植物表达外源蛋白研究进展及展望

doi:10.13560/j.cnki.biotech.bull.1985.2023-0662

摘要/Abstract

摘要：

植物为宿主表达外源蛋白的系统称之为分子农场，通过农杆菌将外源基因导入植物进行表达具有高效、安全、廉价的优点，特别是植物具备一系列翻译后修饰功能，因此该系统能弥补原核表达系统的缺陷。本综述首先介绍了近些年在烟草叶片瞬时表达和水稻胚乳组织特异性表达上取得的进展，特别是一些利用分子农场进行医用蛋白表达、药物合成、疫苗制备等典型案例。在优化生物反应器、提高表达效率策略上，本综述重点探讨了蛋白翻译后水平上的调控，包括蛋白酶抑制剂的作用、糖基化修饰环节以及分子伴侣共表达等对外源蛋白表达的影响。最后，围绕外源蛋白大量囤积于内质网可能引发内质网胁迫的问题，展望了通过优化内质网环境来提高外源蛋白表达效率的可行性。

关键词: 植物生物反应器, 分子农场, 叶片瞬时表达, 水稻种子反应器, 内质网优化

Abstract:

The system using plants as hosts to express foreign proteins is called molecular farming. The production of foreign proteins in plants via Agrobacterium tumefaciens-mediated transformation has the advantages of high efficiency, safety and low cost. Especially, protein post-translational modification in plants can make up for the defects of the prokaryotic expression system. In this review, we firstly introduce advances in tobacco leaf transient expression or rice endosperm specific expression, especially some typical examples of producing pharmaceutical proteins, medical compounds, and vaccines in molecular farming. In terms of optimizing bioreactors and improving expression efficiency, we then focus the regulation in protein post-translation, including the role of protease inhibitors, and the effects of glycosylation modification processes, and molecular chaperones co-expression on the expressions of foreign proteins. Finally, based on the foreign proteins accumulation in the endoplasmic reticulum（ER）would induce stress to ER, we propose the feasibility of increasing expression efficiency of foreign proteins by optimizing the ER environment.

Key words: plant bioreactor, molecular farming, transient expression in tobacco leaves, rice endosperm bioreactors, ER environment optimization

蒋铭轩, 李康, 罗亮, 刘建祥, 芦海平. 植物表达外源蛋白研究进展及展望[J]. 生物技术通报, 2023, 39(11): 110-122.

JIANG Min-xuan, LI Kang, LUO Liang, LIU Jian-xiang, LU Hai-ping. Advances on the Expressions of Foreign Proteins in Plants[J]. Biotechnology Bulletin, 2023, 39(11): 110-122.

