Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (04): 524-530.doi: 10.3724/SP.J.1006.2015.00524


Enhancing Expression and Accumulation of Foreign Proteins by Using the Signal Peptide of Glutelin GluA-2 in Endosperm of Transgenic Rice

WANG Hong-Mei1,ZHANG Chang-Quan1,LI Qian-Feng1, SUN Samuel Sing-Min2,LIU Qiao-Quan1,*,XU Ming-Liang1,3   

  1. 1 Jiangsu Key Laboratory for Crop Genetics and Physiology / Key Laboratory of Plant Functional Genomics of Ministry of Education /Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China; 2 Department of Biology,The Chinese University of Hong Kong, Hong Kong, China; 3 College of Agriculture and Biotechnology, Chinese Agricultural University, Beijing 100193, China
  • Received:2014-11-01 Revised:2015-02-06 Online:2015-04-12 Published:2015-03-03


It is one of the key important techniques to enhance the expression of foreign proteins in target tissue/organ of transgenic plants. Glutelin is the major component of storage proteins in rice seeds, and its expression was tightly temporal and tissuespecific, which is controlled by several mechanisms. To further reveal the function of the Glutelin signal peptide on expression of target gene, in present study, we isolated the promoter and signal peptide-coding sequences of the glutelin GluA-2 gene, and fused them transcriptionally to the GUS coding sequences. Beside, the construct without the GluA-2 signal peptide-coding sequences was also generated as a control. Both constructs with the GUS chimeric genes, named as p13GSG and p13GG, were introduced into the same rice variety by Agrobacterium-mediated transformation. More than twenty independent transgenic lines were generated for each construct, and the integration of the GUS chimeric gene was confirmed by PCR technique. The results from Northern blot analysis showed that, after fusing the GluA-2 signal peptide coding sequences between the GluA-2 promoter and the GUS coding sequence, the transcription of GUS chimeric gene could be dramatically increased. Then, Western blot was carried out by using the GUS-specific antibody, and the results obviously revealed that the accumulation of foreign proteins was significantly
enhanced in the endosperm of transgenic rice with the signal peptide. However, there was no or very low GUS activity in the endosperm of transgenic rice plants with the signal peptide. These results were very useful to improve the grain quality of rice via genetic engineering, especially produce foreign proteins in the seeds of rice as bioreactor.

Key words: Transgenic rice, Signal peptide, Glutelin, Endosperm, Gene expression

[1] Li X, Wu Y, Zhang D Z, Gallikin J W, Franceschi V R, Okita T W.Rice prolamine protein body biogenesis: a BiP mediated process.Science, 1993, 242: 1054–1056

[2] Okita T W, Li X, Roberts M W. Targeting of mRNAs to domains of the endoplasmic reticulum. Trends Cell Biol, 1994, 4: 91–96

[3] Choi S B, Wang C, Muench D G, Ozawa K, Franceschi V R, Wu Y, Okita T W. Messenger RNA targeting of rice seed storage proteins to specific ER subdomains. Nature, 2000, 407: 765–767

[4] Coleman C E, Lopes M A, Gillikei J W, Boston R S, Larkins B A.A defective signal peptide in the maize high-lysine mutant fleury-2. Proc Natl Acad Sic USA, 1995, 92: 6828–6831

[5] Boehm R, Susanne S, Klaus S, Li S M, Lutz H D. Active expression of the ubiA gene from E. coli in tobacco: influence of plant ER-specific signal peptides on the expression of a membranebound prenyltransferase in plant cells. Transgenic Res, 2000, 9:477–486

[6] Yamagata H, Sugimoto T, Tanaka K, Kasai Z. Biosynthesis of storage proteins in developing rice seeds. Plant Physiol, 1982, 70: 1094–1100

[7] 刘巧泉, 周丽慧, 王红梅, 顾铭洪. 水稻种子贮藏蛋白合成的分子生物学研究进展. 分子植物育种, 2008, 6: 1–15

Liu Q Q, Zhou L H, Wang H M, Gu M H. Advances on biosynthesis of rice seed storage proteins in molecular biology. Mol Plant Breed, 2008, 6: 1–15 (in Chinese with English abstract)

[8] Ren Y L, Wang Y H, Liu F, Zhou K N, Ding Y, Zhou F, Wang Y,Liu K, Gan L, Ma W W, Han X H, Zhang X, Guo X P, Wu F Q,Cheng Z J, Wang J L, Lei C L, Lin Q B, Jiang L, Wu C Y, Bao Y Q, Wang H Y, Wan J M. GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm.

