|
|
|
| Deletion Analysis on The Peanut (Arachis hypogaea) Promoter of β-1,3-glucanase Function Region |
|
|
|
|
Abstract The plant β-1,3-glucanase belongs to one of the important pathogenesis-related proteins (PR-proteins), which could be accumulated when induced by exogenous signal molecule. The Arachis hypogaea promoter of β-1,3-glucanase (Ah-Glu-Pro) has been proved to be an inducible promoter, and there might contain some cis-regulatory elements which could respond to exogenous signal molecule. In order to clarify the promoter's function and further analyze and confirm the key cis-regulatory elements within the promoter, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was conducted with peanut genomic DNA as template, and a 970 bp fragment was obtained successfully, named by Ah-Glu-Pro. Online function prediction by PLACE and PlantCARE showed that Ah-Glu-Pro contained some typical cis-elements, such as TATA box and CAAT box, some cis-acting elements which could respond to pathogens and salicylic acid (SA), such as GRWAAW, GT1-motif, W-box, RAV1AAT, INRNTPSADB, AMMORESIVDCRNIA1 and BIHD1OS. Five forward primers Glu-F, Glu-P4, Glu-P3, Glu-P2 and Glu-P1 at the 5' end with the XbaⅠrestriction enzyme cutting site and one reverse primer Glu-R at the 3' end with the BglⅡrestriction enzyme cutting site were designed and synthesized according to predictive results. PCR was conducted with Glu-F/ Glu-R, Glu-P4/Glu-R, Glu-P3/Glu-R, Glu-P2/Glu-R and Glu-P1/Glu-R, and Ah-Glu-P and 4 deleted fragments Ah-Glu-P4~Ah-Glu-P1 with the length of 931, 767, 650, 376 and 217 bp were obtained successfully, respectively. The 5 fragments were then transferred to plasmid pCAMBIA1301-xylA, replacing the Cauliflower mosaic virus 35S promoter (CaMV35S), and 5 corresponding plant expression vectors pCAMBIA1301-xylA-Glu-P~ pCAMBIA1301-xylA-Glu-P1 with xylose isomerase gene (xylA) as safe maker and with β-glucuronidase gene (GUS) as reporter gene were constructed successfully. These plant expression vectors were then transformed to Agrobacterium tumefaciens EHA105 using freezing and thawing method, and further transformed to onion (Allium cepa) epidermal cells by Agrobacterium mediated transformation. GUS histochemical staining and GUS enzyme activity in these transgenic onions were detected. GUS staining on the onion epidermal cells transformed by Ah-Glu-Pro showed that the cells appeared blue when were induced by 5.0 mmol/L SA, while appeared light blue without induction by SA. Those onion epidermal cells transformed by pCAMBIA1301-xylA-Glu-P4~pCAMBIA1301-xylA-Glu-P1 appeared blue when induced by 5.0 mmol/L SA. Those onion epidermal cells transformed by Ah-Glu-P~Ah-Glu-P4 appeared much deep blue than those cells transformed by Ah-Glu-P3~Ah-Glu-P1. GUS enzyme activity in onions transformed by Ah-Glu-P~Ah-Glu-P3 were increased of 1.45, 2.16 and 1.27 times, respectively, after SA induction than that without SA induction. GUS enzyme activity in onions transformed by Ah-Glu-P2~Ah-Glu-P1 had no significant difference between with and without SA induction. Consideration of the predictive cis-regulatory elements showed that RAV1AAT, MYBCOREATCYCB1 and INRNTPSADB within Ah-Glu-P, Ah-Glu-P4 and Ah-Glu-P3 might be positive cis-elements, and GT1-motif and AMMORESIVDCRNIA1 between Ah-Glu-P and Ah-Glu-P4 might be negative cis-elements. The further function confirmation on these cis-regulatory elements will provide theoretical basis for regulating the peanut endogenous β-1,3-glucanase gene efficient expression, improving the disease resistance of peanut, and effective utilization of inducible promoter in the process of peanut genetic transformation.