图/表 4

参考文献 111

[1]	Fraley RT, Rogers SG, Horsch RB, et al. Expression of bacterial genes in plant cells[J]. Proc Natl Acad Sci USA, 1983, 80(15): 4803-4807. doi: 10.1073/pnas.80.15.4803 pmid: 6308651
[2]	Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants[J]. Nature, 1989, 342(6245): 76-78. doi: 10.1038/342076a0
[3]	Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones[J]. Plant Biotechnol J, 2015, 13(8): 1121-1135. doi: 10.1111/pbi.2015.13.issue-8 URL
[4]	Buyel JF. Plant molecular farming - integration and exploitation of side streams to achieve sustainable biomanufacturing[J]. Front Plant Sci, 2019, 9: 1893. doi: 10.3389/fpls.2018.01893 URL
[5]	Schöb H, Kunz C, Meins F Jr. Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing[J]. Mol Gen Genet, 1997, 256(5): 581-585. doi: 10.1007/s004380050604 URL
[6]	Kapila J, De Rycke R, Van Montagu M, et al. An Agrobacterium-mediated transient gene expression system for intact leaves[J]. Plant Sci, 1997, 122(1): 101-108. doi: 10.1016/S0168-9452(96)04541-4 URL
[7]	Sainsbury F, Lomonossoff GP. Transient expressions of synthetic biology in plants[J]. Curr Opin Plant Biol, 2014, 19(100): 1-7. doi: 10.1016/j.pbi.2014.02.003 URL
[8]	Holtz BR, Berquist BR, Bennett LD, et al. Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals[J]. Plant Biotechnol J, 2015, 13(8): 1180-1190. doi: 10.1111/pbi.12469 pmid: 26387511
[9]	Marsian J, Lomonossoff GP. Molecular pharming - VLPs made in plants[J]. Curr Opin Biotechnol, 2016, 37: 201-206. doi: 10.1016/j.copbio.2015.12.007 URL
[10]	Fausther-Bovendo H, Kobinger G. Plant-made vaccines and therapeutics[J]. Science, 2021, 373(6556): 740-741. doi: 10.1126/science.abf5375 pmid: 34385382
[11]	Sainsbury F. Innovation in plant-based transient protein expression for infectious disease prevention and preparedness[J]. Curr Opin Biotechnol, 2020, 61: 110-115. doi: 10.1016/j.copbio.2019.11.002 URL
[12]	Miettinen K, Dong LM, Navrot N, et al. The seco-iridoid pathway from Catharanthus roseus[J]. Nat Commun, 2014, 5: 3606. doi: 10.1038/ncomms4606 pmid: 24710322
[13]	Lau W, Sattely ES. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone[J]. Science, 2015, 349(6253): 1224-1228. doi: 10.1126/science.aac7202 pmid: 26359402
[14]	Rajniak J, Barco B, Clay NK, et al. A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defence[J]. Nature, 2015, 525(7569): 376-379. doi: 10.1038/nature14907
[15]	Polturak G, Breitel D, Grossman N, et al. Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants[J]. New Phytol, 2016, 210(1): 269-283. doi: 10.1111/nph.13796 pmid: 26683006
[16]	Andersen-Ranberg J, Kongstad KT, Nafisi M, et al. Synthesis of C-glucosylated octaketide anthraquinones in Nicotiana benthamiana by using a multispecies-based biosynthetic pathway[J]. Chembiochem, 2017, 18(19): 1893-1897. doi: 10.1002/cbic.201700331 pmid: 28719729
[17]	Reed J, Osbourn A. Engineering terpenoid production through transient expression in Nicotiana benthamiana[J]. Plant Cell Rep, 2018, 37(10): 1431-1441. doi: 10.1007/s00299-018-2296-3
[18]	Mechtcheriakova IA, Eldarov MA, Nicholson L, et al. The use of viral vectors to produce hepatitis B virus core particles in plants[J]. J Virol Methods, 2006, 131(1): 10-15. pmid: 16112207
[19]	Peyret H, Gehin A, Thuenemann EC, et al. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins[J]. PLoS One, 2015, 10(4): e0120751. doi: 10.1371/journal.pone.0120751 URL
[20]	Matić S, Masenga V, Poli A, et al. Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods[J]. Plant Biotechnol J, 2012, 10(4): 410-421. doi: 10.1111/j.1467-7652.2011.00671.x pmid: 22260326
[21]	Love AJ, Chapman SN, Matic S, et al. In planta production of a candidate vaccine against bovine papillomavirus type 1[J]. Planta, 2012, 236(4): 1305-1313. doi: 10.1007/s00425-012-1692-0 pmid: 22718313
[22]	Thuenemann EC, Meyers AE, Verwey J, et al. A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles[J]. Plant Biotechnol J, 2013, 11(7): 839-846. doi: 10.1111/pbi.12076 pmid: 23647743
[23]	Mason HS, Ball JM, Shi JJ, et al. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice[J]. Proc Natl Acad Sci USA, 1996, 93(11): 5335-5340. doi: 10.1073/pnas.93.11.5335 pmid: 8643575
[24]	Mathew LG, Herbst-Kralovetz MM, Mason HS. Norovirus Narita 104 virus-like particles expressed in Nicotiana benthamiana induce serum and mucosal immune responses[J]. Biomed Res Int, 2014, 2014: 807539.
