Article(id=1276213356415553664, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276213295170323272, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.04.011, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1674921600000, receivedDateStr=2023-01-29, revisedDate=1676736000000, revisedDateStr=2023-02-19, acceptedDate=null, acceptedDateStr=null, onlineDate=1782202575067, onlineDateStr=2026-06-23, pubDate=1713974400000, pubDateStr=2024-04-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782202575067, onlineIssueDateStr=2026-06-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782202575067, creator=13701087609, updateTime=1782202575067, updator=13701087609, issue=Issue{id=1276213295170323272, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='4', pageStart='653', pageEnd='871', issueExtLink='null', onlineDate='null', pubDate='1713974400000', pubDateStr='2024-04-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782202560465, creator='13701087609', updateTime=1782203706550, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1276218103419761358, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276213295170323272, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1276218103419761359, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276213295170323272, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=752, endPage=760, ext={EN=ArticleExt(id=1276213358118441090, articleId=1276213356415553664, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Proteasomes Involved in Seed Germination in Rice Aleurone Layer, columnId=1236256434120348225, journalTitle=Chinese Journal of Tropical Crops, columnName=Plant Cultivation, Physiology & Biochemistry, runingTitle=null, highlight=null, articleAbstract=

Seed germination is a key link for plants to enter the vegetative growth stage. During the germination of rice (Oryza sativa L.) seeds, the aleurone layers synthesize and secrete a series of acid hydrolases (such as α-amylase, protease) to degrade the storage substances in the endosperm and provide nutrition for the growth of rice seedlings. The degradation of storage proteins is inseparable from the role of proteasome and protease in rice seeds. In order to explore the involvement of 26S proteasome, 20S proteasome and its subunit PBA1 in the regulation of GA on the storage proteins in the aleurone layers of germinated rice seeds, the rice hybrid Teyou 128 was used as the experimental material. The activity of 26S proteasome, 20S proteasome and PBA1, the transcription level of OsPBA1 and the change of total protein content were detected by enzyme-linked immunosorbent assay and real-time fluorescence quantitative PCR. GA significantly promoted the degradation of storage proteins in aleurone cells and accelerated the germination of rice seeds. When GA synthesis was blocked, the degradation process of storage protein in aleurone layer cells was delayed, thus delaying the germination of rice seeds. Under normal culture conditions, the peak values of 26S proteasome activity and 20S proteasome activity in the aleurone layers of rice seeds appeared at 6 d and 7 d, respectively, while the OsPBA1 expression level and PBA1 activity showed peaks at 5 d and 8 d. Further studies showed that GA could induce the activity of PBA1, 20S proteasome and 26S proteasome, thereby accelerating the process of seed germination. The 26S proteasome activity inhibitor MG132 inhibited the growth of germinated rice seeds, but increased the activity of 20S proteasome and PBA1. The above studies preliminarily revealed the role of 26S proteasome in GA regulating the degradation of storage proteins in rice aleurone layers and its effect on the germination of rice seeds.

, authors=null, authorsList=Wenyi CHEN, Huiping CHEN, authorCompany=null, correspAuthors=Huiping CHEN, 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=1276213362623123598, articleId=1276213356415553664, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=水稻糊粉层蛋白酶体参与种子萌发的研究, columnId=1236256434313286224, journalTitle=热带作物学报, columnName=作物栽培与生理生化, runingTitle=null, highlight=null, articleAbstract=