Plant Cell, 2014, 26: 410–425

[9] Zhao W M, Gatehouse J A, Boulter D. The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains: homology to pea legumin. FEBS Lett, 1983,162: 96–102

[10] Wen T N, Luthe D S. Biochemical characterization of rice glutelin. Plant Physiol, 1985, 78: 172–177

[11] Takaiwa F, Oono K, Wing D, Kato A. Sequence of three members and expression of a new major subfamily of glutelin genes from rice. Plant Mol Biol, 1991, 17: 875–885

[12] Okita T W, Hwang Y S, Hnilo J, Kim W T, Aryan A P, Larson R, Krishnan H B. Structure and expression of the rice glutelin multigene family. J Biol Chem, 1989, 264: 12573–12581

[13] Takaiwa F, Ebinuma H, Kikuchi S, Oono K. Nucleotide sequence of a rice glutelin gene. FEBS Lett, 1987, 221: 43–47

[14] Takaiwa F, Kikuchi S, Oono K. A rice glutelin family: a major type of glutelin mRNAs can be divided into two classes. Mol Gen Genet, 1987, 208: 15–22

[15] Takaiwa F, Oono K. Genomic DNA sequences of two new genes for new storage protein glutelin in rice. Jpn J Genet, 1991, 66:161–171

[16] 刘巧泉, 于恒秀, 张文娟, 龚志云, 顾铭洪. 番茄rbcS3A 启动子控制的外源基因在转基因水稻中的表达特性. 植物生理与分子生物学学报, 2007, 33: 251–257

Liu Q Q, Yu H X, Zhang W J, Gong Z Y, Gu M H. Expression of the GUS fusion gene controlled by the tomato rbcS3A promoter in transgenic rice. J Plant Physiol Mol Biol, 2007, 33: 251–257(in Chinese with English abstract)

[17] 刘巧泉, 张景六, 王宗阳, 洪孟民, 顾铭洪. 根癌农杆菌介导的水稻高效转化系统的建立, 植物生理学报, 1998, 24:259–271

Liu Q Q, Zhang J L, Wang Z Y, Hong M M, Gu M H. A highly efficient transformation mediated by Agrobacterium in rice. Acta Phytophysiol Sin, 1998, 24: 259–271 (in Chinese with English abstract)

[18] Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acid Res, 1980, 8: 4321–4325

[19] 郑霏琴, 王宗阳, 高继平. 水稻胚乳中核糖核酸的分离. 植物生理学通讯, 1993, 29: 438–440

Zheng F Q, Wang Z Y, Gao J P. Isolation of nucleic acids from rice endosperm. Plant Physiol Commun, 1993, 29: 438–440 (inChinese with English abstract)

[20] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989

[21] Bradford H M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248–254

[22] Jefferson R A. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5: 387–405

[23] Fukuda M, Wen L, Satoh-Cruz M, Kawagoe Y, Nagamura Y,Okita T W, Washida H, Sugino A, Ishino S, Ishino Y, Ogawa M,Sunada M, Ueda T, Kumamaru T. A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm. Plant Physiol, 2013, 162: 663–674

[24] Mitsukawa N, Konishi R, Uchiki M, Masumura T, Tanaka K. Molecular cloning and characterization of a cysteine-rich16.6-kDa prolamin in rice seeds. Biosci Biotechnol Biochem,

1999, 63: 1851–1858

[25] Boehm R, Susanne S, Klaus S, Li S M, Lutz H D. Active expression of the ubiA gene from E. coli in tobacco: influence of plant ER-specific signal peptides on the expression of a membrane- bound prenyltransferase in plant cells. Transgenic Res,2000, 9: 477–486

[26] Wright K E, Prior F, Sardana R, Altosaar I, Dudani A K, Ganz P R, Tackaberry E S. Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco. Transgenic Res, 2001, 10: 177–181

[27] 范云六, 张春义. 迎接21世纪农作物生物技术的挑战, 生物技术通报, 1999, (5): 1–6

Fan Y L, Zhang C Y. Greeting the challenges of crop biotechnology in the 21st century. Biotechnol Inf, 1999, (5): 1–6 (in Chinese with English abstract)

[28] Ye X D, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science,

2000, 287: 303–305

[29] Sun S S M, Liu Q Q. Transgenic approaches to improve the nutritional quality of plant proteins. In Vitro Cell Dev Biol-Plant, 2004, 40: 155–162

[30] Fischer R, Stoger E, Schillberg S, Christou P, Twyman R M. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol, 2004, 7: 152–158

[31] Rybicki E P. Plant-made vaccines for humans and animals. Plant Biotech J, 2010, 8: 620–637

[32] Yang Z Q, Liu Q Q, Pan Z M, Yu H X, Jiao X A. Expression of the fusion glycoprotein of newcasstle disease virus in transgenic rice and its immunogenicity in mice. Vaccine, 2007, 25: 591–598

[33] Cheung S C K, Liu L Z, Sun S S M, Liu Q Q, Lan L L, Chan J,Tong P. Inhibition of human MCF-7 breast cancer cells and HT-29 colon cancer cells by rice-produced recombinant human insulin-like growth binding protein-3 (rhIGFBP-3). PLoS One,2013, 8: e77516