[25]	D'Aoust MA, Lavoie PO, Couture MMJ, et al. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice[J]. Plant Biotechnol J, 2008, 6(9): 930-940. doi: 10.1111/pbi.2008.6.issue-9 URL
[26]	Landry N, Ward BJ, Trépanier S, et al. Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza[J]. PLoS One, 2010, 5(12): e15559. doi: 10.1371/journal.pone.0015559 URL
[27]	He Y, Ning TT, Xie TT, et al. Large-scale production of functional human serum albumin from transgenic rice seeds[J]. Proc Natl Acad Sci USA, 2011, 108(47): 19078-19083. doi: 10.1073/pnas.1109736108 pmid: 22042856
[28]	Zhang LP, Shi JN, Jiang DM, et al. Expression and characterization of recombinant human alpha-antitrypsin in transgenic rice seed[J]. J Biotechnol, 2012, 164(2): 300-308. doi: 10.1016/j.jbiotec.2013.01.008 pmid: 23376844
[29]	An N, Ou JQ, Jiang DM, et al. Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds[J]. Int J Mol Sci, 2013, 14(2): 3556-3567. doi: 10.3390/ijms14023556 pmid: 23434658
[30]	Takaiwa F, Yang LJ, Takagi H, et al. Development of rice-seed-based oral allergy vaccines containing hypoallergenic Japanese cedar pollen allergen derivatives for immunotherapy[J]. J Agric Food Chem, 2019, 67(47): 13127-13138. doi: 10.1021/acs.jafc.9b05421 URL
[31]	Saito S, Takagi H, Wakasa Y, et al. Safety and efficacy of rice seed-based oral allergy vaccine for Japanese cedar pollinosis in Japanese monkeys[J]. Mol Immunol, 2020, 125: 63-69. doi: S0161-5890(20)30397-7 pmid: 32650161
[32]	Nahampun HN, Bosworth B, Cunnick J, et al. Expression of H3N2 nucleoprotein in maize seeds and immunogenicity in mice[J]. Plant Cell Rep, 2015, 34(6): 969-980. doi: 10.1007/s00299-015-1758-0 pmid: 25677970
[33]	Shi Y, Habibi P, Haq ANU, et al. Seed-based system for cost-effective production of vaccine against chronic respiratory disease in chickens[J]. Mol Biotechnol, 2023, 65(4): 570-580.
[34]	Mirzaee M, Holásková E, Mičúchová A, et al. Long-lasting stable expression of human LL-37 antimicrobial peptide in transgenic barley plants[J]. Antibiotics, 2021, 10(8): 898. doi: 10.3390/antibiotics10080898 URL
[35]	Russell DA, Spatola LA, Dian T, et al. Host limits to accurate human growth hormone production in multiple plant systems[J]. Biotechnol Bioeng, 2005, 89(7): 775-782. pmid: 15696512
[36]	Perrin Y, Vaquero C, Gerrard I, et al. Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment(scFV)antibody used in cancer diagnosis and therapy[J]. Mol Breed, 2000, 6(4): 345-352. doi: 10.1023/A:1009657701588 URL
[37]	Marcondes J, Hansen E. Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg[J]. Braz J Infect Dis, 2008, 12(6): 469-471. doi: S1413-86702008000600004 pmid: 19287831
[38]	Matsui T, Takita E, Sato T, et al. Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease[J]. Transgenic Res, 2011, 20(4): 735-748. doi: 10.1007/s11248-010-9455-9 URL
[39]	Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, et al. Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague[J]. Planta, 2010, 232(2): 409-416. doi: 10.1007/s00425-010-1176-z pmid: 20461403
[40]	Kim YS, Kim BG, Kim TG, et al. Expression of a cholera toxin B subunit in transgenic lettuce(Lactucasativa L.) using Agrobac-terium-mediated transformation system[J]. Plant Cell Tissue Organ Cult, 2006, 87(2): 203-210. doi: 10.1007/s11240-006-9156-5 URL
[41]	Li HY, Ramalingam S, Chye ML. Accumulation of recombinant SARS-CoV spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines[J]. Exp Biol Med, 2006, 231(8): 1346-1352. doi: 10.1177/153537020623100808 URL
[42]	Huy NX, Yang MS, Kim TG. Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce(Lactuca sativa L.)[J]. Mol Biotechnol, 2011, 48(3): 201-209. doi: 10.1007/s12033-010-9359-1 URL
[43]	Tremblay R, Feng M, Menassa R, et al. High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems[J]. Transgenic Res, 2011, 20(2): 345-356. doi: 10.1007/s11248-010-9419-0 pmid: 20559869
[44]	Chong DK, Langridge WH. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants[J]. Transgenic Res, 2000, 9(1): 71-78. pmid: 10853271
[45]	Luchakivskaya Y, Kishchenko O, Gerasymenko I, et al. High-level expression of human interferon alpha-2b in transgenic carrot(Daucus carota L.) plants[J]. Plant Cell Rep, 2011, 30(3): 407-415. doi: 10.1007/s00299-010-0942-5 pmid: 21046110