种子萌发是植物进入营养生长阶段的关键性环节,在水稻(Oryza sativa L.)种子萌发过程中,糊粉层会合成并分泌一系列的酸性水解酶(如α-淀粉酶、蛋白酶)用于降解胚乳中的贮藏物质,为水稻幼苗生长提供营养。水稻种子贮藏蛋白的降解离不开蛋白酶体及蛋白酶的作用。为探究26S蛋白酶体、20S蛋白酶体及其亚基PBA1参与GA对萌发水稻种子糊粉层中贮藏蛋白的调节过程,本研究以水稻杂交种特优128为试验材料,通过采用酶联免疫吸附技术、实时荧光定量PCR技术等方法,检测26S蛋白酶体、20S蛋白酶体及PBA1的活性和OsPBA1转录水平,以及总蛋白含量的变化。结果表明:GA明显促进糊粉层细胞中贮藏蛋白的降解,加快水稻种子的萌发;而在GA合成受阻时,糊粉层细胞中贮藏蛋白的降解进程延缓,从而延迟水稻种子的萌发。正常培养条件下,水稻种子糊粉层中26S蛋白酶体活性和20S蛋白酶体活性的峰值分别出现在6 d和7 d,而OsPBA1表达水平及PBA1活性则在5 d和8 d均呈现峰值。进一步研究表明,GA可以诱导PBA1、20S蛋白酶体及26S蛋白酶体的活性,从而加快种子萌发的进程;而在26S蛋白酶体活性抑制剂MG132处理下,抑制了萌发水稻种子的生长,但提高了20S蛋白酶体和PBA1的活性。以上研究初步揭示了26S蛋白酶体在GA调节水稻种子糊粉层贮藏蛋白降解中的作用及其对种子萌发的影响。

, authors=

陈文奕(1997—),男,硕士研究生,研究方向:植物生长发育与调控。

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* 陈惠萍(CHEN Huiping),E-mail:
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陈文奕(1997—),男,硕士研究生,研究方向:植物生长发育与调控。

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陈文奕(1997—),男,硕士研究生,研究方向:植物生长发育与调控。

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Molecular and Cellular Biology, 2005, 25(11): 4662-4675., articleTitle=Identification and characterization of a drosophila proteasome regulatory network, refAbstract=null)], funds=[Fund(id=1276466485384053633, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, awardId=31960453, language=CN, fundingSource=国家自然科学基金项目(31960453), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1276466473262515038, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, xref=null, ext=[AuthorCompanyExt(id=1276466473270903647, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, companyId=1276466473262515038, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=College of Life Science, Hainan University, Haikou, Hainan 570228, China), AuthorCompanyExt(id=1276466473279292256, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, companyId=1276466473262515038, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=海南大学生命科学学院,海南海口 570228)])], figs=[ArticleFig(id=1276466482045387637, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Fig. 1, caption=Changes of growth (A) and total protein contents of aleurone layers (B) in germinating rice seeds under different treatments

Different lowercase letters indicate significant difference among treatments (P<0.05).

, figureFileSmall=yAbkhzblJ0h5YGV6LNhBag==, figureFileBig=WK5rO5lamCCaLEyuUIX2eQ==, tableContent=null), ArticleFig(id=1276466482125079414, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=图1, caption=不同处理下萌发水稻种子长势(A)及糊粉层中总蛋白含量(B)变化

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=yAbkhzblJ0h5YGV6LNhBag==, figureFileBig=WK5rO5lamCCaLEyuUIX2eQ==, tableContent=null), ArticleFig(id=1276466482502566775, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Fig. 2, caption=Activity changes of 26S proteasome (A) and 20S proteasome (B) in aleurone layers of germinating rice seeds

Different lowercase letters indicate significant difference among treatments (P<0.05).

, figureFileSmall=i0WEXQjSoRlDVMUsyCtojg==, figureFileBig=EVog/0VvAd0Mqn5ZxpxQGg==, tableContent=null), ArticleFig(id=1276466482863276920, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=图2, caption=萌发水稻种子糊粉层26S蛋白酶体活性(A)及20S蛋白酶体活性(B)变化

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=i0WEXQjSoRlDVMUsyCtojg==, figureFileBig=EVog/0VvAd0Mqn5ZxpxQGg==, tableContent=null), ArticleFig(id=1276466482942968697, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Fig. 3, caption=Level changes of OsPBA1 transcript (A) and PBA1 activity (B) in aleurone layers of germinating rice seeds

Different lowercase letters indicate significant difference among treatments (P<0.05).

, figureFileSmall=cVsia3WoqPinvDcMKmQCAg==, figureFileBig=V4f8SUjNRxnurfAkQ8FdaA==, tableContent=null), ArticleFig(id=1276466483295290234, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=图3, caption=萌发水稻种子糊粉层的OsPBA1表达水平(A)及PBA1活性(B)的变化

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=cVsia3WoqPinvDcMKmQCAg==, figureFileBig=V4f8SUjNRxnurfAkQ8FdaA==, tableContent=null), ArticleFig(id=1276466483744080763, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Fig. 4, caption=Activities changes of 26S proteasome (A), 20S proteasome (B) and PBA1 (C) in aleurone layers of germination rice seeds treated with GA and Uni, respectively.

