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Article(id=1276844414429425937, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276844393709568941, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.10.006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1709740800000, receivedDateStr=2024-03-07, revisedDate=1713456000000, revisedDateStr=2024-04-19, acceptedDate=null, acceptedDateStr=null, onlineDate=1782353031021, onlineDateStr=2026-06-25, pubDate=1729785600000, pubDateStr=2024-10-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782353031021, onlineIssueDateStr=2026-06-25, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782353031021, creator=13701087609, updateTime=1782353031021, updator=13701087609, issue=Issue{id=1276844393709568941, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='10', pageStart='1999', pageEnd='2242', issueExtLink='null', onlineDate='null', pubDate='1729785600000', pubDateStr='2024-10-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782353026082, creator='13701087609', updateTime=1782355588483, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276855141311574992, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276844393709568941, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276855141311574993, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276844393709568941, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2054, endPage=2058, ext={EN=ArticleExt(id=1276844414735610131, articleId=1276844414429425937, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Screening of MeMinD Interacting Protein in Cassava, columnId=1236256430337085821, journalTitle=Chinese Journal of Tropical Crops, columnName=Omics & Biotechnology, runingTitle=null, highlight=null, articleAbstract=

MinD is involved in the fine regulation of plastid division and plays a key role in maintaining plastid morphology. Previous laboratory research found that the MeMinD protein is involved in the division of cassava plastids, but the related proteins that work in conjunction with MeMinD to regulate the division of cassava plastids have not been clearly identified. This study constructed the pGBKT7-MeMinD vector and, through yeast two-hybrid library screening, a total of 8 candidate interacting proteins of MeMinD were obtained; after point-to-point verification using the yeast two-hybrid system, it was found that the nicotinate phosphoribosyltransferase 2 (MeNAPRT2) interacts with MeMinD. The results of this study would provide new information for further analysis of the mechanism of MeMinD's participation in the regulation of cassava plastid splitting.

, authors=null, authorsList=Mu PAN, Xiaohua LU, Yuan YAO, Yinhua CHEN, Jianchun GUO, Yajie WANG, authorCompany=null, correspAuthors=Jianchun GUO, Yajie WANG, 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=1276844417784869153, articleId=1276844414429425937, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=木薯MeMinD的互作蛋白筛选, columnId=1236256430517440904, journalTitle=热带作物学报, columnName=组学与生物技术, runingTitle=null, highlight=null, articleAbstract=

MinD参与质体分裂的精细调控，在质体形态维持中起着关键作用。实验室前期研究发现MeMinD蛋白参与了木薯质体的分裂，然而与MeMinD蛋白协同调控木薯质体分裂的相关蛋白尚未明确。本研究构建了pGBKT7-MeMinD载体，通过酵母双杂交文库筛选，共获得MeMinD蛋白的8个候选互作蛋白，点对点验证后发现烟酸磷酸核糖转移酶2（MeNAPRT2）与MeMinD存在互作关系。该研究结果为进一步解析MeMinD参与调控木薯质体分裂的相关机制提供新的信息。

, authors=

* 同等贡献作者

潘沐（1997—），女，硕士，研究方向：作物分子育种

陆小花（1994—），女，博士研究生，研究方向：作物分子育种。

, authorsList=潘沐, 陆小花, 姚远, 陈银华, 郭建春, 王亚杰, authorCompany=null, correspAuthors=郭建春, 王亚杰, authorNote=null, correspAuthorsNote=
** 郭建春（GUO Jianchun），E-mail：
王亚杰（WANG Yajie），E-mail：。
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2.中国热带农业科学院热带生物技术研究所/农业农村部热带作物生物学与遗传资源利用重点实验室，海南海口　571101, bio={"content":"

潘沐（1997—），女，硕士，研究方向：作物分子育种

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潘沐（1997—），女，硕士，研究方向：作物分子育种

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陆小花（1994—），女，博士研究生，研究方向：作物分子育种。

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陆小花（1994—），女，博士研究生，研究方向：作物分子育种。

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M: DL2000 DNA marker.

