作物学报 ›› 2015, Vol. 41 ›› Issue (04): 524-530.doi: 10.3724/SP.J.1006.2015.00524
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
WANG Hong-Mei1,ZHANG Chang-Quan1,LI Qian-Feng1, SUN Samuel Sing-Min2,LIU Qiao-Quan1,*,XU Ming-Liang1,3
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。
[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: 251–257Liu 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, 24:259–271Liu 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: 438–440Zheng 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): 1–6Fan 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 antimicrobialpeptide 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. |
|