Different lowercase letters indicate significant difference among treatments (P<0.05).

, figureFileSmall=g1El+2lXem3NBGWM56Z5Uw==, figureFileBig=WwcnKf9NufXxZjMGrRtP6g==, tableContent=null), ArticleFig(id=1276466483857326972, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=图4, caption=GA、Uni处理5 d的26S蛋白酶体(A)、20S蛋白酶体(B)及PBA1活性(C)变化

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=g1El+2lXem3NBGWM56Z5Uw==, figureFileBig=WwcnKf9NufXxZjMGrRtP6g==, tableContent=null), ArticleFig(id=1276466484184482685, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Fig. 5, caption=Effects of MG132 on growths (A, B, C) and activities of 26S proteasome (D), 20S proteasome (E), and PBA1 (F) of aleurone layers in the germination of rice seeds

Different lowercase letters indicate significant difference among treatments (P<0.05).

, figureFileSmall=zIqyg6aJ5XgsxVfOFcQMzw==, figureFileBig=qS9f6G6O7NPd+b0ZxXUF4A==, tableContent=null), ArticleFig(id=1276466484553581438, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=图5, caption=MG132处理对萌发水稻种子生长(A、B、C)及26S蛋白酶体活性(D)、20S蛋白酶体活性(E)和PBA1活性(F)的影响

不同小写字母表示处理间差异显著(P<0.05)。

, figureFileSmall=zIqyg6aJ5XgsxVfOFcQMzw==, figureFileBig=qS9f6G6O7NPd+b0ZxXUF4A==, tableContent=null), ArticleFig(id=1276466484662633343, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=EN, label=Tab. 1, caption=

Primer sequences used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
基因Gene登录号Accession number引物序列(5′-3′)Primers sequence (5′-3′)
OsActinNM_001057621F: TCTCTCTGTATGCCAGTGGTCGT
R: TCATAGTCCAGGGCGATGTAGG
OsPBA1XM_015795772F: GGCTCATCAATTATTGCACTG
R: GCTCCACGCTCTTGTATCG
), ArticleFig(id=1276466484956234624, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276213356415553664, language=CN, label=表1, caption=

本研究所用引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因Gene登录号Accession number引物序列(5′-3′)Primers sequence (5′-3′)
OsActinNM_001057621F: TCTCTCTGTATGCCAGTGGTCGT
R: TCATAGTCCAGGGCGATGTAGG
OsPBA1XM_015795772F: GGCTCATCAATTATTGCACTG
R: GCTCCACGCTCTTGTATCG
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水稻糊粉层蛋白酶体参与种子萌发的研究
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陈文奕 , 陈惠萍 *
热带作物学报 | 作物栽培与生理生化 2024,45(4): 752-760
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热带作物学报 |作物栽培与生理生化 2024 , 45 (4) : 752 -760
水稻糊粉层蛋白酶体参与种子萌发的研究
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陈文奕, 陈惠萍*
作者信息
  • 海南大学生命科学学院,海南海口 570228
通讯作者:
* 陈惠萍(CHEN Huiping),E-mail:
Proteasomes Involved in Seed Germination in Rice Aleurone Layer
Wenyi CHEN, Huiping CHEN*
Affiliations
  • College of Life Science, Hainan University, Haikou, Hainan 570228, China
出版时间: 2024-04-25 doi: 10.3969/j.issn.1000-2561.2024.04.011
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种子萌发是植物进入营养生长阶段的关键性环节,在水稻(Oryza sativa L.)种子萌发过程中,糊粉层会合成并分泌一系列的酸性水解酶(如α-淀粉酶、蛋白酶)用于降解胚乳中的贮藏物质,为水稻幼苗生长提供营养。水稻种子贮藏蛋白的降解离不开蛋白酶体及蛋白酶的作用。为探究26S蛋白酶体、20S蛋白酶体及其亚基PBA1参与GA对萌发水稻种子糊粉层中贮藏蛋白的调节过程,本研究以水稻杂交种特优128为试验材料,通过采用酶联免疫吸附技术、实时荧光定量PCR技术等方法,检测26S蛋白酶体、20S蛋白酶体及PBA1的活性和OsPBA1转录水平,以及总蛋白含量的变化。结果表明:GA明显促进糊粉层细胞中贮藏蛋白的降解,加快水稻种子的萌发;而在GA合成受阻时,糊粉层细胞中贮藏蛋白的降解进程延缓,从而延迟水稻种子的萌发。正常培养条件下,水稻种子糊粉层中26S蛋白酶体活性和20S蛋白酶体活性的峰值分别出现在6 d和7 d,而OsPBA1表达水平及PBA1活性则在5 d和8 d均呈现峰值。进一步研究表明,GA可以诱导PBA1、20S蛋白酶体及26S蛋白酶体的活性,从而加快种子萌发的进程;而在26S蛋白酶体活性抑制剂MG132处理下,抑制了萌发水稻种子的生长,但提高了20S蛋白酶体和PBA1的活性。以上研究初步揭示了26S蛋白酶体在GA调节水稻种子糊粉层贮藏蛋白降解中的作用及其对种子萌发的影响。