, figureFileSmall=HIr9K4oR30uhO6vBdxF6oQ==, figureFileBig=QWoGj1xhJQoQpFikKNerTQ==, tableContent=null), ArticleFig(id=1276844427679232348, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=EN, label=Fig. 2, caption=Construction of pGBKT7-MeMinD Vector

M: DL2000 DNA marker; 1-5: Coliforms.

, figureFileSmall=HpGg+Iz374ofuq+tqLm1gQ==, figureFileBig=FcDn0pRkYgj1uOx/z4KrEw==, tableContent=null), ArticleFig(id=1276844427922501981, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=CN, label=图2, caption=pGBKT7-MeMinD载体的构建

M：DL2000 DNA marker；1~5：大肠杆菌。

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“+”: Positive control; “–”: Negative control.

, figureFileSmall=mJ6/k5KQoYwnRPAiHyU0bw==, figureFileBig=9zr7NS9QP+49nppjXLRbiA==, tableContent=null), ArticleFig(id=1276844428413235555, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=CN, label=图5, caption=酵母双杂交筛选阳性克隆

“+”：阳性对照；“–”：阴性对照。

, figureFileSmall=mJ6/k5KQoYwnRPAiHyU0bw==, figureFileBig=9zr7NS9QP+49nppjXLRbiA==, tableContent=null), ArticleFig(id=1276844428488733028, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=EN, label=Fig. 6, caption=Growth of yeast transforming bacteria on defective plates, figureFileSmall=CuBJJSBMAZQ3TeZZ/QzJXQ==, figureFileBig=dGSgyabu6Lig6na6R+4R8w==, tableContent=null), ArticleFig(id=1276844428560036197, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=CN, label=图6, caption=酵母转化菌在缺陷平板上的生长情况, figureFileSmall=CuBJJSBMAZQ3TeZZ/QzJXQ==, figureFileBig=dGSgyabu6Lig6na6R+4R8w==, tableContent=null), ArticleFig(id=1276844428614562150, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=EN, label=Tab. 1, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列（5′-3′）Primer sequence（5′-3′）
pGBKT7-MeMinD-Fgccatggaggccgaattc
ATGATATCTCTCAAACCCCTTCC
pGBKT7-MeMinD-Rggccgctgcaggtcgac
TTACACTCCAAACAAGGAAAAAAAC
pGADT7-MeFBA1-1-Fcatggaggccagtgaattc
ATGGCCTCAGCATCACTTCTCA
pGADT7-MeFBA1-1-Rctcgagctcgatggatcc
TTAGTACACGTAGCCCTTGACGAAC
pGADT7-MeFBA1-2-Fcatggaggccagtgaattc
ATGGCCTCTGCTTCTCTCCTC
pGADT7-MeFBA1-2-Rctcgagctcgatggatcc
CTAGTAAACATAGCCCTTCACAAAC
pGADT7-MeNAPRT2-Fcatggaggccagtgaattc
ATGGAGGTGAAAGACTATGGTCT
pGADT7-MeNAPRT2-Rctcgagctcgatggatcc
CTATTGTAGCTCTCCAACAGGC
pGADT7-MeALDH2B4-Fcatggaggccagtgaattc
ATGGCAGCTCGCAGGAC
pGADT7-MeALDH2B4-Rcagctcgagctcgatggatcc
TTATAACCAGGCTGGATTCTTTAATGG
), ArticleFig(id=1276844428685865319, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=CN, label=表1, caption=

