大豆硫转运蛋白基因GmSULTR1;2b启动子的克隆及活性分析

【目的】硫转运蛋白（sulfate transporter，SULTR）参与根系对外界环境中硫酸根（SO₄^2-）的吸收与转运。大豆硫转运蛋白基因GmSULTR1;2b在根中特异表达，其功能是将外界的SO₄^2-吸收转运到植物根系中。文章克隆大豆硫转运蛋白GmSULTR1;2b的启动子，研究该启动子的驱动活性和组织表达情况，从而了解GmSULTR1;2b的调控机制，为提高大豆含硫氨基酸含量提供分子依据。【方法】根据NCBI中GmSULTR1;2b的序列，分析预测该基因上游2 259 bp为启动子，并利用在线数据库PLACE和Plant-CARE预测该启动子序列的调控元件。以大豆品种南农N2899的DNA为模板，进行普通PCR扩增，将克隆的启动子序列与GUS连接构建植物重组表达载体pSULTR1;2b∷GUS。利用冻融法将重组质粒转入农杆菌EHA105中，通过农杆菌介导的遗传转化法转化大豆进行瞬时表达，以GUS为报告基因对启动子的活性进行分析。另外，将重组质粒转入发根农杆菌K599中进行大豆毛状根转化试验，借助于GUS报告基因，通过体视镜观察毛状根的横切面，分析启动子在根中的表达情况。最后以转化的阳性毛状根为材料，通过GUS酶活试验（GUS activity）分析启动子的活性。【结果】克隆大豆品种南农N2899的GmSULTR1;2b启动子与NCBI序列基本一致。通过在线预测分析启动子的调控元件发现该启动子具有真核生物启动子必须的核心元件TATA-box外，还含有激素应答元件ERE（乙烯响应元件）、ABRE（脱落酸响应元件）等，胁迫应答元件TC-rich repeats（干旱胁迫以及病虫害胁迫）、AT-rich element（AT-rich的DNA与蛋白结合位点）和MYB等。重组载体pSULTR1;2b∷GUS经PCR和测序鉴定，证实已构建成功。大豆瞬时表达后进行X-gluc染色显示，重组载体侵染的大豆显蓝色，说明GmSULTR1;2b启动子能够驱动下游GUS的表达。对转化的毛状根染色之后，体视镜下观察阳性根的横切面，发现GUS主要在根毛、根表皮和中柱内表达，表明GmSULTR1;2b启动子主要在根毛、根表皮和中柱内表达。对转化毛状根进行GUS酶活试验（GUS activity）说明该启动子的启动活性比CaMV35S启动子的启动活性弱。【结论】克隆了GmSULTR1;2b启动子序列，该启动子具有驱动下游GUS的表达的功能，而且该启动子在根毛、根表皮和中柱内表达。

Cloning and Activity Analysis of the Promoter of Sulfate Transporter Gene GmSULTR1;2b

【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.