GA  /  蛋白酶体  /  水稻糊粉层  /  贮藏蛋白  /  种子萌发

Seed germination is a key link for plants to enter the vegetative growth stage. During the germination of rice (Oryza sativa L.) seeds, the aleurone layers synthesize and secrete a series of acid hydrolases (such as α-amylase, protease) to degrade the storage substances in the endosperm and provide nutrition for the growth of rice seedlings. The degradation of storage proteins is inseparable from the role of proteasome and protease in rice seeds. In order to explore the involvement of 26S proteasome, 20S proteasome and its subunit PBA1 in the regulation of GA on the storage proteins in the aleurone layers of germinated rice seeds, the rice hybrid Teyou 128 was used as the experimental material. The activity of 26S proteasome, 20S proteasome and PBA1, the transcription level of OsPBA1 and the change of total protein content were detected by enzyme-linked immunosorbent assay and real-time fluorescence quantitative PCR. GA significantly promoted the degradation of storage proteins in aleurone cells and accelerated the germination of rice seeds. When GA synthesis was blocked, the degradation process of storage protein in aleurone layer cells was delayed, thus delaying the germination of rice seeds. Under normal culture conditions, the peak values of 26S proteasome activity and 20S proteasome activity in the aleurone layers of rice seeds appeared at 6 d and 7 d, respectively, while the OsPBA1 expression level and PBA1 activity showed peaks at 5 d and 8 d. Further studies showed that GA could induce the activity of PBA1, 20S proteasome and 26S proteasome, thereby accelerating the process of seed germination. The 26S proteasome activity inhibitor MG132 inhibited the growth of germinated rice seeds, but increased the activity of 20S proteasome and PBA1. The above studies preliminarily revealed the role of 26S proteasome in GA regulating the degradation of storage proteins in rice aleurone layers and its effect on the germination of rice seeds.

