒转入农杆菌GV3101，瞬时转化至本氏烟草叶片组织中，制作玻片，在激光共聚焦显微镜下观察、拍照，以空载体转化的农杆菌作为对照。结果如图7所示，烟草叶片亚细胞器内质网区域出现了绿色的荧光（荧光的线条并不是完全光滑的曲线，有模糊的边缘和毛刺，不认为定位于细胞膜上；细胞核并不是完全亮的，核膜一圈更亮一些，这种围绕细胞核亮的情况极大可能是定位在内质网上），与仅含GFP的空载体相比，荧光强度略强。据此推断，Tai6.33665蛋白可能定位于内质网。
2.3　Tai6.33665和ib-miR52的表达模式分析
使用植物总RNA提取试剂盒提取甘薯块根总RNA，12个样品的RNA浓度在686.86~1 394.47 ng/μL之间，A260/A280在2.15~2.19之间。对所有样品进行荧光定量检测获得Ct值，采用2-ΔΔCt方法进行数据分析并作图。结果显示，ib-miR52的表达量与甘薯块根花青素的含量呈正相关关系；Tai6.33665的表达量与ib-miR52恰好相反，与甘薯块根花青素的含量呈负相关关系（图8）。推测ib-miR52通过靶向Tai6.33665正调控甘薯块根中花青素的积累，Tai6.33665负调控甘薯块根中花青素的积累。
3　讨论
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本研究通过整合生物信息学、亚细胞定位及表达分析，初步解析了甘薯Tai6.33665基因在花青素合成中的功能与调控作用。生物信息学分析显示，Tai6.33665编码的蛋白具有典型的bHLH结构域，并与花青素合成相关的转录因子（如QMP81308.1）高度同源。bHLH转录因子在植物次生代谢调控中具有高度保守性，如在番茄中，SlAN2-like通过结合MYB蛋白激活花青素合成基因（DFR、ANS）的表达[10]。然而，本研究发现Tai6.33665的表达量与花青素积累呈显著负相关，这一现象提示其可能作为负调控因子抑制花青素合成。类似地，拟南芥中的MYC2通过竞争性结合MYB蛋白抑制花青素合成[11]，表明负调控机制在植物中具有普遍性。
亚细胞定位试验表明，Tai6.33665蛋白可能定位于内质网。内质网不仅是蛋白质合成的场所，还参与信号转导和胁迫响应[12]。如拟南芥中的内质网定位蛋白BIP3通过调控未折叠蛋白反应（UPR）影响次生代谢[13]。Tai6.33665的定位特性可能表明其通过内质网相关通路间接调控花青素合成，如通过调控内质网应激信号或蛋白修饰（如磷酸化）。此外，内质网与细胞核之间的膜接触位点（MCSs）可能介导转录因子的快速转运[14]，未来需进一步验证其定位与功能关联。
本研究发现Tai6.33665的表达与花青素含量呈负相关，支持其负调控作用的假设。然而，在118L组中Tai6.33665的表达变化未达到显著水平。因白肉甘薯（118L）花青素含量极低，其块根细胞中花青素合成相关的基因网络可能整体表达水平很低或变异较大。这可能导致：（1）Tai6.33665本身在白肉中的基础表达模式与紫肉品种存在差异，其变化幅度相对较小；（2）低表达背景下，技术检测（如qPCR）的变异相对增大，使得较小的表达差异更难达到统计显著性；（3）其调控的下游基因可能在白肉中本就不表达或表达量极低，使得Tai6.33665的调控作用失去了‘着力点’，导致其表达变化在统计学和生物学意义上均显得不突出。
本研究发现ib-miR52与Tai6.33665的表达呈负相关。miRNA通过靶向降解mRNA或抑制翻译参与基因表达调控[15]。如苹果中的miR858靶向MYB转录因子调控花色苷积累[16]，而甘薯中ib-miR52可能通过类似机制抑制Tai6.33665的转录活性。此外，miRNA与转录因子的互作网络具有物种特异性，如水稻中的miR529通过靶向OsSPL14调控分蘖[17]，提示需通过试验验证ib-miR52与Tai6.33665的直接靶向关系。
负调控基因的发现为分子育种提供了新策略。如CRISPR/Cas9技术已被成功用于敲除甘薯中的淀粉合成抑制基因，显著提高块根淀粉含量[18]。若Tai6.33665被证实为花青素合成的负调控因子，其敲除或表达抑制可能显著提升紫肉甘薯的花青素含量。此外，基于miRNA的调控技术（如人工miRNA设计）也可用于定向调控目标基因表达[16]。在葡萄中，VvMYC1通过与VvMYBA形成复合体激活花青素合成[19]，而甘薯中的IbMYC1则显示正调控作用[7]。本研究发现Tai6.33665的负调控功能，同一基因家族成员在不同物种或组织中的功能分化。这种功能多样性可能源于基因复制后的亚功能化或新功能化[4]，需通过比较基因组学进一步解析。
4　结论
收起
本研究揭示了甘薯Tai6.33665基因作为bHLH转录因子家族成员，通过保守的蛋白结构域参与花青素合成的负调控机制：其表达量与花青素积累呈显著负相关，且受ib-miR52靶向抑制；亚细胞定位分析表明该蛋白定位于内质网，推测其可能通过内质网相关通路调控翻译后修饰或信号转导。这些发现不仅拓展了植物次生代谢调控网络的分子机制认知，也为利用基因编辑技术（如CRISPR/Cas9）靶向调控Tai6.33665或ib-miR52、培育高花青素甘薯品种提供理论依据和应用方向。
基金
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  • 广西重点研发计划项目(桂农科AB241484015)
  • 广西农业科学院基本科研业务专项(桂农科2022JM37; 桂农科2021YT060)
文献
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2025年第46卷第11期
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doi: 10.3969/j.issn.1000-2561.2025.11.001
  • 接收时间：2025-06-19
  • 首发时间：2026-06-24
  • 出版时间：2025-11-25
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  • 收稿日期：2025-06-19
  • 录用日期：2025-08-05
基金
广西重点研发计划项目(桂农科AB241484015)
广西农业科学院基本科研业务专项(桂农科2022JM37; 桂农科2021YT060)
作者信息
    广西壮族自治区农业科学院经济作物研究所，广西南宁　530007

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* 韦民政（WEI Minzheng），E-mail：
唐秀桦（TANG Xiuhua），E-mail：。
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https://castjournals.cast.org.cn/joweb/rdzwxb/CN/10.3969/j.issn.1000-2561.2025.11.001
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
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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