菊苣质体同源片段的克隆及非抗生素标记的质体定点整合表达载体构建

摘要/Abstract

摘要： 依据烟草、玉米、拟南芥和大豆质体基因组rps7基因和16S rDNA基因的保守序列设计引物,以菊苣质体基因组DNA为模板,PCR扩增到1段4.8 kb的片段。序列分析表明,该片段全长4 751 bp(GenBank登录号为GQ199478),包括449 bp的 rps7基因5′端序列、793 bp的rps12基因、72 bp的trnV基因、1 315 bp的16S rDNA基因5′端以及大部分的基因间隔区,该序列与烟草对应区段的相似性为92%,与莴苣对应区段的相似性为97%。以此片段作为定点整合外源基因的同源重组片段,构建了非抗生素标记的菊苣质体定点整合表达载体pJBADH-GFP,酶切分析及PCR检测证明构建的载体符合预期设计。该载体的构建为建立高效、稳定的菊苣质体转化和无抗生素筛选体系奠定基础,为进一步通过质体基因工程手段将更多感兴趣的基因导入菊苣进行遗传改良,或以菊苣质体作生物反应器生产动物口服疫苗等搭建技术平台。

Abstract: A pair of primers used in amplifying plastid fragments of chicory was designed based on the conservative sequences of both rps7 and 16S rDNA genes in the tobacco, maize, Arabidopsis thaliana and soybean plastid genomes. The 4.8 kb fragment was cloned from the chicory plastid genome. Sequencing analysis indicated that this fragment was 4751 bp in size (GenBank Accession Number GQ199478), and consisted of 449 bp 5′-end of the rps7 gene, 793 bp sequence of rps12 gene, 72 bp sequence of the trnV gene, 1 315 bp 5′-end of the 16S rDNA gene and most gene intergenic regions. The BLAST results showed that the similarity of this fragment with tobacco and lettuce were 92% and 97% respectively. Using this fragment as a homologous targeting sequence, we constructed the site-specific integration chicory-specific plastid expression vector pJBADH-GFP that carries the multicistron expression cassette of prrn-BADH-gfp-psbA-3′. The results of restriction analysis and PCR detection confirmed this. This plastid expression vector is desirable both for use in establishment of a chicory antibiotic-free plastid transformation system and traits improvement of chicory or for use as a bioreactor to produce edible vaccines for animals via plastid genetic transformation.

中图分类号:

王玉华^*,郝建国,贾敬芬^*. 菊苣质体同源片段的克隆及非抗生素标记的质体定点整合表达载体构建[J]. 草业学报, 2009, 18(6): 72-81.

WANG Yu-hua, HAO Jian-guo, JIA Jing-fen. Cloning of homologous targeting sequences and construction of antibiotic-freeplastid site-specific integration expression vector of chicory[J]. Acta Prataculturae Sinica, 2009, 18(6): 72-81.