GA  /  proteasome  /  aleurone layers  /  storage protein  /  seed germination
陈文奕, 陈惠萍. 水稻糊粉层蛋白酶体参与种子萌发的研究. 热带作物学报, 2024 , 45 (4) : 752 -760 . DOI: 10.3969/j.issn.1000-2561.2024.04.011
Wenyi CHEN, Huiping CHEN. Proteasomes Involved in Seed Germination in Rice Aleurone Layer[J]. Chinese Journal of Tropical Crops, 2024 , 45 (4) : 752 -760 . DOI: 10.3969/j.issn.1000-2561.2024.04.011
禾谷类种子的胚乳由淀粉性胚乳及外层的糊粉层构成。成熟的糊粉层细胞仍具有活性,其形态为规则的多边形,富含蛋白质、矿物质、淀粉和维生素等物质[1-2]。水稻糊粉层细胞内的贮藏蛋白以清蛋白及球蛋白为主,大多数积累在液泡内从而形成蛋白质贮藏液泡(protein storage vacuoles,PSVs)[3-4]。赤霉素(gibberellic acid,GA)是植物生长和发育过程中重要的调节激素。在禾谷类种子萌发过程中,胚会释放出GA诱导糊粉层细胞合成一系列的酸性水解酶(如α-淀粉酶、核酸酶、蛋白酶等)[5-6],这些酶会在中央大液泡破裂后释放,并用于降解淀粉性胚乳中贮藏的营养物质,为种子萌发提供营养[7]。与此同时,糊粉层细胞内的贮藏蛋白在水稻种子萌发过程被胚生长所利用[8]
26S蛋白酶体是一种依赖ATP,并能水解泛素化蛋白的蛋白酶复合物,主要负责细胞质和细胞核中的蛋白质降解,对于蛋白质稳态至关重要[9-10]。26S蛋白酶体控制着植物体内平衡的各种过程,包括植物发育、光反应、细胞分裂及响应植物激素反应等[9,11-12],并且与植物防御相关[13],其功能的丧失会显著影响植物的生长[14]。研究表明,26S蛋白酶体是种子萌发过程中所必需的,它能降解ABA信号传导及合成的正调节因子,从而打破种子休眠并促进萌发[15];以及维持拟南芥地上部分器官的大小及细胞增殖速率等[16]
DELLA蛋白是GA信号转导的核心,在植物生长调控中充当生长抑制因子[17-18]。外源GA可通过诱导26S蛋白酶体降解DELLA蛋白来促进大麦幼苗的生长[19],而GA生物合成抑制剂及26S蛋白酶体抑制剂均能抑制DELLA蛋白的降解[20]。由此可见,GA信号传导在26S蛋白酶体介导的蛋白质降解中起着重要的作用。
26S蛋白酶体由2个不同功能的亚复合体组成,即20S蛋白酶体和19S调节颗粒[21]。20S蛋白酶体是一个桶形结构,由2个相同的外α环和2个相同的内β环堆叠组成,使20S蛋白酶体具有α1-7β1-7β1-7α1-7的双重对称性结构,14个α亚基和14个β亚基的正确组装确保了20S蛋白酶体的完整性[22-24]。20S蛋白酶体上含有β1、β2和β5三种蛋白酶活性位点,并形成蛋白水解室,而19S调节颗粒的作用是识别泛素化蛋白,并将其转运到20S蛋白酶体的蛋白水解室内进行降解[25-26]。近年的研究发现,多种蛋白质可以被20S蛋白酶体本身降解,而不需要与19S调节颗粒结合后发挥其调节作用[27-28]。20S蛋白酶体介导的蛋白质降解过程不依赖泛素化及ATP,在氧化应激后,20S蛋白酶体活性升高,从而快速降解氧化损伤的蛋白质[29-30]。20S蛋白酶体β亚基1(20S proteasome β subunit1,PBA1)是一种具有催化活性的半胱氨酸蛋白酶,已证实其参与PCD的发生[31]。在银诱导葡萄悬浮细胞的PCD过程中,PBA1专一性抑制剂AC-APnLD-CHO可使26S蛋白酶体功能受损,伴随着多泛素蛋白的增加[32]
本研究通过借助酶联免疫吸附技术和实时荧光定量PCR技术等,探究蛋白酶体及其亚基PBA1参与GA对萌发水稻种子糊粉层中贮藏蛋白的调节过程,为深入研究GA调节泛素-蛋白酶体系统介导水稻糊粉层中蛋白质降解的分子机制提供理论依据。
