CAJ | 학술논문

【目的】硫转运蛋白（sulfate transporter，SULTR）参与根系对外界环境中硫酸根（SO42-）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SO42-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2259 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。【目的】硫轉運蛋白（sulfate transporter，SULTR）參與根繫對外界環境中硫痠根（SO42-）的吸收與轉運。大豆硫轉運蛋白基因GmSULTR1；2b在根中特異錶達，其功能是將外界的SO42-吸收轉運到植物根繫中。文章剋隆大豆硫轉運蛋白GmSULTR1；2b的啟動子，研究該啟動子的驅動活性和組織錶達情況，從而瞭解GmSULTR1；2b的調控機製，為提高大豆含硫氨基痠含量提供分子依據。【方法】根據NCBI中GmSULTR1；2b的序列，分析預測該基因上遊2259 bp為啟動子，併利用在線數據庫PLACE和Plant-CARE預測該啟動子序列的調控元件。以大豆品種南農 N2899的 DNA 為模闆，進行普通 PCR 擴增，將剋隆的啟動子序列與 GUS連接構建植物重組錶達載體pSULTR1；2b∷GUS。利用凍融法將重組質粒轉入農桿菌EHA105中，通過農桿菌介導的遺傳轉化法轉化大豆進行瞬時錶達，以GUS為報告基因對啟動子的活性進行分析。另外，將重組質粒轉入髮根農桿菌K599中進行大豆毛狀根轉化試驗，藉助于GUS報告基因，通過體視鏡觀察毛狀根的橫切麵，分析啟動子在根中的錶達情況。最後以轉化的暘性毛狀根為材料，通過GUS酶活試驗（GUS activity）分析啟動子的活性。【結果】剋隆大豆品種南農N2899的GmSULTR1；2b啟動子與NCBI序列基本一緻。通過在線預測分析啟動子的調控元件髮現該啟動子具有真覈生物啟動子必鬚的覈心元件 TATA-box 外，還含有激素應答元件 ERE（乙烯響應元件）、ABRE（脫落痠響應元件）等，脅迫應答元件TC-rich repeats（榦旱脅迫以及病蟲害脅迫）、AT-rich element（AT-rich的DNA與蛋白結閤位點）和MYB等。重組載體pSULTR1；2b∷GUS經PCR和測序鑒定，證實已構建成功。大豆瞬時錶達後進行X-gluc染色顯示，重組載體侵染的大豆顯藍色，說明GmSULTR1；2b啟動子能夠驅動下遊GUS的錶達。對轉化的毛狀根染色之後，體視鏡下觀察暘性根的橫切麵，髮現GUS主要在根毛、根錶皮和中柱內錶達，錶明GmSULTR1；2b啟動子主要在根毛、根錶皮和中柱內錶達。對轉化毛狀根進行GUS酶活試驗（GUS activity）說明該啟動子的啟動活性比CaMV35S啟動子的啟動活性弱。【結論】剋隆瞭GmSULTR1；2b啟動子序列，該啟動子具有驅動下遊GUS的錶達的功能，而且該啟動子在根毛、根錶皮和中柱內錶達。
【목적】류전운단백（sulfate transporter，SULTR）삼여근계대외계배경중류산근（SO42-）적흡수여전운。대두류전운단백기인GmSULTR1；2b재근중특이표체，기공능시장외계적SO42-흡수전운도식물근계중。문장극륭대두류전운단백GmSULTR1；2b적계동자，연구해계동자적구동활성화조직표체정황，종이료해GmSULTR1；2b적조공궤제，위제고대두함류안기산함량제공분자의거。【방법】근거NCBI중GmSULTR1；2b적서렬，분석예측해기인상유2259 bp위계동자，병이용재선수거고PLACE화Plant-CARE예측해계동자서렬적조공원건。이대두품충남농 N2899적 DNA 위모판，진행보통 PCR 확증，장극륭적계동자서렬여 GUS련접구건식물중조표체재체pSULTR1；2b∷GUS。이용동융법장중조질립전입농간균EHA105중，통과농간균개도적유전전화법전화대두진행순시표체，이GUS위보고기인대계동자적활성진행분석。령외，장중조질립전입발근농간균K599중진행대두모상근전화시험，차조우GUS보고기인，통과체시경관찰모상근적횡절면，분석계동자재근중적표체정황。최후이전화적양성모상근위재료，통과GUS매활시험（GUS activity）분석계동자적활성。【결과】극륭대두품충남농N2899적GmSULTR1；2b계동자여NCBI서렬기본일치。통과재선예측분석계동자적조공원건발현해계동자구유진핵생물계동자필수적핵심원건 TATA-box 외，환함유격소응답원건 ERE（을희향응원건）、ABRE（탈락산향응원건）등，협박응답원건TC-rich repeats（간한협박이급병충해협박）、AT-rich element（AT-rich적DNA여단백결합위점）화MYB등。중조재체pSULTR1；2b∷GUS경PCR화측서감정，증실이구건성공。대두순시표체후진행X-gluc염색현시，중조재체침염적대두현람색，설명GmSULTR1；2b계동자능구구동하유GUS적표체。대전화적모상근염색지후，체시경하관찰양성근적횡절면，발현GUS주요재근모、근표피화중주내표체，표명GmSULTR1；2b계동자주요재근모、근표피화중주내표체。대전화모상근진행GUS매활시험（GUS activity）설명해계동자적계동활성비CaMV35S계동자적계동활성약。【결론】극륭료GmSULTR1；2b계동자서렬，해계동자구유구동하유GUS적표체적공능，이차해계동자재근모、근표피화중주내표체。
[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(SO42-). 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.