[46]	Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, et al. Transgenic carrot tap roots expressing an immunogenic F1-V fusion protein from Yersinia pestis are immunogenic in mice[J]. J Plant Physiol, 2011, 168(2): 174-180.
[47]	Sun QY, Ding LW, Lomonossoff GP, et al. Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture[J]. J Biotechnol, 2011, 155(2): 164-172. doi: 10.1016/j.jbiotec.2011.06.033 URL
[48]	Qiu XG, Wong G, Audet J, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp[J]. Nature, 2014, 514(7520): 47-53. doi: 10.1038/nature13777
[49]	Mor TS. Molecular pharming's foot in the FDA's door: Protalix's trailblazing story[J]. Biotechnol Lett, 2015, 37(11): 2147-2150. doi: 10.1007/s10529-015-1908-z pmid: 26149580
[50]	Zhu QL, Tan JT, Liu YG. Molecular farming using transgenic rice endosperm[J]. Trends Biotechnol, 2022, 40(10): 1248-1260. doi: 10.1016/j.tibtech.2022.04.002 pmid: 35562237
[51]	Ou JQ, Guo ZB, Shi JN, et al. Transgenic rice endosperm as a bioreactor for molecular pharming[J]. Plant Cell Rep, 2014, 33(4): 585-594. doi: 10.1007/s00299-013-1559-2 pmid: 24413763
[52]	Wakasa Y, Takagi H, Hirose S, et al. Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis[J]. Plant Biotechnol J, 2013, 11(1): 66-76. doi: 10.1111/pbi.12007 pmid: 23066780
[53]	Liu WX, Liu HL, Qu LQ. Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds[J]. Theor Appl Genet, 2013, 126(9): 2289-2297. doi: 10.1007/s00122-013-2135-4 pmid: 23748707
[54]	Yin ZJ, Liu HL, Dong XB, et al. Increasing α-linolenic acid content in rice bran by embryo-specific expression of ω3/Δ15-desaturase gene[J]. Mol Breed, 2014, 33(4): 987-996. doi: 10.1007/s11032-013-0014-y URL
[55]	Wang XT, Karki U, Abeygunaratne H, et al. Plant cell-secreted stem cell factor stimulates expansion and differentiation of hematopoietic stem cells[J]. Process Biochem, 2021, 100: 39-48. doi: 10.1016/j.procbio.2020.09.029 pmid: 33071562
[56]	Diamos AG, Mason HS. Chimeric 3' flanking regions strongly enhance gene expression in plants[J]. Plant Biotechnol J, 2018, 16(12): 1971-1982. doi: 10.1111/pbi.12931 pmid: 29637682
[57]	Rosenthal SH, Diamos AG, Mason HS. An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves[J]. Plant Mol Biol, 2018, 96(4-5): 429-443. doi: 10.1007/s11103-018-0708-y pmid: 29429129
[58]	Peyret H, Brown JKM, Lomonossoff GP. Improving plant transient expression through the rational design of synthetic 5' and 3' untranslated regions[J]. Plant Methods, 2019, 15: 108. doi: 10.1186/s13007-019-0494-9 pmid: 31548848
[59]	Yanez RJR, Lamprecht R, Granadillo M, et al. LALF_32-51-E7, a HPV-16 therapeutic vaccine candidate, forms protein body-like structures when expressed in Nicotiana benthamiana leaves[J]. Plant Biotechnol J, 2018, 16(2): 628-637. doi: 10.1111/pbi.2018.16.issue-2 URL
[60]	Thomas DR, Walmsley AM. Improved expression of recombinant plant-made hEGF[J]. Plant Cell Rep, 2014, 33(11): 1801-1814. doi: 10.1007/s00299-014-1658-8 pmid: 25048022
[61]	Jutras PV, Dodds I, van der Hoorn RA. Proteases of Nicotiana benthamiana: an emerging battle for molecular farming[J]. Curr Opin Biotechnol, 2020, 61: 60-65. doi: 10.1016/j.copbio.2019.10.006 URL
[62]	Grosse-Holz F, Kelly S, Blaskowski S, et al. The transcriptome, extracellular proteome and active secretome of agroinfiltrated Nicotiana benthamiana uncover a large, diverse protease repertoire[J]. Plant Biotechnol J, 2018, 16(5): 1068-1084. doi: 10.1111/pbi.12852 pmid: 29055088
[63]	Robert S, Jutras PV, Khalf M, et al. Companion protease inhibitors for the in situ protection of recombinant proteins in plants[J]. Methods Mol Biol, 2016, 1385: 115-126.
[64]	Jutras PV, Marusic C, Lonoce C, et al. An accessory protease inhibitor to increase the yield and quality of a tumour-targeting MAb in Nicotiana benthamiana leaves[J]. PLoS One, 2016, 11(11): e0167086. doi: 10.1371/journal.pone.0167086 URL
[65]	Jutras PV, Grosse-Holz F, Kaschani F, et al. Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana[J]. Plant Biotechnol J, 2019, 17(8): 1670-1678. doi: 10.1111/pbi.2019.17.issue-8 URL
[66]	Sainsbury F, Jutras PV, Vorster J, et al. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants[J]. Front Plant Sci, 2016, 7: 141. doi: 10.3389/fpls.2016.00141 pmid: 26913045
[67]	Grosse-Holz F, Madeira L, Zahid MA, et al. Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana[J]. Plant Biotechnol J, 2018, 16(10): 1797-1810. doi: 10.1111/pbi.12916 pmid: 29509983