本研究所用引物

, figureFileSmall=null, figureFileBig=null, tableContent=
引物名称Primer name引物序列（5′-3′）Primer sequence（5′-3′）
pGBKT7-MeMinD-Fgccatggaggccgaattc
ATGATATCTCTCAAACCCCTTCC
pGBKT7-MeMinD-Rggccgctgcaggtcgac
TTACACTCCAAACAAGGAAAAAAAC
pGADT7-MeFBA1-1-Fcatggaggccagtgaattc
ATGGCCTCAGCATCACTTCTCA
pGADT7-MeFBA1-1-Rctcgagctcgatggatcc
TTAGTACACGTAGCCCTTGACGAAC
pGADT7-MeFBA1-2-Fcatggaggccagtgaattc
ATGGCCTCTGCTTCTCTCCTC
pGADT7-MeFBA1-2-Rctcgagctcgatggatcc
CTAGTAAACATAGCCCTTCACAAAC
pGADT7-MeNAPRT2-Fcatggaggccagtgaattc
ATGGAGGTGAAAGACTATGGTCT
pGADT7-MeNAPRT2-Rctcgagctcgatggatcc
CTATTGTAGCTCTCCAACAGGC
pGADT7-MeALDH2B4-Fcatggaggccagtgaattc
ATGGCAGCTCGCAGGAC
pGADT7-MeALDH2B4-Rcagctcgagctcgatggatcc
TTATAACCAGGCTGGATTCTTTAATGG
), ArticleFig(id=1276844428773945704, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=EN, label=Tab. 2, caption=

Annotation of MeMinD candidate interacting proteins

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.GeneBank登录号GenBank login number基因注释Gene annotation蛋白名称Protein name
1XP_021592298.1NADH dehydrogenase（ubiquinone）1 alpha subcomplex subunit 5MeMT-ND
2XP_021609330.1Fructose-bisphosphate aldolase 1MeFBA1-1
3XP_021604034.1Fructose-bisphosphate aldolase 1MeFBA1-2
4XP_021628049.117.4 kDa class III heat shock proteinMeHSP
5XP_043811339.1Eukaryotic translation initiation factor 3 subunit EMeEIF3
6XP_043809748.1Ribulose bisphosphate carboxylase small subunitMeRubisco
7XP_021631093.1Nicotinate phosphoribosyltransferase 2MeNAPRT2
8XP_021592138.1Aldehyde dehydrogenase family 2 member B4MeALDH2B4
), ArticleFig(id=1276844428836860265, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, language=CN, label=表2, caption=

MeMinD候选互作蛋白注释

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.GeneBank登录号GenBank login number基因注释Gene annotation蛋白名称Protein name
1XP_021592298.1NADH dehydrogenase（ubiquinone）1 alpha subcomplex subunit 5MeMT-ND
2XP_021609330.1Fructose-bisphosphate aldolase 1MeFBA1-1
3XP_021604034.1Fructose-bisphosphate aldolase 1MeFBA1-2
4XP_021628049.117.4 kDa class III heat shock proteinMeHSP
5XP_043811339.1Eukaryotic translation initiation factor 3 subunit EMeEIF3
6XP_043809748.1Ribulose bisphosphate carboxylase small subunitMeRubisco
7XP_021631093.1Nicotinate phosphoribosyltransferase 2MeNAPRT2
8XP_021592138.1Aldehyde dehydrogenase family 2 member B4MeALDH2B4
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木薯MeMinD的互作蛋白筛选
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潘沐 1, 2 , 陆小花 1, 2 , 姚远 2 , 陈银华 1, 3 , 郭建春 2, ** , 王亚杰 2, **
热带作物学报 | 组学与生物技术 2024,45(10): 2054-2058
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热带作物学报 |组学与生物技术 2024 , 45 (10) : 2054 -2058
木薯MeMinD的互作蛋白筛选
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[Author(id=1276844418367877423, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1276844418464346419, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, authorId=1276844418367877423, language=EN, stringName=Mu PAN, firstName=Mu, middleName=null, lastName=PAN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1.School of Life and Health Sciences, Hainan University, Haikou, Hainan 570228, China
2.Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs, Haikou, Hainan 571101, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1276844418716004660, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276844414429425937, authorId=1276844418367877423, language=CN, stringName=潘沐, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1.海南大学生命健康学院，海南海口　570228
2.中国热带农业科学院热带生物技术研究所/农业农村部热带作物生物学与遗传资源利用重点实验室，海南海口　571101, bio={"content":"

潘沐（1997—），女，硕士，研究方向：作物分子育种

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潘沐（1997—），女，硕士，研究方向：作物分子育种

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陆小花（1994—），女，博士研究生，研究方向：作物分子育种。

