水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

doi:10.3724/SP.J.1006.2013.00816

摘要/Abstract

摘要：

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。

关键词: 水稻, 蛋白二硫键异构酶, 基因沉默, RNAi载体构建, 高温胁迫, 结实特性

Abstract:

To clarify the effect of PDI gene silence on rice yield traits and grain quality, we sub-cloned a 450 bp RNAi fragment from Nipponbare by RT-PCR, and successfully constructed a binary-vector pTCK303-RiOsPDI containing intron hpRNA (ihpRNA), then transformed it into the callus of wild type Nipponbare mediated by EHA105. The T-DNA region for PDI RNA interference in regenerating rice plants was integrated with rice genome via single copy in T₀ generation transgenic plants, and showed a 3:1 genetic mode in T₁ transgenic population, which could be conformed by PCR amplifying analysis and Southern blotting identification. The PCR and qRT-PCR for PDI gene expression in different organs showed that there were much lower level of PDI expression in grains, leaves, stem and sheath for transgenic plants compared with those for wild type Nipponbare, with almost 80% of the dropping in transgenic seeds. The influence of high temperature stress on seed setting traits, panicle agronomic traits and grain quality and also its relation to PDI gene expression in developing grains was further examined with T₁ generation of transgenic plants imposed to two temperature treatments under the controlled temperature chambers, the results indicated that PDI silence transgenic plants had a remarkable lower seed setting rate, somewhat lower grain weight compared with its wild phenotype under the high temperature treatment in despite of no obviously varying with its wild phenotype under normal developmental condition, suggesting that PDI gene should be probably responsible for rice tolerance to high temperature stress. Moreover, there were lower translucence and higher chalky degree for transgenic plants than for Nipponbare, although no significant difference were observed in grain total protein and amylose content between PDI silence transgenic plant and its CK.

Key words: Rice (Oryza sativa L.), Protein disulfide isomerase (PDI), Gene silence, RNA interference, High temperature stress, Seed setting characteristics

刘光快,曹珍珍,韦克苏,潘刚,苏达,张春娇,程方民. 水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析[J]. 作物学报, 2013, 39(05): 816-826.

IU Guang-Kuai,CAO Zhen-Zhen,WEI Ke-Su,PAN Gang,SU Da,ZHANG Chun-Jiao,CHENG Fang-Min. RNAi Vector Construction for Protein Disulfide Isomerase Gene and Seed Setting Characteristics in Offspring of Transgenic Rice under High Temperature Treatment[J]. Acta Agron Sin, 2013, 39(05): 816-826.

参考文献

[1]Noiva R, Lennarz W J. Protein disulfide isomerase: a multifunctional protein resident in the lumen of the endoplasmic reticulum. J Biol Chem, 1992, 267: 3553−3556

[2]Wilkinson B, Gilbert H F. Protein disulfide isomerase. Biochim Biophys Acta, 2004, 1699: 35−44

[3]Urade R. Cellular response to unfolded proteins in the endoplasmic reticulum of plants. FEBS J, 2007, 274: 1152–1171

[4]Kaufman R J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev, 1999, 13: 1211–1233

[5]Tian G, Xiang S, Noiva R, Lennarz W J, Schindelin H. The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell, 2006, 124: 61–73

[6]Puig A, Gilbert H F. Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme. J Biol Chem, 1994, 269: 7764−7771

[7]Lebeche D, Lucero H A, Kaminer B. Calcium binding properties of rabbit liver protein disulfide isomerase. Biochem Biophys Res Commun, 1994, 202: 556−561

[8]Wetterau J R, Combs K A, Spinner S N, Joiner B J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex. J Biol Chem, 1990, 265: 9800−9807

[9]Gilbert H F. Protein chaperones and protein folding. Curr Opin Biotechnol, 1994, 5: 534−540

[10]Wadahama H, Kamauchi S, Ishimoto M, Kawada T, Urade R. Protein disulfide isomerase family proteins involved in soybean protein biogenesis. FEBS J, 2007, 274: 687−703

[11]Edman J C, Ellis L, Blacher R W. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature, 1985, 317: 267−270

[12]Yamauchi K, Lmabert N, Fredman R B. Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulfide isomerase. Biochem. and Biophys Res Commun, 1987, 146: 1485−1490

[13]Walker K W, Gilbert H F. Scanning and escape during protein-disulfide isomerase-assisted protein folding. J Biol Chem, 1997, 272: 8845–8848

[14]Van Lith M, Karala A R, Bown D, Gatehouse J A, Ruddock L W, Saunders P T K, Benham A M. A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells. Mol Biol Cell, 2007, 18: 2795–2804

[15]Xu Z J, Ueda K, Kiyoshi M, Ono M, Inoue M. Molecular characterization of a novel protein disulfide isomerase in carrot. Gene, 2002, 284: 225–231