[68]	Hatsugai N, Kuroyanagi M, Yamada K, et al. A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death[J]. Science, 2004, 305(5685): 855-858. doi: 10.1126/science.1099859 pmid: 15297671
[69]	Zauner FB, Dall E, Regl C, et al. Crystal structure of plant legumain reveals a unique two-chain state with pH-dependent activity regulation[J]. Plant Cell, 2018, 30(3): 686-699. doi: 10.1105/tpc.17.00963 URL
[70]	Gu C, Shabab M, Strasser R, et al. Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana[J]. PLoS One, 2012, 7(3): e32422. doi: 10.1371/journal.pone.0032422 URL
[71]	Jutras PV, D’Aoust MA, Couture MMJ, et al. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants[J]. Biotechnol J, 2015, 10(9): 1478-1486. doi: 10.1002/biot.201500056 pmid: 25914077
[72]	Jutras PV, Goulet MC, Lavoie PO, et al. Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent[J]. Plant Biotechnol J, 2018, 16(11): 1928-1938. doi: 10.1111/pbi.12928 pmid: 29618167
[73]	Mandal MK, Ahvari H, Schillberg S, et al. Tackling unwanted proteolysis in plant production hosts used for molecular farming[J]. Front Plant Sci, 2016, 7: 267. doi: 10.3389/fpls.2016.00267 pmid: 27014293
[74]	Mandal MK, Fischer R, Schillberg S, et al. Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2(BY-2)suspension cells[J]. Biotechnol J, 2014, 9(8): 1065-1073. doi: 10.1002/biot.201300424 pmid: 24828029
[75]	Duwadi K, Chen L, Menassa R, et al. Identification, characterization and down-regulation of cysteine protease genes in tobacco for use in recombinant protein production[J]. PLoS One, 2015, 10(7): e0130556. doi: 10.1371/journal.pone.0130556 URL
[76]	Kallolimath S, Castilho A, Strasser R, et al. Engineering of complex protein sialylation in plants[J]. Proc Natl Acad Sci USA, 2016, 113(34): 9498-9503. doi: 10.1073/pnas.1604371113 pmid: 27444013
[77]	Jeong IS, Lee SM, Bonkhofer F, et al. Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies[J]. Plant J, 2018, 94(1): 131-145. doi: 10.1111/tpj.2018.94.issue-1 URL
[78]	Strasser R. Plant protein glycosylation[J]. Glycobiology, 2016, 26(9): 926-939. pmid: 26911286
[79]	Castilho A, Beihammer G, Pfeiffer C, et al. An oligosaccharyltransferase from Leishmania major increases the N-glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana[J]. Plant Biotechnol J, 2018, 16(10): 1700-1709. doi: 10.1111/pbi.2018.16.issue-10 URL
[80]	Strasser R, Stadlmann J, Schähs M, et al. Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure[J]. Plant Biotechnol J, 2008, 6(4): 392-402. doi: 10.1111/j.1467-7652.2008.00330.x pmid: 18346095
[81]	Jansing J, Sack M, Augustine SM, et al. CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1, 2-xylose and core α-1, 3-fucose[J]. Plant Biotechnol J, 2019, 17(2): 350-361. doi: 10.1111/pbi.12981 pmid: 29969180
[82]	Parsons J, Altmann F, Arrenberg CK, et al. Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants[J]. Plant Biotechnol J, 2012, 10(7): 851-861. doi: 10.1111/j.1467-7652.2012.00704.x pmid: 22621344
[83]	Shin YJ, Castilho A, Dicker M, et al. Reduced paucimannosidic N-glycan formation by suppression of a specific β-hexosaminidase from Nicotiana benthamiana[J]. Plant Biotechnol J, 2017, 15(2): 197-206. doi: 10.1111/pbi.2017.15.issue-2 URL
[84]	Liu JX, Howell SH. Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants[J]. Plant Cell, 2010, 22(9): 2930-2942. doi: 10.1105/tpc.110.078154 URL
[85]	杨正婷, 刘建祥. 植物内质网胁迫应答研究进展[J]. 生物技术通报, 2016, 32(10): 84-96. doi: 10.13560/j.cnki.biotech.bull.1985.2016.10.005
	Yang ZT, Liu JX. Endoplasmic reticulum stress response in plants[J]. Biotechnol Bull, 2016, 32(10): 84-96.
[86]	Sun JL, Li JY, Wang MJ, et al. Protein quality control in plant organelles: current progress and future perspectives[J]. Mol Plant, 2021, 14(1): 95-114. doi: 10.1016/j.molp.2020.10.011 URL
[87]	Balchin D, Hayer-Hartl M, Hartl FU. In vivo aspects of protein folding and quality control[J]. Science, 2016, 353(6294): aac4354. doi: 10.1126/science.aac4354 URL
[88]	De Wilde K, De Buck S, Vanneste K, et al. Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response[J]. Plant Physiol, 2013, 161(2): 1021-1033. doi: 10.1104/pp.112.209718 URL
[89]	Nuttall J, Vine N, Hadlington JL, et al. ER-resident chaperone interactions with recombinant antibodies in transgenic plants[J]. Eur J Biochem, 2002, 269(24): 6042-6051. doi: 10.1046/j.1432-1033.2002.03302.x pmid: 12473100
