[1]宫宇,毛卓卓,史贵霞,等.大豆E3泛素连接酶基因GmPUB1的克隆及功能研究[J].南京农业大学学报,2020,43(5):820-828.[doi:10.7685/jnau.201912063]
 GONG Yu,MAO Zhuozhuo,SHI Guixia,et al.Cloning and functional analysis of soybean E3 ubiquitin ligase gene GmPUB1[J].Journal of Nanjing Agricultural University,2020,43(5):820-828.[doi:10.7685/jnau.201912063]
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大豆E3泛素连接酶基因GmPUB1的克隆及功能研究()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
43卷
期数:
2020年5期
页码:
820-828
栏目:
植物科学
出版日期:
2020-09-15

文章信息/Info

Title:
Cloning and functional analysis of soybean E3 ubiquitin ligase gene GmPUB1
作者:
宫宇 毛卓卓 史贵霞 杨中义 喻德跃 黄方
南京农业大学国家大豆改良中心/作物遗传与种质创新国家重点实验室/农学院, 江苏 南京 210095
Author(s):
GONG Yu MAO Zhuozhuo SHI Guixia YANG Zhongyi YU Deyue HUANG Fang
National Center for Soybean Improvement/State Key Laboratory of Crop Genetics and Germplasm Enhancement/College of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
关键词:
大豆E3泛素连接酶GmPUB1基因功能分析
Keywords:
soybean[Glycine max(L.)Mree]E3 ubiquitin ligaseGmPUB1 genefunctional analysis
分类号:
S565.1
DOI:
10.7685/jnau.201912063
摘要:
[目的] 本研究通过对大豆E3泛素连接酶基因GmPUB1的克隆及在拟南芥中异源表达,探究E3泛素连接酶在大豆生长发育和非生物胁迫中的作用。[方法] 克隆大豆E3泛素连接酶基因GmPUB1,并对其进行生物信息学分析。利用qRT-PCR技术分析该基因在不同组织中以及不同胁迫处理下的表达模式。通过洋葱表皮细胞瞬时表达系统对GmPUB1蛋白进行亚细胞定位分析。在拟南芥中异源表达GmPUB1并对其表型进行分析。[结果] GmPUB1基因(Glyma.14g212200)的CDS序列为1 320 bp,编码439个氨基酸且该蛋白相对分子质量和等电点分别为48.63×103和8.35。qRT-PCR分析发现GmPUB1在开花后30 d的种子中表达最高。在PEG3350、NaCl、4℃及JA等非生物胁迫诱导下,GmPUB1均上调表达。GmPUB1蛋白在整个细胞内分布。过表达GmPUB1的转基因拟南芥株系的千粒质量显著提高,氨基酸含量发生改变,尤其甲硫氨酸含量显著升高,种子萌发率降低。转基因拟南芥株系具有耐盐性,但对干旱以及ABA敏感。[结论] GmPUB1基因可参与调控种子生长发育并响应逆境胁迫。
Abstract:
[Objectives] In this study,the mechanism of E3 ubiquitin ligase in soybean growth and abiotic stress was explored by cloning the soybean E3 ubiquitin ligase gene GmPUB1 and heterologous expression in Arabidopsis thaliana.[Methods] The E3 ubiquitin ligase gene GmPUB1 was cloned and analyzed by bioinformatics. The expression pattern of the gene under different tissues and different stress treatments was analyzed by qRT-PCR. Subcellular localization analysis of GmPUB1 protein was performed by the onion epidermal cell transient expression system. Heterologous expression of GmPUB1 in A.thaliana and analysis of its phenotype were conducted.[Results] The CDS sequence of GmPUB1 (Glyma.14g212200) was 1 320 bp,encoding 439 amino acids. What’s more,the molecular weight and isoelectric point of the protein were 48.63×103 and 8.35,respectively. qRT-PCR analysis showed that GmPUB1 was expressed with the highest level in seeds 30 days after flowering. GmPUB1 was up-regulated under the induction of PEG3350,NaCl,4℃ and JA abiotic stress. GmPUB1 protein was lacalized into cells. The 1 000-grain weight of the transgenic lines increased significantly,and the amino acid content changed,especially the methionone significantly increased,but the germination rate decreased.Transgenic A.thaliana lines were salt tolerant but sensitive to drought and ABA.[Conclusions] GmPUB1 gene can be involved in regulating seed growth and development and respond to abiotic stress.

参考文献/References:

[1] 宋素胜,谢道昕. 泛素蛋白酶体途径及其对植物生长发育的调控[J]. 植物学通报,2006(5):564-577. Song S S,Xie D X. The ubiquitin-proteosome pathway and plant development[J]. Chinese Bulletin of Botany,2006(5):564-577(in Chinese with English abstract).
[2] 陈默,于丽杰,金晓霞,等. 植物泛素/26S蛋白酶体途径的研究进展[J]. 中国生物工程杂志,2014,34(4):118-126. Chen M,Yu L J,Jin X X,et al. The progress on the ubiquitin/26S proteasome pathway in plants[J]. China Biotechnology,2014,34(4):118-126(in Chinese with English abstract).
[3] Zhou B J,Zeng L R. Conventional and unconventional ubiquitination in plant immunity[J]. Molecular Plant Pathology,2017,18(9):1313-1330.
[4] Qin T,Liu S M,Zhang Z N,et al. GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17[J]. Plant Molecular Biology,2019,99(4/5):379-393.
[5] Sharma B,Joshi D,Yadav P K,et al. Role of ubiquitin-mediated degradation system in plant biology[J]. Frontiers in Plant Science,2016,7:806.
[6] Miller C,Wells R,McKenzie N,et al. Variation in expression of the HECT E3 ligase UPL3 modulates LEC2 levels,seed size and crop yield in Brassica napus[J]. The Plant cell,2019,31:2370-2385.
[7] Ryu M Y,Cho S K,Hong Y,et al. Classification of barley U-box E3 ligases and their expression patterns in response to drought and pathogen stresses[J]. BMC Genomics,2019,20:326.
[8] Park G G,Park J J,Yoon J,et al. A RING finger E3 ligase gene,Oryza sativa Delayed Seed Germination 1(OsDSG1),controls seed germination and stress responses in rice[J]. Plant Molecular Biology,2010,74(4/5):467-478.
[9] Wang W L,Wang W Q,Wu Y Z,et al. The involvement of wheat(Triticum aestivum L.)U-box E3 ubiquitin ligase TaPUB1 in salt stress tolerance[J]. Journal of Integrative Plant Biology,2019,62(5):631-651.
[10] Ohi M D,Vander Kooi C W,Rosenberg J A,et al. Structural insights into the U-box,a domain associated with multi-ubiquitination[J]. Nature Structural Biology,2003,10(4):250-255.
[11] 黄喆,王保云,刘箐,等. 植物U-box蛋白在抗病抗逆反应中的功能研究进展[J/OL]. 基因组学与应用生物学,2019. http://kns.cnki.net/kcms/detail/45.1369.Q.20190610.1154.004.html. Huang Z,Wang B Y,Liu J,et al. Research progress on the function of plant U-box protein in disease resistance and stress resistance[J/OL]. Genomics and Applied Biology(in Chinese with English abstract).
[12] Han P L,Dong Y H,Jiang H,et al. Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance[J]. Journal of Integrative Agriculture,2019,18(7):1604-1612.
[13] Wang N,Liu Y P,Cong Y H,et al. Genome-wide identification of soybean U-box E3 ubiquitin ligases and roles of GmPUB8 in negative regulation of drought stress response in Arabidopsis[J]. Plant and Cell Physiology,2016,57(6):1189-1209.
[14] Zhang M,Zhao J F,Li L,et al. The Arabidopsis U-box E3 ubiquitin ligase PUB30 negatively regulates salt tolerance by facilitating BRI1 kinase inhibitor 1(BKI1)degradation[J]. Plant,Cell & Environment,2017,40(11):2831-2843.
[15] Qin Q,Wang Y X,Huang L Y,et al. A U-box E3 ubiquitin ligase OsPUB67 is positively involved in drought tolerance in rice[J]. Plant Molecular Biology,2020,102(1/2):89-107.
[16] 张沿政,陈龙,李永光,等. 大豆RING/U-box蛋白Glyma.13G115900的克隆及其对非生物胁迫的应答[J]. 大豆科学,2017,36(6):851-856. Zhang Y Z,Chen L,Li Y G,et al. Cloning and expression analysis of a RING/U-box protein of Glyma.13G115900 from soybean under abiotic stress[J]. Soybean Science,2017,36(6):851-856(in Chinese with English abstract).
[17] Shi G X,Huang F,Gong Y,et al. RNA-Seq analysis reveals that multiple phytohormone biosynthesis and signal transduction pathways are reprogrammed in curled-cotyledons mutant of soybean[Glycine max(L.)Merr.] [J]. BMC Genomics,2014,15(1):510.
[18] Seo D H,Ryu M Y,Jammes F,et al. Roles of four Arabidopsis U-box E3 ubiquitin ligases in negative regulation of abscisic acid-mediated drought stress responses[J]. Plant Physiology,2012,160(1):556-568.
[19] Liu Y C,Wu Y R,Huang X H,et al. AtPUB19,a U-box E3 ubiquitin ligase,negatively regulates abscisic acid and drought responses in Arabidopsis thaliana[J]. Molecular Plant,2011,4(6):938-946.
[20] Park J J,Yi J,Yoon J,et al. OsPUB15,an E3 ubiquitin ligase,functions to reduce cellular oxidative stress during seedling establishment[J]. The Plant Journal,2011,65(2):194-205.
[21] Braumann I,Urban W,Preuβ A,et al. Semi-dwarf barley(Hordeum vulgare L.)brh2 and ari-l mutants are deficient in a U-box E3 ubiquitin ligase[J]. Plant Growth Regulation,2018,86(2):223-234.
[22] Li W,Ahn I P,Ning Y S,et al. The U-box/ARM E3 ligase PUB13 regulates cell death,defense,and flowering time in Arabidopsis[J]. Plant Physiology,2012,159(1):239-250.
[23] Zhang Y S,Yin X Y,Yang A F,et al. Stability of inheritance of transgenes in maize(Zea mays L.)lines produced using different transformation methods[J]. Euphytica,2005,144(1/2):11-22.
[24] Bent A. Arabidopsis thaliana floral dip transformation method[J]. Methods in Molecular Biology,2006,343:87-103.

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备注/Memo

备注/Memo:
收稿日期:2019-12-30。
基金项目:国家自然科学基金项目(31871649)
作者简介:宫宇,硕士研究生。
通信作者:黄方,教授,博导,研究方向为大豆遗传育种,E-mail:fhang@njau.edu.cn。
更新日期/Last Update: 1900-01-01