CN103667315A - Salt-tolerant and drought-resistant gene TaDHN1 of wheat, recombinant plasmid and application - Google Patents
Salt-tolerant and drought-resistant gene TaDHN1 of wheat, recombinant plasmid and applicationDownload PDFInfo
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Translated fromChinese技术领域technical field
本发明属于植物基因工程技术领域，尤其涉及小麦耐盐、抗旱基因TaDHN1、重组质粒及应用。 The invention belongs to the technical field of plant genetic engineering, and in particular relates to a wheat salt-tolerant and drought-resistant geneTaDHN1, a recombinant plasmid and its application.
背景技术Background technique
近年来，各种非生物胁迫严重影响作物的产量，其中盐和干旱胁迫对植物的影响尤为突出。通过基因工程培育耐逆新品种是有效利用盐碱地、提高植物的抗旱能力，增加粮食产量，保障粮食安全的重要手段。目前利用基因工程技术开展植物耐盐、抗旱方面的研究已取得了较大的的进展。一些实验表明，将植物本身以及其他生物中与耐盐相关的基因转入植物中，其异源转录和翻译产物可以使转基因植物的耐盐能力提高。 In recent years, various abiotic stresses have seriously affected the yield of crops, among which salt and drought stress have particularly prominent effects on plants. Cultivating stress-tolerant new varieties through genetic engineering is an important means to effectively utilize saline-alkali land, improve plant drought resistance, increase food production, and ensure food security. At present, great progress has been made in the study of plant salt tolerance and drought resistance by using genetic engineering technology. Some experiments have shown that the salt-tolerance-related genes in the plant itself and other organisms are transferred into plants, and their heterologous transcription and translation products can improve the salt-tolerance ability of transgenic plants. the
小麦是全世界35%以上人口的主食，也是山东省主要粮食作物之一。但小麦属甜土植物，在盐渍化土壤中生长极差，因此克隆耐盐基因，培育耐盐小麦新品种非常重要。 Wheat is the staple food of more than 35% of the world's population, and it is also one of the main food crops in Shandong Province. However, wheat is a sweet-soil plant that grows extremely poorly in saline soils. Therefore, it is very important to clone salt-tolerant genes and breed new salt-tolerant wheat varieties. the
脱水素（又称为第二类胚发育后期丰富蛋白）是ABA依赖的盐胁迫信号转导通路下游的功能基因，其最早发现于胚发育后期，在种子成熟后期的脱水过程中大量表达。后来研究表明，除了参与植物正常的生长发育外，脱水素基因主要参与了植物对高盐、干旱、冷等各种非生物胁迫的抗性。 Dehydrin (also known as the second type of post-embryonic abundant protein) is a functional gene downstream of the ABA-dependent salt stress signal transduction pathway. It was first discovered in the late embryo development and expressed in large quantities during the dehydration process of the late seed maturity. Later studies showed that, in addition to participating in normal plant growth and development, dehydrin genes are mainly involved in plant resistance to various abiotic stresses such as high salt, drought, and cold. the
发明内容Contents of the invention
本发明的目的是提供一种耐盐、抗旱基因——小麦脱水素基因TaDHN1、重组质粒及其应用。我们在前期芯片杂交实验中发现一个代表脱水素基因的探针在盐处理后的小麦根中显著上调表达，本研究在前期研究的基础上，克隆了该脱水素基因，功能研究表明，过表达该基因可提高植物的耐盐和抗旱能力。 The purpose of the present invention is to provide a salt-tolerant and drought-resistant gene—wheat dehydrin geneTaDHN1, recombinant plasmid and application thereof. In the previous microarray hybridization experiments, we found that a probe representing a dehydrin gene was significantly up-regulated in wheat roots after salt treatment. Based on the previous research, this study cloned the dehydrin gene. Functional studies showed that overexpression The gene can improve the salt tolerance and drought resistance of plants.