[90]	Zhang LP, Jiang DM, Pang JL, et al. The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death[J]. Plant Mol Biol, 2013, 83(1-2): 153-161. doi: 10.1007/s11103-013-0056-x pmid: 23564402
[91]	Wakasa Y, Yasuda H, Takaiwa F. Secretory type of recombinant thioredoxin h induces ER stress in endosperm cells of transgenic rice[J]. J Plant Physiol, 2013, 170(2): 202-210.
[92]	Klabunde J, Kleebank S, Piontek M, et al. Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha[J]. FEMS Yeast Res, 2007, 7(7): 1168-1180. pmid: 17617219
[93]	Song Y, Sata J, Saito A, et al. Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes[J]. J Biochem, 2001, 130(6): 757-764. doi: 10.1093/oxfordjournals.jbchem.a003046 pmid: 11726275
[94]	Robinson AS, Hines V, Wittrup KD. Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae[J]. Biotechnology(N Y), 1994, 12(4): 381-384.
[95]	Vad R, Nafstad E, Dahl LA, et al. Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone[J]. J Biotechnol, 2005, 116(3): 251-260. doi: 10.1016/j.jbiotec.2004.12.004 URL
[96]	Powers SL, Robinson AS. PDI improves secretion of redox-inactive beta-glucosidase[J]. Biotechnol Prog, 2007, 23(2): 364-369. doi: 10.1021/bp060287p URL
[97]	Damasceno LM, Anderson KA, Ritter G, et al. Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2007, 74(2): 381-389. doi: 10.1007/s00253-006-0652-7 pmid: 17051412
[98]	Butz JA, Niebauer RT, Robinson AS. Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast[J]. Biotechnol Bioeng, 2003, 84(3): 292-304. pmid: 12968283
[99]	Wakasa Y, Hayashi S, Takaiwa F. Expression of OsBiP₄ and OsBiP₅ is highly correlated with the endoplasmic reticulum stress response in rice[J]. Planta, 2012, 236(5): 1519-1527. doi: 10.1007/s00425-012-1714-y pmid: 22824965
[100]	Liu JX, Srivastava R, Che P, et al. An endoplasmic reticulum stress response in Arabidopsis is mediated by proteolytic processing and nuclear relocation of a membrane-associated transcription factor, bZIP28[J]. Plant Cell, 2007, 19(12): 4111-4119. doi: 10.1105/tpc.106.050021 URL
[101]	Deng Y, Humbert S, Liu JX, et al. Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis[J]. Proc Natl Acad Sci USA, 2011, 108(17): 7247-7252. doi: 10.1073/pnas.1102117108 pmid: 21482766
[102]	Nagashima Y, Mishiba KI, Suzuki E, et al. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor[J]. Sci Rep, 2011, 1: 29. doi: 10.1038/srep00029 pmid: 22355548
[103]	Hayashi S, Wakasa Y, Takahashi H, et al. Signal transduction by IRE1-mediated splicing of bZIP50 and other stress sensors in the endoplasmic reticulum stress response of rice[J]. Plant J, 2012, 69(6): 946-956. doi: 10.1111/tpj.2012.69.issue-6 URL
[104]	Lu SJ, Yang ZT, Sun L, et al. Conservation of IRE1-regulated bZIP74 mRNA unconventional splicing in rice(Oryza sativa L.) involved in ER stress responses[J]. Mol Plant, 2012, 5(2): 504-514. doi: 10.1093/mp/ssr115 pmid: 22199238
[105]	Srivastava R, Chen YN, Deng Y, et al. Elements proximal to and within the transmembrane domain mediate the organelle-to-organelle movement of bZIP28 under ER stress conditions[J]. Plant J, 2012, 70(6): 1033-1042. doi: 10.1111/tpj.2012.70.issue-6 URL
[106]	Sun L, Lu SJ, Zhang SS, et al. The lumen-facing domain is important for the biological function and organelle-to-organelle movement of bZIP28 during ER stress in Arabidopsis[J]. Mol Plant, 2013, 6(5): 1605-1615. doi: 10.1093/mp/sst059 pmid: 23558471
[107]	Humbert S, Zhong SH, Deng Y, et al. Alteration of the bZIP60/IRE1 pathway affects plant response to ER stress in Arabidopsis thaliana[J]. PLoS One, 2012, 7(6): e39023. doi: 10.1371/journal.pone.0039023 URL
[108]	Valkonen M, Ward M, Wang HM, et al. Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response[J]. Appl Environ Microbiol, 2003, 69(12): 6979-6986. doi: 10.1128/AEM.69.12.6979-6986.2003 URL
[109]	Valkonen M, Penttilä M, Saloheimo M. Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae[J]. Appl Environ Microbiol, 2003, 69(4): 2065-2072. doi: 10.1128/AEM.69.4.2065-2072.2003 URL
[110]	Cain K, Peters S, Hailu HN, et al. A CHO cell line engineered to express XBP1 and ERO1-Lα has increased levels of transient protein expression[J]. Biotechnol Prog, 2013, 29(3): 697-706. doi: 10.1002/btpr.v29.3 URL
[111]	Tigges M, Fussenegger M. Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells[J]. Metab Eng, 2006, 8(3): 264-272. pmid: 16635796