试验材料为水稻(Oryza sativa L.)杂交种特优128,购自海南省儋州市种子公司。
选取去除颖壳后的水稻种子,用0.1%高锰酸钾消毒10 min,用纯水将其洗干净后,分别置于添加不同处理液的培养皿中。根据实验需求,萌发水稻种子分别在浸泡1~9 d后取出,使用手术刀片分离水稻种子糊粉层用于检测相应的指标。
本研究设置2个处理组。处理1:(1)CK(纯水);(2)GA(50 μmol/L);(3)GA合成抑制剂烯效唑(Uniconazole,Uni,75 μmol/L)。处理2:(1)CK(DMSO);(2)26S蛋白酶体活性抑制剂MG132(50 μmol/L)。
本实验采用Solarbio公司的植物蛋白提取试剂盒(BC3720)进行水稻糊粉层总蛋白的提取。称取100 mg的水稻糊粉层放入液氮中速冻(最好过夜),取出糊粉层于含液氮的研钵中研磨成粉末,加入1 mL裂解液后,置于4 ℃裂解20 min。4 ℃,14 000 r/min离心30 min后收集上清,上清即为水稻糊粉层总蛋白。采用BCA法测定上清液中总蛋白含量,每组样品重复3次。
称取糊粉层100 mg,使用液氮研磨成粉末,加入900 μL 0.01 mol/L PBS缓冲液(pH 7.2~7.4),在4 ℃下5000 r/min离心15 min,吸取上清液至预冷离心管中备用。本试验分别采用江苏酶免实业有限公司的植物26S蛋白酶体、20S蛋白酶体及20S蛋白酶体β亚基1(PBA1)的ELISA检测试剂盒进行检测,使用全自动酶标分析仪(AMR-100)测定其在450 nm处的吸光度。分别将原倍标准品稀释后,制作出26S蛋白酶体、20S蛋白酶体及PBA1的标准曲线,依照其标准曲线计算出酶活性。每组样品重复3次。
根据实验要求选取水稻糊粉层50 mg,采用天根生化公司产品(DP441)植物总RNA提取试剂盒进行总RNA的提取,按照试剂盒说明书进行操作,并稍加修改。制取1.2%的琼脂糖凝胶进行电泳15 min,利用凝胶成像分析系统检测所提取的RNA完整性。使用微量核酸检测仪检测RNA纯度及浓度,然后将RNA定量为100 ng/μL,以进行逆转录反应。
以定量的水稻总RNA为模板,使用TaKaRa公司的产品PrimeScript RT reagent Kit with gDNA Eraser(RR047A)合成cDNA。总反应体系为20 μL。
本实验采用biosharp公司的产品荧光定量试剂盒SYBR Green Master Mix(BL705A)。以cDNA为模板,OsActin为内参基因,OsPBA1为目的基因检测的表达水平,每个样品重复3次。相应基因引物序列如表1所示。
在不同处理中随机选取培养5 d的萌发水稻种子10粒,使用直尺测量其胚根长度和胚芽长度,并取3次独立实验所测量的平均值进行差异显著性分析。
实验数据采用Excel软件进行处理,并用SPSS 23.0软件进行差异显著性分析(P<0.05)。
图1A为水稻种子萌发1~9 d的长势,与纯水培养相比,GA处理明显促进水稻种子的萌发,而GA合成抑制剂Uni处理后,水稻种子的萌发受到抑制。如图1B所示,纯水、GA和GA合成抑制剂Uni处理的水稻糊粉层在种子萌发过程中,总蛋白含量均随着时间的增加而逐渐降低。在2~9 d,与对照相比,GA处理的糊粉层中,总蛋白含量显著降低;而GA合成抑制剂Uni处理的糊粉层中总蛋白含量一直维持在较高水平。以上结果表明,外源GA可加快糊粉层中蛋白质的降解,从而促进水稻种子的萌发,而GA合成抑制剂Uni则减缓糊粉层中蛋白质的降解,抑制水稻种子的萌发。
图2A可知,26S蛋白酶体活性在水稻种子萌发1~6 d呈现上升的趋势,并在6 d时达到最高活性水平;在7~9 d时,26S蛋白酶体活性水平逐渐下降。该结果表明在水稻种子萌发过程中糊粉层细胞中的26S蛋白酶体活性具有依赖时间的效应。
图2B所示,在水稻种子萌发1~7 d时,糊粉层中20S蛋白酶体活性逐渐上升,并在第7天时达到峰值,随后其活性逐渐下降。表明20S蛋白酶体活性在水稻种子萌发过程中同样具有依赖时间的效应。
综合以上结果,在水稻种子萌发过程中,糊粉层细胞中含有活性的26S蛋白酶体和20S蛋白酶体,二者的活性均随着萌发时间的增加而变化,且20S蛋白酶体活性比26S蛋白酶体活性迟1 d达到最高峰。