|
|
Received: 03 December 2015
Published: 06 May 2016
|
|
Corresponding Authors:
lixian qiao
E-mail: lxqiao73@163.com
|
|
|
|
| [1]何江峰, 韩冰, 赵宏鑫.植物β-, -葡聚糖酶的研究进展[J].内蒙古农业科技, 2007, 05期(05):21-25[2] 李春娟, 单世华, 许婷婷, 等.几丁质酶和β-1, 3-葡聚糖酶基因研究进展[J].生物技术通讯, 2004, 15(5):502-505[3]Kim Y J, Hwang B K.Isolation of a basic 34 kiloDalton β-1,3-glucanase with inhibitory activity against Phytophthora capsici from pepper stems[J].Physiological & Molecular Plant Pathology, 1997, 50(2):103-115[4]聂丽娜, 夏兰琴, 徐兆师, 等.植物基因启动子的克隆及其功能研究进展[J].植物遗传资源学报, 2008, 9(3):385-391[5]李云锋, 胡川, 曹燕, 等.转基因水稻中β-, -葡聚糖酶基因启动子缺失体的激发子诱导活性[J].植物病理学报, 2009, 39(2):147-152[6]Moreno A B, Pe?as G, Rufat M, et al.Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice[J].Molecular plant-microbe interactions : MPMI, 2005, 18(9):960-72[7]朱丽萍, 于壮, 邹翠霞, 等.植物逆境相关启动子及功能[J].遗传, 2010, 32(3):229-234[8]Castresana C, Carvalho F D, Gheysen G, et al.Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene[J].Plant Cell, 1990, 2(12):1131-1143[9]Samac D A, Shah D M.Developmental and Pathogen-Induced Activation of the Arabidopsis Acidic Chitinase Promoter[J].Plant Cell, 1991, 3(10):1063-1072[10]Pontier D, Balagué C, Bezombes-Marion I, et al.Identification of a novel pathogen-responsive element in the promoter of the tobacco gene HSR203J,a molecular marker of the hypersensitive response[J].Plant Journal for Cell & Molecular Biology, 2001, 26(5):495-507[11]Chang J Q, Wang X L, Yang Y, et al.Cloning of Cold-Induced Promoter CBF 3 from Arabidopsis thaliana and Its Activity Detection in Transgenic Tobacco[J].Biotechnology, 2009, 19(5):5-7[12]Zuo J, Chua N H.Chemical-inducible systems for regulated expression of plant genes[J].Current Opinion in Biotechnology, 2000, 11(2):146-151[13] 王合春, 陈新利, 隋炯明, 等.花生β-1, 3-葡聚糖酶基因启动子的克隆及功能分析[J]. 植物遗传资源学报, 2013, 14(5).[J].植物遗传资源学报, 2013, 14(5):864-870[14]Kanzaki H, Ryohei T.Regulation of expression of rice thaumatin-like protein: inducibility by elicitor requires promoter W-box elements[J].Plant Cell Reports, 2008, 27(9):1521-1528[15]Bradford M M.A Rapid Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,AnalBiochem. 72,248-254[J].Analytical Biochemistry, 1976, 72(1):248-254[16]牟少亮, 蔡金森, 严雁, 等.水稻-启动子的克隆及其功能分析[J].核农学报, 2013, 27(12):1803-1808[17] 李燕.紫花苜蓿诱导表达启动子MsZPP的克隆及功能分析[D]. 北京畜牧兽医研究所, 2012, 39-41.[18]Chen X L, Song R T, Yu M Y, et al.Cloning and functional analysis of the chitinase gene promoter in peanut[J].Genetics & Molecular Research, 2015, 14(4):12710-12722[19]Agius F, Amaya I, Botella M A, et al.Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression[J].Journal of Experimental Botany, 2005, 56(409):37-46[20]Loppes R, Radoux M.Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtii[J].Plant Mol Biol, 2001, 45(2):215-227[21] 邵元华.杨树NPR1基因的克隆及功能验证[D]. 安徽农业大学, 2012.[22]Nakamura M, Tsunoda T, Obokata J.Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator[J].Plant Journal for Cell & Molecular Biology, 2002, 29(1):1-10 |
|
|
|