生物反应器Bioreactor	外源蛋白Foreign protein	载体Vector	表达宿主Host	参考文献Reference
烟草叶片生物反应器 Tobacco leaf bioreactor	乙型肝炎核心抗原Hepatitis B core antigen	pEAQ-HT	烟草叶片Tobacco leaf	[18-19]
	人乳头瘤病毒-8抗原 Human papillomaviruse-8 antigen	pEAQ-HT	烟草叶片Tobacco leaf	[20]
	牛乳头瘤病毒-1抗原 Bovine papillomaviruse-1 antigen	pEAQ-HT-DEST	烟草叶片Tobacco leaf	[21]
	蓝舌病毒-8抗原Bluetongue virus antigen	pEAQ-HT	烟草叶片Tobacco leaf	[22]
	诺如病毒Norwalk抗原 Norwalk virus antigen	pBI201	烟草叶片Tobacco leaf	[23]
	诺如病毒Narita抗原Narita virus antigen	pICH10990	烟草叶片Tobacco leaf	[24]
	流感病毒H1N1抗原Influenza virus H5N1 antigen	pCAMBIA2300	烟草叶片Tobacco leaf	[25]
	流感病毒H5N1抗原Influenza virus H5N1 antigen	pCAMBIA2300	烟草叶片Tobacco leaf	[26]
	新型冠状病毒抗原COVID Covifenz^®	Unknown	烟草叶片Tobacco leaf	Medicago, Canada
种子生物反应器 Seed bioreactor	人血清白蛋白Human serum albumin	pOsPMP	水稻种子Rice seed	[27]
	人抗胰蛋白酶Human alpha-antitrypsin	pOsPMP	水稻种子Rice seed	[28]
	碱性成纤维细胞生长因子Basic fibroblast growth factor	pOsPMP	水稻种子Rice seed	[29]
	雪松花粉敏感蛋白CRYJ1/2 Cryptomeria japonica 1/2	pCSPmALSAg7-GW	水稻种子Rice seed	[30-31]
	流感病毒H3N2抗原Influenza virus H3N2 antigen	pHN05	玉米种子Maize seed	[32]
	TM-1疫苗 TM-1 gene of Mycoplasma gallisepticum antigen	pPZP211	小麦种子Wheat seed	[33]
	抗菌肽Human LL-37 antimicrobial peptide	Unknow	大麦种子Barley seed	[34]
	人生长激素Human growth hormone	pwrg4803	大豆种子Soybean seed	[35]
	单链Fv片段抗体 Single-chain Fv fragment（scFV）antibody	pGEM3zf	豌豆种子Pea seed	[36]
其他生物反应器 Other bioreactor	乙型肝炎疫苗 Hepatitis B virus antigen HBsAg antigen	pG35SHBsAg	生菜植株Lettuce Seedling	[37]
	猪水肿病疫苗Double repeated B subunit of Shiga toxin 2e antigen	pBI121	生菜植株Lettuce Seedling	[38]
	鼠疫杆菌抗原F1-V from Yersinia pestis antigen	pBI121	生菜植株Lettuce Seedling	[39]
	霍乱疫苗Cholera toxin B subunit antigen	pCAMBIA3300	生菜植株Lettuce Seedling	[40]
	严重急性呼吸综合征冠状病毒疫苗SARS-CoV spike protein antigen	pCRII-SI	生菜植株Lettuce Seedling	[41]
	猪流行性腹泻病毒疫苗 Porcine epidemic diarrhea virus antigen	pMYV514	生菜植株Lettuce Seedling	[42]
	大豆凝集素Soybean agglutinin	pBI-101	马铃薯块茎Potato tubers	[43]
	人源酪蛋白Human lactoferrin	pPCV701	马铃薯块茎Potato tubers	[44]
	人干扰α-2b Human interferon alpha-2b	pCBV16	胡萝卜根Carrot root	[45]
	鼠疫杆菌抗原F1-V from Yersinia pestis antigen	pBI-FV	胡萝卜根Carrot root	[46]
	人血清白蛋白Human serum albumin	pCAMBIA1300	烟草BY-2细胞 Tobacco BY-2 cells	[47]
	埃博拉病毒抗体Ebola virus antibody	pCAMBIA1300	烟草BY-2细胞 Tobacco BY-2 cells	[48]
	葡萄糖脑苷脂酶Human b-glucocerebrosidase	Unknown	胡萝卜细胞Carrot cells	[49]