本发明的技术方案在于：首先根据小麦的表达谱芯片数据选出在小麦幼苗根中显著受盐诱导表达的脱水素基因（探针），然后根据探针序列设计基因特异性引物，从小麦幼苗根的全长cDNA文库中克隆脱水素基因TaDHN1全长cDNA。测序获得序列后，构建包含TaDHN1基因完整ORF的稳定过表达载体，最后在拟南芥中进行功能验证。 The technical scheme of the present invention is: firstly select the dehydrin gene (probe) that is significantly induced by salt in wheat seedling roots according to the wheat expression profile chip data, and then design gene-specific primers according to the probe sequence, from wheat seedlings The full-length cDNA of the dehydrin geneTaDHN1 was cloned from the root full-length cDNA library. After the sequence was obtained by sequencing, a stable overexpression vector containing the complete ORF of theTaDHN1 gene was constructed, and finally its function was verified in Arabidopsis.
本发明提供的小麦耐盐、抗旱基因名称为TaDHN1，所述基因cDNA的核苷酸序列如SEQ ID No.1所示，其氨基酸序列为SEQ ID No.2所示。 The name of the wheat salt-tolerant and drought-resistant gene provided by the present invention isTaDHN1 , the nucleotide sequence of the gene cDNA is shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID No.2.
本发明还提供了含上述小麦基因TaDHN1的重组质粒pCAMBIA-super1300/TaDHN1：通过引物向SEQ ID No.1序列两侧引入XbaI和SacI双酶切位点，然后由XbaI和SacI双酶切植物表达载体pCAMBIA-super1300和SEQ ID No.1所示的基因片断，回收载体大片段和目标基因片段再连接获得， The present inventionalso provides the recombinant plasmid pCAMBIA-super1300/TaDHN1 containing the above-mentioned wheat geneTaDHN1 : introduceXba I andSac I double restriction sites on both sides of the sequence of SEQ ID No. Digest the plant expression vector pCAMBIA-super1300 and the gene fragment shown in SEQ ID No.1, recover the large fragment of the vector and reconnect the target gene fragment,
所述引物序列为：D1ORF5: 5’-TCTAGAATGGAGTACCAGGGGCAGCAC-3’（XbaI）The primer sequence is: D1ORF5: 5'-TCTAGAATGGAGTACCAGGGGCAGCAC-3' (Xba I)
D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’(SacI)。D1ORF3: 5'-GAGCTCTCAGTGCTGTCCGCCGGG-3' (Sac I).
本发明所述重组质粒的特征在于外源基因的表达框中含有TaDHN1基因的完整的编码框，即全长ORF（open reading frame）。 The recombinant plasmid of the present invention is characterized in that the expression frame of the exogenous gene contains a complete coding frame of theTaDHN1 gene, that is, a full-length ORF (open reading frame).
本发明提供了一种小麦耐盐、抗旱基因TaDHN1在培育耐盐和抗旱植物中的应用， The invention provides the application of a wheat salt-tolerant and drought-resistant geneTaDHN1 in cultivating salt-tolerant and drought-resistant plants,
本发明所述耐盐和抗旱植物是小麦、玉米和水稻单子叶植物。The salt-tolerant and drought-resistant plants of the present invention are monocotyledonous plants of wheat, corn and rice.
重组质粒pCAMBIA-super1300/TaDHN1在培育耐盐、抗旱植物中的应用。 Application of recombinant plasmid pCAMBIA-super1300/TaDHN1 in cultivating salt-tolerant and drought-resistant plants. the
本发明提供了提高含有基因TaDHN1植物耐盐和抗旱性的方法，是将基因TaDHN1导入宿主植物细胞、组织或植株个体，得到具有耐盐、抗旱性能的植株，所述的宿主植物为小麦、玉米和水稻单子叶植物。 The invention provides a method for improving the salt tolerance and drought resistance of plants containing the geneTaDHN1 . The method is to introduce the geneTaDHN1 into host plant cells, tissues or plant individuals to obtain plants with salt tolerance and drought resistance. The host plants are wheat and corn. and rice monocots.
本发明的有益效果：本发明首次克隆得到了小麦脱水素基因TaDHN1，并通过根瘤农杆菌介导的方法将该基因转入拟南芥，经过比较分析证明，转基因植株的耐盐、抗旱能力明显提高。 Beneficial effects of the present invention: the present invention clones the wheat dehydrin geneTaDHN1 for the first time, and transfers the gene into Arabidopsis thaliana through the method mediated by Agrobacterium tumefaciens. It is proved by comparative analysis that the transgenic plants have obvious salt tolerance and drought resistance improve.
