欢迎访问作物学报,今天是

作物学报 ›› 2015, Vol. 41 ›› Issue (04): 524-530.doi: 10.3724/SP.J.1006.2015.00524

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

以谷蛋白GluA-2 信号肽增强外源蛋白在转基因水稻胚乳中的表达与积累

王红梅1,张昌泉1,李钱峰1,辛世文2,刘巧泉1,*,徐明良1,3   

  1. 1 扬州大学农学院 / 植物功能基因组学教育部重点实验室 / 江苏省作物遗传生理重点实验室 / 粮食作物现代产业技术协同创新中心,江苏扬州225009; 2 香港中文大学生物系, 香港; 3 中国农业大学农学与生物技术学院, 北京100193
  • 收稿日期:2014-11-01 修回日期:2015-02-06 出版日期:2015-04-12 网络出版日期:2015-03-03
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2014ZX08001006-005, 2014ZX08009-024B), 江苏省杰出青年基金项目(BK2012010),江苏省高校优势学科建设工程和“青蓝工程”等项目资助。

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 Published:2015-04-12 Published online:2015-03-03

摘要:

提高外源蛋白在特定目标组织器官中的表达量是转基因植物研究与开发的核心技术之一。谷蛋白是水稻种子中最主要的贮藏蛋白, 其表达具有严格的时空特异性。为进一步研究谷蛋白信号肽序列在指导基因表达中的作用,本研究克隆了水稻谷蛋白GluA-2 基因的启动子及其信号肽编码序列, 并与GUS 报告基因编码区融合, 构建了分别含有和不含有信号肽的表达载体p13GG 和p13GSG; 经农杆菌介导法分别转入同一水稻品种中, 获得了20 多个独立转化子, PCR 证明外源基因都已整合进了水稻基因组中。Northern 杂交结果表明, 融合GluA-2 信号肽编码序列可显著提高GUS 基因在水稻胚乳中的转录; 利用GUS 特异的抗体进行Western 杂交分析, 显示该信号肽序列可显著提高外源蛋白在转基因水稻胚乳中的积累, 但是其所指导表达的GUS 蛋白在水稻胚乳中并没有表现出相应的活性, 其机制有待进一步深入解析。相关结果对于水稻品质改良基因工程研究以及以水稻种子作为生物反应器高效表达外源蛋白具有重要的指导作用。

关键词: 转基因水稻, 信号肽, 谷蛋白, 胚乳, 基因表达

Abstract:

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] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[2] 姚晓华, 王越, 姚有华, 安立昆, 王燕, 吴昆仑. 青稞新基因HvMEL1 AGO的克隆和条纹病胁迫下的表达[J]. 作物学报, 2022, 48(5): 1181-1190.
[3] 杨谨, 白爱宁, 白雪, 陈娟, 郭林, 刘春明. 水稻胚胎和胚乳双缺陷突变体eed1的表型与遗传分析[J]. 作物学报, 2022, 48(2): 292-303.
[4] 渠建洲, 冯文豪, 张兴华, 徐淑兔, 薛吉全. 基于全基因组关联分析解析玉米籽粒大小的遗传结构[J]. 作物学报, 2022, 48(2): 304-319.
[5] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[6] 王艳朋, 凌磊, 张文睿, 王丹, 郭长虹. 小麦B-box基因家族全基因组鉴定与表达分析[J]. 作物学报, 2021, 47(8): 1437-1449.
[7] 宋天晓, 刘意, 饶莉萍, Soviguidi Deka Reine Judesse, 朱国鹏, 杨新笋. 甘薯细胞壁蔗糖转化酶基因IbCWIN家族成员鉴定及表达分析[J]. 作物学报, 2021, 47(7): 1297-1308.
[8] 解盼, 刘蔚, 康郁, 华玮, 钱论文, 官春云, 何昕. 甘蓝型油菜CBF基因家族的鉴定和表达分析[J]. 作物学报, 2021, 47(12): 2394-2406.
[9] 李鹏, 刘彻, 宋皓, 姚盼盼, 苏沛霖, 魏跃伟, 杨永霞, 李青常. 烟草非特异性脂质转移蛋白基因家族的鉴定与分析[J]. 作物学报, 2021, 47(11): 2184-2198.
[10] 黄素华, 林席跃, 雷正平, 丁在松, 赵明. 强再生力水稻品种碳氮营养与激素生理特征研究[J]. 作物学报, 2021, 47(11): 2278-2289.
[11] 马硕, 焦悦, 杨江涛, 王旭静, 王志兴. 基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征[J]. 作物学报, 2020, 46(11): 1703-1710.
[12] 米文博, 方园, 刘自刚, 徐春梅, 刘高阳, 邹娅, 徐明霞, 郑国强, 曹小东, 方新玲. 白菜型冬油菜温敏不育系PK3-12S育性转换的差异蛋白质组学分析[J]. 作物学报, 2020, 46(10): 1507-1516.
[13] 靳舒荣,王艳玫,常悦,王月华,李加纳,倪郁. 不同收获指数甘蓝型油菜β-淀粉酶活性及其基因家族成员的表达分析[J]. 作物学报, 2019, 45(8): 1279-1285.
[14] 董玉凤 王旭静 宋亚亚 靳 茜 王志兴. 利用基因拆分技术培育耐草甘膦转基因水稻的研究 [J]. 作物学报, 2019, 45(3): 344-353.
[15] 冯韬,官春云. 甘蓝型油菜光敏色素互作因子4 (BnaPIF4)基因克隆和功能分析[J]. 作物学报, 2019, 45(2): 204-213.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!