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潘沐1, 2, 陆小花1, 2, 姚远2, 陈银华1, 3, 郭建春2, ** , 王亚杰2, **
作者信息
  • 1.海南大学生命健康学院，海南海口　570228
  • 2.中国热带农业科学院热带生物技术研究所/农业农村部热带作物生物学与遗传资源利用重点实验室，海南海口　571101
  • 3.海南大学热带农林学院，海南海口　570228
通讯作者:
** 郭建春（GUO Jianchun），E-mail：
王亚杰（WANG Yajie），E-mail：。
Screening of MeMinD Interacting Protein in Cassava
Mu PAN1, 2, Xiaohua LU1, 2, Yuan YAO2, Yinhua CHEN1, 3, Jianchun GUO2, ** , Yajie WANG2, **
Affiliations
  • 1.School of Life and Health Sciences, Hainan University, Haikou, Hainan 570228, China
  • 2.Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs, Haikou, Hainan 571101, China
  • 3.School of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan 570228, China
出版时间: 2024-10-25 doi: 10.3969/j.issn.1000-2561.2024.10.006
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摘要
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MinD参与质体分裂的精细调控，在质体形态维持中起着关键作用。实验室前期研究发现MeMinD蛋白参与了木薯质体的分裂，然而与MeMinD蛋白协同调控木薯质体分裂的相关蛋白尚未明确。本研究构建了pGBKT7-MeMinD载体，通过酵母双杂交文库筛选，共获得MeMinD蛋白的8个候选互作蛋白，点对点验证后发现烟酸磷酸核糖转移酶2（MeNAPRT2）与MeMinD存在互作关系。该研究结果为进一步解析MeMinD参与调控木薯质体分裂的相关机制提供新的信息。

木薯  /  MeMinD  /  MeNAPRT2  /  酵母双杂交  /  质体

MinD is involved in the fine regulation of plastid division and plays a key role in maintaining plastid morphology. Previous laboratory research found that the MeMinD protein is involved in the division of cassava plastids, but the related proteins that work in conjunction with MeMinD to regulate the division of cassava plastids have not been clearly identified. This study constructed the pGBKT7-MeMinD vector and, through yeast two-hybrid library screening, a total of 8 candidate interacting proteins of MeMinD were obtained; after point-to-point verification using the yeast two-hybrid system, it was found that the nicotinate phosphoribosyltransferase 2 (MeNAPRT2) interacts with MeMinD. The results of this study would provide new information for further analysis of the mechanism of MeMinD's participation in the regulation of cassava plastid splitting.