附图说明Description of drawings
图1 为TaDHN1基因全长cDNA 序列的扩增结果， Figure 1 is the amplification result of the full-length cDNA sequence ofTaDHN1 gene,
其中M 为λDNA/(EcoRⅠ+ Hind Ⅲ)Marker；下同，Where M is λDNA/(Eco RI+Hin d Ⅲ)Marker; the same below,
图2为 ABA和NaCl处理后TaDHN1基因在小麦幼苗根和叶中的RT-PCR分析：Fig. 2 is the RT-PCR analysis ofTaDHN1 gene in wheat seedling root and leaf after ABA and NaCl process:
图2-a 为TaDHN1在ABA处理的小麦幼苗叶中的表达情况；Figure 2-a shows the expression ofTaDHN1 in ABA-treated wheat seedling leaves;
图2-b为TaDHN1在ABA处理的小麦幼苗根中的表达情况；Figure 2-b shows the expression ofTaDHN1 in ABA-treated wheat seedling roots;
图2- c为TaDHN1在NaCl处理的小麦幼苗叶中的表达情况；Figure 2-c is the expression ofTaDHN1 in NaCl-treated wheat seedling leaves;
图2-d为TaDHN1在NaCl处理的小麦幼苗根中的表达情况，Figure 2-d shows the expression ofTaDHN1 in NaCl-treated wheat seedling roots,
图3 为构建的植物表达载体pCAMBIA-super1300/TaDHN1的XbarI、SacI双酶切验证结果，Figure 3 is the result ofXbar I andSac I double enzyme digestion verification of the constructed plant expression vector pCAMBIA-super1300/TaDHN1,
图4为转表达载体pCAMBIA-super1300/TaDHN1的农杆菌菌液PCR检测结果，Fig. 4 is the PCR detection result of the Agrobacterium liquid of the expression vector pCAMBIA-super1300/TaDHN1,
图5 为转基因拟南芥株系的表型鉴定：Figure 5 is the phenotypic identification of transgenic Arabidopsis lines:
图5-A：野生型Col-0及转基因拟南芥株系在不同浓度NaCl处理下初生根根长统计结果，Figure 5-A: Statistical results of root length of primary roots of wild-type Col-0 and transgenic Arabidopsis lines treated with different concentrations of NaCl,
其中：WT：未转化的Col-0野生型拟南芥；8、20为转pCAMBIA-super1300/TaDHN1的不同转基因拟南芥株系；CK为对照，即在正常的MS培养基上培养；﹡：P﹤0.05，﹡﹡：P﹤0.01，Among them: WT: untransformed Col-0 wild-type Arabidopsis; 8 and 20 are different transgenic Arabidopsis lines transformed with pCAMBIA-super1300/TaDHN1; CK is the control, that is, cultured on normal MS medium; : P﹤0.05,﹡﹡: P﹤0.01,
图5-B：野生型Col-0及转基因拟南芥株系在不同甘露醇浓度处理下初生根根长统计结果，Figure 5-B: Statistical results of root length of primary roots of wild-type Col-0 and transgenic Arabidopsis lines under different mannitol concentrations,
图5-C：正常生长3周的拟南芥及正常生长3周然后干旱处理15天的拟南芥生长情况。Figure 5-C: Arabidopsis thaliana grown normally for 3 weeks and Arabidopsis grown normally for 3 weeks and then drought-treated for 15 days.
具体实施方式Detailed ways
实施例1Example 1
TaDHN1基因cDNA序列的克隆Cloning of cDNA sequence ofTaDHN1 gene
1．1 TaDHN1基因全长cDNA序列的克隆和序列测定1.1 Cloning and sequencing of the full-length cDNA sequence ofTaDHN1 gene
1．引物序列1. Primer sequence
根据芯片探针序列设计基因特异性的下游引物，与文库的5′端锚定引物进行配对,以小麦幼根的全长cDNA文库为模板扩增基因的全长cDNA。Gene-specific downstream primers were designed according to the chip probe sequence, paired with the 5′-end anchor primer of the library, and the full-length cDNA of the gene was amplified using the full-length cDNA library of wheat young roots as a template.