[16]Cao Y-Y(曹云英), Duan H(段骅), Yang L-N(杨立年), Wang Z-Q(王志琴), Liu L-J(刘立军), Yang J-C(杨建昌). Effect of high temperature during heading and early grain filling on grain yield of indica rice cultivars differing in heat-tolerance and its physiological mechanism. Acta Agron Sin (作物学报), 2009, 35(3): 512−521 (in Chinese with English abstract)

[17]Wei K-S(韦克苏), Cheng F-M(程方民), Dong H-T(董海涛), Zhang Q-F(张其芳), Liu K-G(刘奎刚), Cao Z-Z(曹珍珍). Microarray analysis of gene expression profile to grain storage metabolism in rice endosperm as affected by high temperature at filling stage. Sci Agric Sin (中国农业科学), 2010, 43(1): 1−11 (in Chinese with English abstract)

[18]Yasuda H, Hirose S, Kawakatsu T, Wakasa Y, Takaiwa F. Overexpression of BiP has inhibitory effects on the accumulation of seed storage proteins in endosperm cells of rice. Plant Cell Physiol, 2009, 50: 1532–1543

[19]Wei K-S(韦克苏), Zhang Q-F(张其芳), Cheng F-M(程方民), Chen N(陈能), Xie L-H(谢黎红). Expression profiles of rice soluble starch synthase (SSS) genes in response to high temperature stress at filling stage. Acta Agron Sin (作物学报), 2009, 35(1): 18−24 (in Chinese with English abstract)

[20]Zakaria S, Mastuda T, Tajima S, Nitta Y. Effect of high temperature at ripening stage on the reserve accumulation in seed in some rice cultivars. Plant Prod Sci, 2002, 5: 160−168

[21]Auer C, Frederick R. Crop improvement using small RNAs: applications and predictive ecological risk assessments. Trends Biotechnol, 2009, 27: 644−651

[22]Hamilton A J, Baulcombe D C. A novel species of small antisense RNA in post-transcripional gene silencing. Science, 1999, 286: 950−952

[23]Boston R S, Viitanen P V, Vierling E. Molecular chaperones and protein folding in plants. Plant Mol Biol, 1996, 32: 191−222

[24]Ko H S, Uehara T, Nomur Y. Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death. J Biol Chem, 2002, 277: 35386−35392

[25]Yang K Z, Xia C, Liu X L, Dou X Y, Wang W, Chen L Q, Zhang X Q, Xie L F, He L Y, Ma X, Ye D. A mutation in THERMOSENSITIVE MALE STERILE 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis. Plant J, 2009, 57: 870–882

[26]Fatima C A, Sonia M B, Carlos M, Wagner C O, Elizabeth P B F. Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiol, 2001, 126:1042–1054

[27]Wakasa Y, Yasuda H, Ono Y, Kawakatsu T, Hirose S, Takahashi H, Hayashi S, Yang L J, Takaiwa F. Expression of ER quality control-related genes in response to changes in BiP1 levels in developing rice endosperm. Plant J, 2011, 65: 675–689

[28]Houston N L, Fan C, Xiang Q Y, Schulze J M, Jung R, Boston R S. Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins. Plant Physiol, 2005, 137: 762–778

[29]Onda Y, Kumamaru T, Kawagoe Y. ER membrane localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm. Proc Natl Acad Sci USA, 2009, 106: 14156−14161

[30]Koh A, Nishimura K, Urade R. Relationship between endogenous protein disulfide isomerase family proteins and glutenin macropolymer. J Agric Food Chem, 2010, 58: 12970–12975

[31]Li C P, Larkins B A. Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant. Plant Mol Biol, 1996, 30: 873−882

[32]Takemoto Y, Coughlan S J, Okita T W, Satoh H, Ogawa M, Kumamaru T. The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase. Plant Physiol, 2002, 128: 1212−1222

[33]Muench D G, Wu Y, Zhang Y, Li X, Boston R S, Okita T W. Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue. Plant Cell Physiol, 1997, 38: 404–412

[34]Satoh-Cruz M, Crofts A J, Takemoto-Kuno Y, Sugino A, Washida H, Crofts N, Okita T W, Ogawa M, Satoh H, Kumamaru T. Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm. Plant Cell Physiol, 2010, 51: 1581–1593

[35]Majoul I, Ferrari D, Söling H D. Reduction of protein disulfide bonds in an oxidizing environment the disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum. FEBS Lett, 1997, 401: 104−108

[36]Han X H, Wang Y H, Jiang L, Ren Y L, Liu F, Peng C, Li J J, Jin X M, Wu F Q, Wang J L, Guo X P, Zhang X, Cheng Z J, Wan J M. The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice. J Exp Bot, 2012, 63: 121–130

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