cassava  /  MeMinD  /  MeNAPRT2  /  yeast two-hybrid  /  plastid
潘沐, 陆小花, 姚远, 陈银华, 郭建春, 王亚杰. 木薯MeMinD的互作蛋白筛选. 热带作物学报, 2024 , 45 (10) : 2054 -2058 . DOI: 10.3969/j.issn.1000-2561.2024.10.006
Mu PAN, Xiaohua LU, Yuan YAO, Yinhua CHEN, Jianchun GUO, Yajie WANG. Screening of MeMinD Interacting Protein in Cassava[J]. Chinese Journal of Tropical Crops, 2024 , 45 (10) : 2054 -2058 . DOI: 10.3969/j.issn.1000-2561.2024.10.006
正文
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植物质体（叶绿体、淀粉体等）的分裂由一系列蛋白协同完成[1]。MinD参与质体分裂的精细调控，在质体形态维持中起着关键作用。MinD有2个重要的结构域：MinC和MinE结合结构域。MinC基因所编码的MinC蛋白是细胞分裂抑制因子，在MinD的ATPase活性催化下，与其相互作用并结合形成复合物MinCD，之前，充当“钟摆”功能的MinE在两极之间来回移动，驱动MinCD复合物来回振荡，使Z环只能在中部进行组装，促进质体可以均等分裂成2个[2]。当MinD过量表达，会让复合物MinCD遍布质体所有位点，抑制Z环形成，从而抑制质体分裂；而MinD的减少或缺失，使Z环的形成不局限在中部，形成偏离中心或多个FtsZ环，质体分裂发生在不适当的位置[3-4]。
目前关于质体的研究多以叶绿体为对象，过量表达拟南芥AtMinD会导致叶绿体增大[5]，而减少AtMinD表达量或缺失该基因的突变体，引起叶绿体不对称分裂[4]。将AtMinD在烟草过表达，转基因植株的叶绿体大，且数目减少[6]。这些结果都进一步证实了MinD在质体分裂中的关键作用。淀粉体是木薯块根淀粉积累的场所，叶绿体是叶片临时性淀粉合成和降解的细胞器。前期，本实验室克隆了木薯的MeMinD基因，异源表达于大肠杆菌可导致菌体的分裂位点错位和丝状形成，亚细胞定位分析表明MeMinD可定位于叶绿体[7]。通过基因编辑敲除MeMinD基因，突变体木薯的叶绿体和淀粉体显著增大（待发表）。以上结果表明MeMinD蛋白参与调控木薯质体的分裂，然而与MeMinD蛋白协同调控木薯质体分裂的相关蛋白尚未明确。本研究将MeMinD构建至pGBKT7载体，进行酵母双杂交文库筛选实验，挖掘与MeMinD互作的质体分裂相关蛋白，为解析木薯质体分裂调控机制提供新的信息。
1　材料与方法
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1.1　材料
华南8号木薯（SC8，Manihot esculenta Crantz）、木薯cDNA文库、酵母双杂交质粒pGBKT7、pGADT7、pGADT7-T、pGBKT7-lam、pGBKT7-p53由本实验室提供；大肠杆菌DH5α感受态、酵母菌AH109感受态购自上海唯地生物技术有限公司；缺陷培养基SD/-Trp（SD/T）、SD/-Trp-Leu（SD/TL）、SD/-Trp-Leu-His-Ade（SD/TLHA）、X-α-Gal购自北京酷来搏科技有限公司。
1.2　方法
1.2.1　pGBKT7-MeMinD的构建与毒性检测
将酶切纯化的诱饵载体pGBKT7（BD）载体与纯化MeMinD基因片段进行同源重组，转化大肠杆菌DH5α感受态细胞，进行菌落PCR，筛选阳性单克隆，构建酵母双杂交载体pGBKT7-MeMinD。提取pGBKT7-MeMinD重组质粒和pGBKT7（BD）空载质粒并分别转化酵母菌AH109，将阳性克隆用SD/T液体培养基扩大培养，将OD600控制在0.8~1.0，再将菌液按1、10–1、10–2、10–3、10–4浓度梯度进行稀释，分别点在SD/T平板上，30 ℃培养2~3 d，观察菌落生长情况。
1.2.2　自激活检验
将pGBKT7-MeMinD质粒与pGADT7（BD）空载质粒共转入AH109酵母菌株，将阳性酵母液体培养基扩大培养，分别点在SD/TL、SD/TLHA、SD/TLHA+X-α-Gal三种平板上培养，观察实验组（pGADT7+pGBKT7-MeMinD）和对照组（阳性对照：pGBKT7-p53+pGADT7-T，阴性对照：pGBKT7-lam+pGADT7-T）的酵母菌在3种平板上的生长情况，以此确定MeMinD蛋白是否存在自激活现象。
1.2.3　MeMinD互作蛋白筛选