引物序列为：TaDHN1：5′- GAGACGAACAGAGGTAGTACATG -3′， The primer sequence is: TaDHN1: 5′- GAGACGAACAGAGGTAGTACATG -3′,
NT3：5’-ACTAAAGggaACAAAAGCTGG AG-3’。NT3: 5'-ACTAAAGggaACAAAAGCTGG AG-3'.
2．PCR 反应体系（50μL） 2. PCR reaction system (50μL)
2×GC bufferⅠ 10μl2×GC bufferⅠ 10μl
模板cDNA文库 1ulTemplate cDNA library 1ul
dNTPs（2.5mM each） 0.5μldNTPs (2.5mM each) 0.5μl
Primer1 (10μM) 1μlPrimer1 (10μM) 1μl
Primer2(10μM) 1μlPrimer2(10μM) 1μl
LA Taq(TaKaRa) 0.5ulLA Taq(TaKaRa) 0.5ul
ddH2O 加至终体积50μlAdd ddH2 O to a final volume of 50 μl
3．PCR 反应程序为3. The PCR reaction procedure is
94℃ 预变性3min；94℃变性45sec，58℃复性1min，72℃延伸1min，循环35 次；72℃延伸5min。Pre-denaturation at 94°C for 3min; denaturation at 94°C for 45sec, renaturation at 58°C for 1min, extension at 72°C for 1min, cycle 35 times; extension at 72°C for 5min.
4．1％琼脂糖凝胶电泳 4.1% agarose gel electrophoresis
PCR扩增产物用1％琼脂糖凝胶电泳检测后发现在700bp处有一条目的条带（图1）。After the PCR amplification product was detected by 1% agarose gel electrophoresis, a band was found at 700bp (Figure 1).
5．扩增片段的回收、与T载体的链接 5. Recovery of amplified fragments, linking with T vectors
对扩增条带采用Tiangen公司的琼脂糖胶回收试剂盒，步骤按说明书进行。PCR产物与pGEM-T (Promega)载体连接，连接体系为：For the amplified bands, the agarose gel recovery kit from Tiangen Company was used, and the steps were carried out according to the instructions. The PCR product was connected to the pGEM-T (Promega) vector, and the connection system was:
回收的PCR产物 7μl
10×T4连接酶缓冲液 1μl10×T4 ligase buffer 1 μl
pGEM-T载体(50ng/μl) 1μlpGEM-T vector (50ng/μl) 1μl
T4DNA连接酶（3U/μl）(Takara) 1μlT4DNA Ligase (3U/μl) (Takara) 1μl
dH2O至10μldH2O to 10 μl
于16℃水浴连接过夜。Incubate overnight in a water bath at 16°C.