参考文献

[1] Shimizu M, Goto M, Hanai M, et al. Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco[J]. Plant Physiology, 2008, 147(4): 1976-1983.
[2] Arlen P A, Falconer R, Cherukumilli S, et al. Field production and functional evaluation of chloroplast-derived interferon-alpha2b[J]. Plant Biotechnology Journal, 2007, 5(4): 511-525.
[3] Arlen P A, Singleton M, Adamovicz J J, et al. Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts[J]. Infection and Immunity, 2008, 76(8): 3640-3650.
[4] Chebolu S, Daniell H. Stable expression of Gal/GalNac lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis[J]. Plant Biotechnology Journal, 2007, 5(2): 230-239.
[5] Shao H B, Hed M, Qian K X, et al. The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors[J]. Comptes Rendus Biologies, 2008, 331(3): 179-184.
[6] 王婷婷, 徐国荣, 张举仁, 等. 细胞工程技术选育的草地早熟禾新种质的耐热性分析[J]. 草业学报, 2009,18(2):60-65.
[7] Abid M, Palms B, Deryke R, et al. Transformation of chicory and expression of the bacterial uidA and nptⅡ genes in the transgenic regenerants[J]. Journal of Experimental Botany, 1995, 46(284): 337-346.
[8] Piéron S, Watillon B. Expression of a chimeric GUS gene construct as a tool to study nodule morphogenesis in chicory leaves[J]. Plant Cell, Tissue and Organ Culture, 2001, 66(3): 159-165.
[9] 宋书锋, 曹凤, 杨培志, 等. 普那菊苣高效再生体系建立和遗传转化研究[J]. 分子植物育种, 2006, 4(4): 565-570.
[10] Williams F M K, Davey M R, Power J B, et al. Chicory (Cichorium intybus L.) expression the lol1 gene exhibits inhibition of ice recrystallisation[A]. In: van Hintum T J L, Lebeda A, Pink D, et al. Proceedings of the Eucarpia Meeting on Leafy Vegetables Breeding and Genetics, 2003[C], Noordwijkerhout, The Netherlands, 2003. 137-142.
[11] Lim H T, Park E J, Lee J Y, et al. High plant regeneration and ectopic expression of OsMADS1 gene in root chicory (Cichorium intybus L. var. sativus)[J]. Plant Biotechnology Journal, 2003, 5(4): 215-219.
[12] 程林梅, 曹秋芬, 高洪文, 等. 菊苣再生体系建立及转AFL2基因的研究[J]. 草地学报, 2004, 12(3): 119-203.
[13] 程林梅, 孙毅, 王亦学, 等. 菊苣农杆菌介导转化受体系统的研究[J]. 草业学报, 2008, 17(1): 130-134.
[14] 赵宇玮, 王英娟, 步怀宇, 等. AtNHX1基因对菊苣的转化和耐盐性研究[J]. 草业学报, 2009, 18(3): 103-109.
[15] 龚小松, 阎隆飞. 高等植物质体DNA提取方法改进[J]. 科学通报, 1991, 36(6): 467-469.
[16] Chakrabarti S K, Lutzk A, Lertwiriyawong B, et al. Expression of th cry9Aa2 B. T. gene in tobacco chloroplasts confers resistance to potato tuber mot[J]. Transgenic Research, 2006, 15(4): 481-488.
[17] Sikdar S R, Serino G, Chaudhuri S, et al. Plastid transformation in Arabidopsis thaliana[J]. Plant Cell Report, 1998, 18(1): 20-24.
[18] Lee S M, Kang K, Chung H, et al. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny[J]. Moleculars and Cells, 2006, 21(3): 401-410.
[19] Hou B K, Zhou Y H, Wan L H, et al. Chloroplast transformation in oilseed rape[J]. Transgenic Research, 2003, 12(1): 111-114.
[20] Nguyen T T, Nugent G, Cardi T, et al. Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.)[J]. Plant Science, 2005, 168(6): 1495-1500.
[21] Kumar S, Dhingra A, Daniell H. Stable transformtion of the cotton plastid genome and maternal inheritance of transgenes[J]. Plant Molecular Biology, 2004, 56(2): 203-216.
[22] Zubkot M K, Zubkot E I, van Zuilen K, et al. Stable transformation of petunia plastids[J]. Transgenic Research, 2004, 13(6): 523-530.
[23] Dufourmantel N, Tissot G, Goutorbe F, et al. Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensts/cry1Ab protoxin[J]. Plant Molecular Biology, 2005, 58(5): 659-668.
[24] Ruhlman T, Ahangari R, Devine A, et al. Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in non-obese diabetic mice[J]. Plant Biotechnology, 2007, 5(4): 495-510.
[25] Kumar S, Dhingra A, Daniell H. Plastid-expressed bataine aldehyde dehydrogenase gene in carrot cultured cells roots, and leaves confers enhanced salt tolerance[J]. Plant Physiology, 2004, 136(1): 2843-2854.
[26] Wurbs D, Ruf S, Bock R. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome[J]. The Plant Journal, 2007, 49(26): 276-288.
[27] Liu C W, Lin C C, Chen J J, et al. Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment[J]. Plant Cell Report, 2007, 26(10): 1733-1744.
[28] Okumura S, Sawada M, Park Y W, et al. Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts[J]. Transgenic Research, 2006, 15(5): 637-646.
[29] Svab Z, Hajdukiewicz P, Maliga P. Stable transformation of plastids in higher plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87(21): 8526-8530.
[30] Corneille S, Lutz K, Svab Z, et al. Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system[J]. Plant Journal, 2001, 27(2): 171-178.
[31] Svab Z, Maliga P. High-frequency plastid transfomation in tobacco by selection for chimeric aadA gene[J]. Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(3): 913-917.
[32] Carrer H, Hockenberry T N, Svab Z, et al. Kannamycin resistance as a selectable marker for plastid transformation in tobacco[J]. Molecular and General Genetics, 1993, 241(1-2): 49-56.
[33] Iamtham S, Day A. Removal of antibiotic resistance genes from transgenic tobacco plastids[J]. Nature Biotechnology, 2000, 18(11): 1172-1176.
[34] Hajdukiewicz P T, Gilbertson L, Staub J M. Multiple pathways for Cre/lox-mediated recombination in plastids[J]. Plant Journal, 2001, 27(2): 161-170.
[35] 王玉祥, 张博, 王涛. 盐胁迫对苜蓿叶绿素、甜菜碱含量和细胞膜透性的影响[J]. 草业科学, 2009, 26(3): 53-56.
[36] Daniell H, Wiebe P O, Millan A F S. Antibiotic free chloroplast genetic engineering-an environmentally friendly approach[J]. Trends in Plant Science, 2001, 6(6): 237-239.
[37] Jia G X, Zhu Z Q, Chang F Q, et al. Transformation of tomato with the BADH gene from Atriplex improves salt tolerance[J]. Plant Cell Report, 2002, 21(2): 141-146.
[38] 司怀军, 张宁, 王蒂. 转甜菜碱醛脱氢酶基因提高烟草抗旱及耐盐性[J]. 作物学报, 2007, 33(8): 1335-1339.
[39] 徐冰, 韩烈保, 姚娜, 等. 草地早熟禾转CMO-BADH双基因和转CMO基因耐盐性分析[J]. 草地学报, 2008, 16(4): 353-358