OsPBA1在水稻种子萌发过程出现2个表达峰值,OsPBA1的相对表达量在1~5 d随时间的增加而上升,并在第5天达到第一个表达峰值,约为1 d的6.24倍;在6~7 d,其相对表达量开始下降,而第8天时,表达量明显升高,出现第二个表达峰值,其表达量约为1 d的5.4倍,随后糊粉层中OsPBA1的表达量开始下降(图3A)。
图3B所示,在水稻种子萌发过程中,糊粉层细胞中PBA1活性的变化趋势与OsPBA1转录水平的变化趋势类似。在1~5 d,PBA1活性随时间的延长而上升,并在第5天达到第一个峰值;在6~7 d,PBA1活性下降,第8天时出现上升的趋势,并达到第二个峰值,该峰值低于5 d的活性峰值;随后,水稻糊粉层细胞中的PBA1活性开始下降。
以上结果表明,OsPBA1表达水平和PBA1活性随着种子萌发时间的增加而变化,PBA1可能以依赖时间的方式调控水稻种子的萌发过程。
图4A所示,与对照相比,GA处理5 d的水稻糊粉层中26S蛋白酶体的活性升高了9.97%(P<0.05),而GA合成抑制剂Uni处理5 d则使26S蛋白酶体的活性降低了8.67%(P<0.05)。同样,与对照相比,GA处理5 d的水稻糊粉层中20S蛋白酶体和PBA1的活性分别提高了17%(P<0.05)和35.09%(P<0.05),而GA合成抑制剂Uni处理5 d后,20S蛋白酶体和PBA1的活性则分别降低了22.92%(P<0.05)和24.93%(P<0.05)(图4B图4C)。
以上结果表明GA对水稻糊粉层中26S蛋白酶体、20S蛋白酶体和PBA1的活性均具有促进作用,而GA合成抑制剂Uni则起着抑制作用。
使用MG132处理5 d后,水稻种子的生长明显受到抑制,其胚根和胚芽的长度短于对照的长度(图5A图5B图5C)。由图5D可知,与对照相比,MG132处理的水稻糊粉层26S蛋白酶体活性降低了31.75%(P<0.05),该结果表明MG132能够有效抑制26S蛋白酶体的活性,且在种子萌发过程中,26S蛋白酶体的活性与其萌发表型相关。
图5E可知,与对照相比,MG132处理的糊粉层中20S蛋白酶体活性升高了25.63%(P<0.05),表明MG132处理可以推进26S蛋白酶体的解离,导致游离20S蛋白酶体的活性上升。通过进一步检测MG132对PBA1活性的影响发现,在相同处理下,PBA1活性的变化与20S蛋白酶体活性的变化一致。与对照相比,MG132处理的水稻糊粉层中,PBA1活性升高71.37%(P<0.05),表明26S蛋白酶体的正常组装受到抑制后,亚基PBA1的活性会上调(图5F)。
以上结果表明,MG132可抑制萌发水稻种子的生长,且对水稻糊粉层26S蛋白酶体的组装亦具抑制作用,从而导致20S蛋白酶体活性及其亚基PBA1活性的上升。
通过泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)调节蛋白质水平是真核生物中最复杂和最普遍的途径之一,UPS可通过降解受损蛋白质、错误折叠蛋白或短寿命的调节蛋白,从而保证功能蛋白的正常工作[33-35]。UPS对蛋白质的降解是一个多步骤的复杂过程,底物蛋白质首先通过泛素激活酶(ubiquitin-activating enzyme,E1)、泛素结合酶(ubiquitin-conjugating enzyme,E2)和泛素连接酶(ubiquitin-protein ligase,E3)的协同作用被多泛素化,随后泛素化蛋白质被UPS的核心26S蛋白酶体识别并降解为小肽或氨基酸[36-37]。26S蛋白酶体在植物生长发育过程中起着重要的调节作用,可通过选择性降解蛋白质,从而高效控制许多细胞过程[38-39]
研究表明,GA可在种子萌发过程中促进水解酶的分泌,加速了贮藏物质的分解,为种子萌发提供营养[40]。在本研究中,通过观察萌发水稻种子的长势及检测总蛋白的含量发现,使用GA处理水稻种子时,促进糊粉层中贮藏蛋白的降解,从而加快水稻种子的萌发进程;而在GA合成受到抑制时,糊粉层中贮藏蛋白的降解受到阻碍,水稻种子的萌发则受到抑制。26S蛋白酶体在激素信号传导方面是保守的,GA可诱导26S蛋白酶体靶向降解蛋白质,从而促进种子萌发及果实发育等[18,41],但对于其中机制还待进一步研究。