参照仇婷婷[8]的方法进行酵母双杂交文库筛选MeMinD的候选互作蛋白。将变蓝的酵母菌斑进行PCR检测，PCR产物送至生工生物工程（上海）股份有限公司进行测序，再利用NCBI Blast软件进行序列比对，筛选出木薯MeMinD可能互作的蛋白并进行功能注释。
1.2.4　酵母双杂交点对点验证
设计候选互作蛋白的基因扩增引物（表1），克隆其编码区并连接到pGADT7载体。PCR筛选阳性克隆并测序验证。将候选互作蛋白基因的pGADT7重组质粒与pGBKT7-MeMinD质粒共转AH109酵母菌株，进行酵母双杂交点对点验证，筛选MeMinD的互作蛋白。
2　结果与分析
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2.1　诱饵载体pGBKT7-MeMinD的构建
以SC8木薯cDNA为模板，PCR扩增MeMinD的编码区序列。电泳检测结果显示扩增片段与预期大小（987 bp）一致（图1）。将MeMinD基因片段与线性化的pGBKT7载体重组连接，转化大肠杆菌感受态细胞。挑取5个单菌落进行菌落PCR鉴定，获得4个阳性克隆并进行测序验证（图2）。提取测序正确的pGBKT7-MeMinD诱饵载体，用于后续酵母双杂交文库筛选。
2.2　毒性检测和自激活检测
将pGBKT7-MeMinD和pGBKT7空载分别转化酵母感受态AH109，将菌液稀释不同浓度（1、10–1、10–2、10–3、10–4）后在SD/TL培养基上培养。结果显示转pGBKT7-MeMinD和pGBKT7空载的酵母菌生长状态无差异（图3），说明MeMinD对AH109酵母菌无毒性，可进行后续实验。
将pGBKT7-MeMinD和pGADT7空载共转酵母菌株进行自激活检测。在SD/TL培养基上，共转pGBKT7-MeMinD和pGADT7空载的酵母菌、阳性对照（pGBKT7-p53+pGADT7-T）和阴性对照（pGBKT7-lam+pGADT7-T）的酵母菌在SD/TL培养基上均能正常生长，但在SD/TLHA和SD/TLHA+X-α-Gal培养基上只有阳性对照的酵母菌生长，且阳性对照在SD/TLHA+X-α-Gal培养基上的酵母菌落变蓝。此结果表明MeMinD蛋白不具有自激活活性，可用于酵母双杂交筛选木薯cDNA文库（图4）。
2.3　MeMinD互作蛋白筛选
将本实验室前期制备的木薯酵母双杂交质粒文库大规模转化到含有诱饵载体pGBKT7-MeMinD的AH109酵母感受态。转化后的酵母菌液涂布于SD/TLHA固体培养基上培养，获得单克隆酵母菌，并分别点在SD/TLHA+X-α-Gal培养基上进行培养（图5）。将变蓝的阳性菌落用于PCR扩增插入片段并测序，最终获得8个候选互作蛋白（表2）。这些蛋白功能涉及卡尔文循环、细胞代谢、NAD（P）H再生和细胞热耐受等多种生物学过程。
2.4　MeMinD互作蛋白点对点验证
根据候选蛋白的注释结果，挑选出4个候选蛋白进行酵母双杂交点对点验证，包括MeFBA1-1、MeFBA1-2、MeNAPRT2、MeALDH2B4。以SC8木薯品种cDNA为模板，PCR扩增出4个候选互作蛋白基因的CDS序列，将其分别构建到pGADT7载体。将诱饵载体和猎物载体共转酵母。结果显示，pGBKT7-MeMinD+pGADT7-MeFBA1-1、pGBKT7-MeMinD+pGADT7-MeFBA1-2、pGBKT7-MeMinD+pGADT7-MeALDH2B4、pGBKT7-MeMinD+pGADT7-MeNAPRT2以及对照组在SD/TL上均可以长出菌斑并正常生长（图6），说明质粒共转化酵母成功。酵母双杂交验证结果显示，仅有pGBKT7-MeMinD+pGADT7-MeNAPRT2能在SD/TLHA上生长出菌斑且在SD/TLHA+X-α-Gal上变蓝，表明在酵母细胞内MeMinD与MeNAPRT2蛋白存在相互关系。
3　讨论
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淀粉体和叶绿体均属于质体，由前质体发育而来，具有相似的分裂机制。植物质体的分裂过程，包括FtsZ蛋白组装形成Z环，MinD1和MinE1互作形成复合体，Min复合体与ARC3蛋白协同调控Z环精准定位于质体的中部，Z环形成后招募更多的质体分裂相关蛋白，Z环在多种蛋白的作用下引导质膜收缩，最终导致质体分裂。过量或减少拟南芥AtMinD1的表达都会影响叶绿体的分裂，从而影响叶绿体的大小[9]。