[34] Bundó M, Montesinos L, Izquierdo E, Campo S, Mieulet D,Guiderdoni E, Rossignol M, Badosa E, Montesinos E, San Segundo B, Coca M. Production of cecropin A antimicrobial

peptide in rice seed endosperm. BMC Plant Biol, 2014, 14:102

[1] LI Hai-Fen, WEI Hao, WEN Shi-Jie, LU Qing, LIU Hao, LI Shao-Xiong, HONG Yan-Bin, CHEN Xiao-Ping, LIANG Xuan-Qiang. Cloning and expression analysis of voltage dependent anion channel (AhVDAC) gene in the geotropism response of the peanut gynophores [J]. Acta Agronomica Sinica, 2022, 48(6): 1558-1565.
[2] JIN Rong, JIANG Wei, LIU Ming, ZHAO Peng, ZHANG Qiang-Qiang, LI Tie-Xin, WANG Dan-Feng, FAN Wen-Jing, ZHANG Ai-Jun, TANG Zhong-Hou. Genome-wide characterization and expression analysis of Dof family genes in sweetpotato [J]. Acta Agronomica Sinica, 2022, 48(3): 608-623.
[3] YANG Jin, BAI Ai-Ning, BAI Xue, CHEN Juan, GUO Lin, LIU Chun-Ming. Phenotypic and genetic analyses of a rice mutant eed1 with defected embryo and endosperm development [J]. Acta Agronomica Sinica, 2022, 48(2): 292-303.
[4] QU Jian-Zhou, FENG Wen-Hao, ZHANG Xing-Hua, XU Shu-Tu, XUE Ji-Quan. Dissecting the genetic architecture of maize kernel size based on genome-wide association study [J]. Acta Agronomica Sinica, 2022, 48(2): 304-319.
[5] CHEN Xin-Yi, SONG Yu-Hang, ZHANG Meng-Han, LI Xiao-Yan, LI Hua, WANG Yue-Xia, QI Xue-Li. Effects of water deficit on physiology and biochemistry of seedlings of different wheat varieties and the alleviation effect of exogenous application of 5-aminolevulinic acid [J]. Acta Agronomica Sinica, 2022, 48(2): 478-487.
[6] WANG Yan-Peng, LING Lei, ZHANG Wen-Rui, WANG Dan, GUO Chang-Hong. Genome-wide identification and expression analysis of B-box gene family in wheat [J]. Acta Agronomica Sinica, 2021, 47(8): 1437-1449.
[7] SONG Tian-Xiao, LIU Yi, RAO Li-Ping, Soviguidi Deka Reine Judesse, ZHU Guo-Peng, YANG Xin-Sun. Identification and expression analysis of cell wall invertase IbCWIN gene family members in sweet potato [J]. Acta Agronomica Sinica, 2021, 47(7): 1297-1308.
[8] QIN Tian-Yuan, LIU Yu-Hui, SUN Chao, BI Zhen-Zhen, LI An-Yi, XU De-Rong, WANG Yi-Hao, ZHANG Jun-Lian, BAI Jiang-Ping. Identification of StIgt gene family and expression profile analysis of response to drought stress in potato [J]. Acta Agronomica Sinica, 2021, 47(4): 780-786.
[9] LI Peng, LIU Che, SONG Hao, YAO Pan-Pan, SU Pei-Lin, WEI Yao-Wei, YANG Yong-Xia, LI Qing-Chang. Identification and analysis of non-specific lipid transfer protein family in tobacco [J]. Acta Agronomica Sinica, 2021, 47(11): 2184-2198.
[10] HUANG Su-Hua, LIN Xi-Yue, LEI Zheng-Ping, DING Zai-Song, ZHAO Ming. Physiological characters of carbon, nitrogen, and hormones in ratooning rice cultivars with strong regeneration ability [J]. Acta Agronomica Sinica, 2021, 47(11): 2278-2289.
[11] MA Shuo, JIAO Yue, YANG Jiang-Tao, WANG Xu-Jing, WANG Zhi-Xing. Molecular characterization identification by genome sequencing of transgenic glyphosate-tolerant rice G2-7 [J]. Acta Agronomica Sinica, 2020, 46(11): 1703-1710.
[12] MI Wen-Bo, FANG Yuan, LIU Zi-Gang, XU Chun-Mei, LIU Gao-Yang, ZOU Ya, XU Ming-Xia, ZHENG Guo-Qiang, CAO Xiao-Dong, FANG Xin-Ling. Differential proteomics analysis of fertility transformation of the winter rape thermo-sensitive sterile line PK3-12S (Brassica rapa L.) [J]. Acta Agronomica Sinica, 2020, 46(10): 1507-1516.
[13] JIN Shu-Rong,WANG Yan-Mei,CHANG Yue,WANG Yue-Hua,LI Jia-Na,NI Yu. Activity and gene family expression of β-amylase in Brassica napus differing in harvest index [J]. Acta Agronomica Sinica, 2019, 45(8): 1279-1285.
[14] DONG Yu-Feng, WANG Xu-Jing, SONG Ya-Ya, JIN Xi, and WANG Zhi-Xing. Cultivation of herbicide tolerant transgenic rice by gene spliting technique [J]. Acta Agronomica Sinica, 2019, 45(3): 344-353.
[15] Tao FENG,Chun-Yun GUAN. Cloning and characterization of phytochrome interacting factor 4 (BnaPIF4) gene from Brassica napus L. [J]. Acta Agronomica Sinica, 2019, 45(2): 204-213.
Full text



No Suggested Reading articles found!