分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 88-93.

分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建

刘丹, 吴凤芝

东北农业大学园艺学院,哈尔滨 150030

收稿日期:2014-04-28 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:刘丹,女,博士研究生,研究方向：植物分子生物学
基金资助:
国家自然科学基金项目（31172002）

Cloning of Chalcone Isomerase Gene from Potato Onion and Plant Expression Vector Construction

Liu Dan, Wu Fengzhi

Horticulture College,Northeast Agricultural University,Harbin 150030

Received:2014-04-28 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

摘要： 旨在得到分蘖洋葱查尔酮异构酶基因,探明该基因的功能。克隆查尔酮异构酶基因并构建了其超表达和干扰载体,以期得到用于研究查尔酮异构酶基因功能的表达载体。从分蘖洋葱叶片中克隆得到查尔酮异构酶基因cDNA 全长743 bp,GenBank登录号为KJ489062。结果显示,该基因633 bp的开放读码框编码210个氨基酸的多肽序列。将PCR 扩增克隆得到的分蘖洋葱查尔酮异构酶基因片段连接到干扰载体pCAMBIA1301和超表达载体SOL2095中,成功构建了35S 启动子控制的植物表达双元载体pCAMBIA1301-CHI和SOL2095-CHI。

关键词: 分蘖洋葱, 查尔酮异构酶, 基因克隆, 载体构建

Abstract: To clone and analyse the function of the potato onion chalcone isomerase gene, we generated CHI over-expression construct and RNAi construct. Full-length CHI gene is 743 bp, and the GenBank accession number is KJ489062. Results showed that, CHI gene open reading frame contain 633 bp, translating into 210 amino acids. Full-length CHI gene and the fragment of CHI gene for silencing were amplified by PCR on complementary DNA（cDNA）, then PCR fragment was transferred to the binary vector under the control of 35S.

Key words: Potato onion, Chalcone isomerase, Gene cloning, Vetor construction

刘丹, 吴凤芝. 分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建[J]. 生物技术通报, 2014, 0(10): 88-93.

Liu Dan, Wu Fengzhi. Cloning of Chalcone Isomerase Gene from Potato Onion and Plant Expression Vector Construction[J]. Biotechnology Bulletin, 2014, 0(10): 88-93.

参考文献

[1] Cheng H, Li L, Cheng S, et al. Molecular cloning and function assay of a chalcone isomerase gene（GbCHI）from Ginkgo biloba[J]. Plant Cell Reports, 2011, 30（1）：49-62.
[2] 周发俊, 王逸群, 陈由强.植物查尔酮异构酶分子生物学研究进展[J].河北科技师范学院学报, 2008, 22（1）：73-75.
[3] Norimoto S, Toshio A, Shusei S, et al. A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general falavonoids and legume specific5-deoxy（iso） flavonoid in Lotus japonicus[J]. Plant Physiology, 2003, 131（3）：941-951.
[4] Li F, Jin Z, Qu W, et al. Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco[J]. Plant Physiol and Biochem, 2006, 44（7）：455-461.
[5] Van Tunen AJ, Koes RE, Spelt CE, et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida：coordinate, light-regulated and differential expression of flavonoid genes[J]. The EMBO Journal, 1988, 7（5）：1257.
[6] HI, Hirsch AM. Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa（Medicago sativa L.）：highest transcript levels occur in young roots and root tips[J]. Plant Molecular Biology, 1994, 24（5）：767-777.
[7] 安利清, 王涛, 等.百合查尔酮合成酶基因克隆及其转化烟草的花色表达分析[J].西北植物学报, 2012, 32（8）：1511-1517.
[8] Druka A, Kudrna D, Rostoks N, et al. Chalcone isomerase gene from rice（Oryza sativa）and barley（Hordeum vulgare）：physical, genetic and mutation mapping[J]. Gene, 2003, 302：171-178.
[9] Koca U, Berhow MA, Febres VJ, et al. Decreasing unpalatable flavo-noid components in Citrus：the effect of transformation construct[J]. Physiologia Plantarum, 2009, 137（2）：101-114.
[10] 邵国芳.毛葱复种油豆角高产栽培技术[J].作物栽培, 2008, 8：37-39.
[11] 赵廷昌, 刘学敏, 等.转基因作物对土壤微生物多样性影响[J].植物保护, 2007, 33（4）：15-20.
[12] SR, Collins GJ, Robinson S, et al. Overexpression of petunia chalcone isomerase in tomato results in fruits containing increased levels of flavonols[J]. Nat Biotechnol, 2001, 19：470-474.
[13] 赵飞, 冯君伟, 等.转录因子DREB1A基因的克隆与Gateway克隆技术构建植物表达载体[J].生物技术通讯, 2007, 18（2）：205-208.
[14] 陈书霞, 陈巧, 等.利用Gateway技术构建黄瓜HPL基因的RNA干扰载体[J].西北农业学报, 2013, 22（2）：152-158.
[15] Lee S, Kim J, Han JJ, et al. Functional analyses of the flowering time gene OsMADS50, the putative suppressor of over expression of CO 1/AGAMOUS-LIKE 20（SO1/AGL20）ortholog in riee[J]. Plant J, 2004, 38（5）：754-764.
[16] A, Mukhopadhyay N, Arumugam YS, et al. Molecular tagging of erucic acid trait in oilseed mustard（Brassica juncea）by QTL mapping and single nucleotide polymorphisms in FAEI gene[J]. Theor Appl Genet, 2004, 108：743-749.
[17] Y, Higeta D, Suzuki A, et al. Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow flowered carnation（Dianthus caryophyllus）[J]. Plant Cell Physiol, 2002, 43（5）：578-585.