在水稻种子萌发1~9 d,20S蛋白酶体亚基PBA1的活性及其编码基因OsPBA1的表达水平出现2个峰值,在5 d达到第一个峰值,而26S蛋白酶体在其达到峰值的后1 d,26S蛋白酶体的活性才随之达到峰值,由此可知,PBA1水平的升高可促使26S蛋白酶体活性升高。NGUYEN等[42]研究证实,PBA1的基因表达能以特异性方式调节26S蛋白酶体活性。在本研究中,20S蛋白酶体活性在7 d达到峰值,该峰值在26S蛋白酶体活性下降期间,可见,26S蛋白酶体在解离为20S蛋白酶体及19S调节颗粒后,游离20S蛋白酶体水平增加并达到峰值。有研究表明,在植物发育和胁迫反应过程中,调节26S蛋白酶体和20S蛋白酶体之间的比例具有重要意义。在植物衰老过程,26S蛋白酶体活性的下降通常伴随着20S蛋白酶体活性的上升,以增加植物细胞降解氧化蛋白的能力[14,43-44]。本研究中PBA1的活性及OsPBA1的表达水平在8 d达到第二个峰值,该峰值出现在20S蛋白酶体活性下降期间,即20S蛋白酶体活性的降低推动PBA1的活性及OsPBA1的表达水平达到第二个峰值。
在本研究中,使用GA及GA合成抑制剂Uni处理水稻种子,发现GA能够诱导PBA1活性升高,进而上调20S蛋白酶体及26S蛋白酶体活性,而Uni对该过程起着抑制作用。有研究证实,RPX是植物体内的蛋白酶体基因表达调控因子,它与26S蛋白酶体亚基的编码基因启动子结合后,能诱导PBA1的蛋白质水平升高,并调节蛋白酶体的活性增加[42]。而敲除20S蛋白酶体亚基PBE1会影响26S蛋白酶体的组装,致使其活性下降[45]
为了进一步研究在药物抑制26S蛋白酶体活性时,水稻种子生长及20S蛋白酶体和PBA1的活性所受到的影响,本研究使用了26S蛋白酶体活性抑制剂MG132处理水稻种子。当抑制26S蛋白酶体活性后,萌发水稻种子的生长也受到抑制,表明26S蛋白酶体在植物生长过程起着重要作用。CHIU等[15]研究证实,使用MG132处理抑制了拟南芥种子的萌发,直接处理拟南芥胚胎则完全抑制胚胎的生长。当26S蛋白酶体活性受到抑制时,20S蛋白酶体亚基PBA1反馈上调,从而出现了20S蛋白酶体活性的增加,这点得到了他人实验的支持。在19S调节颗粒亚基基因突变或MG132处理导致26S蛋白酶体活性降低的植株中,出现其他亚基基因的反馈上调,伴随着20S蛋白酶体活性的增加[29,46-47]。在哺乳动物中同样观察到26S蛋白酶体活性受抑制后,其亚基基因反馈上调的情况[48]
综合以上结果可知,在水稻种子萌发过程中,GA通过诱导PBA1的活性升高,调节20S蛋白酶体的活性,进而介导26S蛋白酶体的组装,并以此介导26S蛋白酶体依赖性降解贮藏蛋白,加快种子萌发的进程。
  • 国家自然科学基金项目(31960453)
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2024年第45卷第4期
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doi: 10.3969/j.issn.1000-2561.2024.04.011
  • 接收时间:2023-01-29
  • 首发时间:2026-06-23
  • 出版时间:2024-04-25
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  • 收稿日期:2023-01-29
  • 修回日期:2023-02-19
基金
国家自然科学基金项目(31960453)
作者信息
    海南大学生命科学学院,海南海口 570228

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* 陈惠萍(CHEN Huiping),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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