木薯块根富含的淀粉和叶片临时性淀粉的合成和贮存均在质体完成，挖掘鉴定木薯质体分裂相关基因的功能，解析淀粉体和叶绿体的分裂机制将有助于提高木薯产量和淀粉品质。GENG等[10]克隆了木薯FtsZ蛋白的3个基因，MeFtsZ1、MeFtsZ2-1和MeFtsZ2-2。MeFtsZ蛋白均能定位在叶绿体中，并形成Z环结构。MeFtsZ2-1和MeFtsZ2-2在块根的表达量显著高于MeFtsZ1。过表达MeFtsZ2-1和MeFtsZ2-2的拟南芥叶绿体增大，数目减少。WANG等[11]克隆了木薯MeMinE基因，MeMinE-GFP在木薯原生质体中的瞬时表达表明，MeMinE蛋白位于叶绿体中。MeMinE蛋白在木薯叶肉细胞原生质体中的过表达，可导致叶绿体分裂异常，叶绿体数目减少，面积变小。KE等[7]克隆了木薯的MeMinD基因，该基因在叶片中的表达量较高，MeMinD定位于叶绿体，在大肠杆菌异源表达可导致菌体的分裂位点错位和丝状形成。通过基因编辑敲除MeMinD基因，突变体木薯的叶绿体和淀粉体显著增大（数据待发表）。解析MeMinD参与调控木薯质体分裂的机制，将有助于利用分子生物技术培育高产、高淀粉木薯新品种。
目前，与MeMinD蛋白互作，协同调控木薯质体分裂的蛋白尚未明确。本研究采用酵母双杂交技术对MeMinD进行了互作蛋白的筛选，发现MeMinD与MeNAPRT2存在互作关系。NAPRT2（nicotinate phosphoribosyl transferase 2）在植物中参与烟酸（nicotinic acid）的磷酸核糖基转移反应，这是烟酸转化为NAD（烟酰胺腺嘌呤二核苷酸）途径中的关键步骤[12]。NAD是细胞内重要的辅酶，参与多种代谢过程，包括能量代谢和信号传导[13-15]。NAD+（NAD的氧化形式）在氧化还原反应中接受电子和质子，形成NADH。NADH随后在电子传递链中将电子传递给氧气，最终产生ATP。MinD蛋白是一种ATPase，通过ATP的水解，MinD蛋白能够在质膜上形成动态的极性模式，与MinE和MinC蛋白相互作用，共同调控质体的分裂过程。MeMinD与MeNAPRT2的互作表明能量代谢可能参与木薯质体的分裂过程。水稻OsNAPRT1基因突变会导致植株矮化，叶片过早枯萎[16]。目前，尚无NAPRT2参与质体分裂的研究报道。后续将通过双分子荧光互补试验进一步验证MeMinD与MeNAPRT2的互作关系，利用转基因或基因编辑技术探究其在木薯质体分裂过程中的功能。
基金
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  • 海南省自然科学基金项目(320RC709)
  • 国家自然科学基金项目(32301740)
  • 国家木薯产业技术体系项目(CARS-11-HNGJC)
文献
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参考文献 引证文献
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2024年第45卷第10期
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文章信息
doi: 10.3969/j.issn.1000-2561.2024.10.006
  • 接收时间：2024-03-07
  • 首发时间：2026-06-25
  • 出版时间：2024-10-25
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  • 收稿日期：2024-03-07
  • 修回日期：2024-04-19
基金
海南省自然科学基金项目(320RC709)
国家自然科学基金项目(32301740)
国家木薯产业技术体系项目(CARS-11-HNGJC)
作者信息
    1.海南大学生命健康学院，海南海口　570228
    2.中国热带农业科学院热带生物技术研究所/农业农村部热带作物生物学与遗传资源利用重点实验室，海南海口　571101
    3.海南大学热带农林学院，海南海口　570228

通讯作者:

** 郭建春（GUO Jianchun），E-mail：
王亚杰（WANG Yajie），E-mail：。
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https://castjournals.cast.org.cn/joweb/rdzwxb/CN/10.3969/j.issn.1000-2561.2024.10.006
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2种不同金属材料的力学参数
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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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