大豆硫转运蛋白基因GmSULTR1;2b启动子的克隆及活性分析

doi:10.3864/j.issn.0578-1752.2015.08.20

Abstract

Abstract: 【Objective】 Following the nitrogen, phosphorus and potassium, sulfur is the fourth nutrient necessary for the plants. Sulfur-containing organic compounds involved in many important physiological and biochemical reactions in plants, which play an important role in withstanding environmental stress and growth and development of plants. Sulfate transporters (sulfate transporter, SULTR) participate in absorption and transportation of the exogenous sulfate(SO₄^2-). The sulfate transporter gene GmSULTR1;2b of soybean is specifically expressed in the root, which plays a role in transporting sulfate from the environment. Cloning the promoter of GmSULTR1;2b, and studying on its activity and tissue expression will contribute to understanding the regulatory mechanism of GmSULTR1;2b. It can also provide a molecular foundation for improving the content of sulfur amino acid in soybean.【Method】According to the sequence of GmSULTR1;2b in the NCBI, the predicted 2 259 bp upstream was analyzed and predicted as the promoter. The online debases PLACE and Plant-CARE were used to prognose the regulatory elements of the sequence. The sequence was obtained by PCR through taking the soybean cultivars Nannong N2899 DNA as template. The sequence was fused with GUS to construct the plant expression vector pSULTR1;2b::GUS. The binary vector constructs were transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. The transient expression assays were carried out in soybean by Agrobacterium tumefaciens-mediated method, and the activity of the promoter was analyzed by using GUS as the reporter gene. In addition, hairy root transformation experiment was carried out by transforming the binary vector constructs into Agrobacterium rhizogenes K599. By analyzing the transverse section of the hairy roots under the stereoscope, its expression was observed. Finally, the GUS activity was implemented to test the activity of the promoter, which was based on the positive transformed hairy roots.【Result】The cloned promoter sequence of GmSULTR1;2b from Nannong N2899 was basically in line with the sequence in NCBI. Through online prediction analysis of regulatory elements, it was found that the promoter contained not only TATA-box, which was the necessary component of eukaryote, but also contained hormone response element ERE (ethylene response element), ABRE (abscisic acid response element), stress response element TC - rich repeats (diseases and insect pests stress and drought stress), the AT - rich element (the DNA of AT - rich and protein binding sites) and MYB, etc. The successful construction of the recombination vector pSULTR1;2b::GUS was confirmed via PCR and sequencing appraisal. The X-gluc dyeing conducted on the transient expression of soybean showed blue where the soybean was infected by the recombinant vector pSULTR1;2b::GUS. It indicated that the promoter could drive GUS expression downstream. After staining the transformed hairy roots, the transverse section of the positive hairy roots was analyzed under the stereoscope. The GUS was mainly found in the root hair, root epidermis and the stele, which manifested the promoter mainly expressed in the root hair, root epidermis and the stele. The GUS activity test of the transformed hairy roots attested weaker activity than the promoter of CaMV35S.【Conclusion】GmSULTR1;2b promoter was cloned. It could drive GUS in the downstream, and express in the root hairs, root epidermis and the column.

Key words: GmSULTR1;2b, promoter, transient expression, GUS activity

ZHOU Xiao-qiong, DING Yi-qiong, ZUO Li, YU De-yue. Cloning and Activity Analysis of the Promoter of Sulfate Transporter Gene GmSULTR1;2b[J].Scientia Agricultura Sinica, 2015, 48(8): 1650-1659.

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