生物反应器Bioreactor	外源蛋白Foreign protein	载体Vector	表达宿主Host	参考文献Reference
烟草叶片生物反应器 Tobacco leaf bioreactor	乙型肝炎核心抗原Hepatitis B core antigen	pEAQ-HT	烟草叶片Tobacco leaf	[18-19]
	人乳头瘤病毒-8抗原 Human papillomaviruse-8 antigen	pEAQ-HT	烟草叶片Tobacco leaf	[20]
	牛乳头瘤病毒-1抗原 Bovine papillomaviruse-1 antigen	pEAQ-HT-DEST	烟草叶片Tobacco leaf	[21]
	蓝舌病毒-8抗原Bluetongue virus antigen	pEAQ-HT	烟草叶片Tobacco leaf	[22]
	诺如病毒Norwalk抗原 Norwalk virus antigen	pBI201	烟草叶片Tobacco leaf	[23]
	诺如病毒Narita抗原Narita virus antigen	pICH10990	烟草叶片Tobacco leaf	[24]
	流感病毒H1N1抗原Influenza virus H5N1 antigen	pCAMBIA2300	烟草叶片Tobacco leaf	[25]
	流感病毒H5N1抗原Influenza virus H5N1 antigen	pCAMBIA2300	烟草叶片Tobacco leaf	[26]
	新型冠状病毒抗原COVID Covifenz^®	Unknown	烟草叶片Tobacco leaf	Medicago, Canada
种子生物反应器 Seed bioreactor	人血清白蛋白Human serum albumin	pOsPMP	水稻种子Rice seed	[27]
	人抗胰蛋白酶Human alpha-antitrypsin	pOsPMP	水稻种子Rice seed	[28]
	碱性成纤维细胞生长因子Basic fibroblast growth factor	pOsPMP	水稻种子Rice seed	[29]
	雪松花粉敏感蛋白CRYJ1/2 Cryptomeria japonica 1/2	pCSPmALSAg7-GW	水稻种子Rice seed	[30-31]
	流感病毒H3N2抗原Influenza virus H3N2 antigen	pHN05	玉米种子Maize seed	[32]
	TM-1疫苗 TM-1 gene of Mycoplasma gallisepticum antigen	pPZP211	小麦种子Wheat seed	[33]
	抗菌肽Human LL-37 antimicrobial peptide	Unknow	大麦种子Barley seed	[34]
	人生长激素Human growth hormone	pwrg4803	大豆种子Soybean seed	[35]
	单链Fv片段抗体 Single-chain Fv fragment（scFV）antibody	pGEM3zf	豌豆种子Pea seed	[36]
其他生物反应器 Other bioreactor	乙型肝炎疫苗 Hepatitis B virus antigen HBsAg antigen	pG35SHBsAg	生菜植株Lettuce Seedling	[37]
	猪水肿病疫苗Double repeated B subunit of Shiga toxin 2e antigen	pBI121	生菜植株Lettuce Seedling	[38]
	鼠疫杆菌抗原F1-V from Yersinia pestis antigen	pBI121	生菜植株Lettuce Seedling	[39]
	霍乱疫苗Cholera toxin B subunit antigen	pCAMBIA3300	生菜植株Lettuce Seedling	[40]
	严重急性呼吸综合征冠状病毒疫苗SARS-CoV spike protein antigen	pCRII-SI	生菜植株Lettuce Seedling	[41]
	猪流行性腹泻病毒疫苗 Porcine epidemic diarrhea virus antigen	pMYV514	生菜植株Lettuce Seedling	[42]
	大豆凝集素Soybean agglutinin	pBI-101	马铃薯块茎Potato tubers	[43]
	人源酪蛋白Human lactoferrin	pPCV701	马铃薯块茎Potato tubers	[44]
	人干扰α-2b Human interferon alpha-2b	pCBV16	胡萝卜根Carrot root	[45]
	鼠疫杆菌抗原F1-V from Yersinia pestis antigen	pBI-FV	胡萝卜根Carrot root	[46]
	人血清白蛋白Human serum albumin	pCAMBIA1300	烟草BY-2细胞 Tobacco BY-2 cells	[47]
	埃博拉病毒抗体Ebola virus antibody	pCAMBIA1300	烟草BY-2细胞 Tobacco BY-2 cells	[48]
	葡萄糖脑苷脂酶Human b-glucocerebrosidase	Unknown	胡萝卜细胞Carrot cells	[49]