6．回收片段的克隆与测序 6. Cloning and sequencing of recovered fragments
（1）大肠杆菌感受态细胞的制备(1) Preparation of Escherichia coli competent cells
①从-80℃冰箱中拿出保存于甘油中的E.coli DH5a菌株（购自天为时代生物公司），放在冰上缓慢解冻；①Take out theE.coli DH5a strain preserved in glycerol (purchased from Tianwei Times Biotechnology Co., Ltd.) from the -80°C refrigerator, and slowly thaw it on ice;
②在超净工作台上用接种环划线（LB固体培养基，不含Amp）；② Streak a line with an inoculation loop on an ultra-clean workbench (LB solid medium, without Amp);
③将平板于37℃恒温培养箱倒置培养过夜；③Incubate the plate upside down in a constant temperature incubator at 37°C overnight;
④挑取平板上的单菌落接种于含5 ml LB液体培养基的试管中，37℃振荡培养14－16小时；④Pick a single colony on the plate and inoculate it into a test tube containing 5 ml of LB liquid medium, and culture with shaking at 37°C for 14-16 hours;
⑤取0.5 ml菌液接种于含50 ml LB液体培养基的250ml的三角瓶中，37℃振荡（260rpm）培养2－3小时（OD260=0.5）；⑤ Inoculate 0.5 ml of bacterial liquid into a 250ml Erlenmeyer flask containing 50 ml of LB liquid medium, and incubate at 37°C for 2-3 hours with shaking (260rpm) (OD260=0.5);
⑥将培养的菌液置冰上1小时；⑥Put the cultured bacteria solution on ice for 1 hour;
⑦4℃离心4分钟（4000rpm），去上清；⑦ Centrifuge at 4°C for 4 minutes (4000rpm), remove the supernatant;
⑧用25 ml冰预冷的溶液A将菌体轻轻悬浮起来，再在冰上放置40－45分钟；⑧Use 25 ml of ice-precooled solution A to gently suspend the bacteria, and then place it on ice for 40-45 minutes;
⑨重复步骤（8）；⑨Repeat step (8);
⑩用2.5ml冰预冷的溶液B将菌体轻轻悬浮起来，然后将菌液分装到1.5ml离心管中（每管100μl），放入-80℃冰箱备用。⑩Use 2.5ml ice-cooled solution B to gently suspend the bacteria, then divide the bacteria solution into 1.5ml centrifuge tubes (100μl per tube), and put them in a -80°C refrigerator for later use.
（2）连接产物的转化 (2) Conversion of ligation products
①从-80℃冰箱中取出1管（100μl）感受态细胞，置冰上缓缓解冻30分钟；①Take out 1 tube (100 μl) of competent cells from the -80°C refrigerator, and place it on ice to slowly thaw for 30 minutes;
②超净工作台上向管中加入5μl连接反应物，轻轻摇匀，置冰上30分钟；② Add 5 μl of the ligation reaction to the tube on the ultra-clean bench, shake gently, and place on ice for 30 minutes;
③42℃水浴热激90秒，迅速置冰上3－5分钟；③ Heat shock in a water bath at 42°C for 90 seconds, then quickly place on ice for 3-5 minutes;
④在超净工作台上向离心管中加入1ml LB液体培养基（不含Amp），混匀后37℃振荡培养1小时（260rpm）；④Add 1ml of LB liquid medium (without Amp) to the centrifuge tube on the ultra-clean workbench, mix well and incubate with shaking at 37°C for 1 hour (260rpm);
⑤室温下5000rpm离心6分钟，弃掉900μl上清液，将剩余100μl上清液重新悬浮菌体，加入40μl的X-gal和4μl的IPTG，混匀，然后用涂布器将其均匀涂到准备的平板上，放置30分钟；⑤Centrifuge at 5000rpm for 6 minutes at room temperature, discard 900μl of the supernatant, resuspend the bacteria in the remaining 100μl of the supernatant, add 40μl of X-gal and 4μl of IPTG, mix well, and then spread it evenly on the Place on the prepared plate for 30 minutes;
⑥倒置平板于37℃恒温培养箱中过夜，待出现明显单菌落时拿出；⑥Put the plate upside down in a constant temperature incubator at 37°C overnight, and take it out when there are obvious single colonies;
⑦放入4℃冰箱数小时，使蓝白斑颜色分明；⑦ Put it in the refrigerator at 4°C for several hours to make the blue and white spots clear;
⑧用灭菌的牙签挑取白斑于装有10ml LB液体培养基（含60μg/mlAmp）的试管中，37℃摇菌过夜。⑧Use a sterilized toothpick to pick the white spot and place it in a test tube filled with 10ml LB liquid medium (containing 60μg/ml Amp), shake the bacteria overnight at 37°C.
（3）重组质粒的PCR鉴定及测序 (3) PCR identification and sequencing of recombinant plasmids
本实验所用的pGEM-T载体的克隆位点的上游有T7启动子，下游有SP6启动子，所以可以用这两个启动子序列做引物对插入片段进行扩增，对重组质粒进行鉴定；The pGEM-T vector used in this experiment has a T7 promoter upstream and a SP6 promoter downstream of the cloning site, so these two promoter sequences can be used as primers to amplify the insert and identify the recombinant plasmid;
①PCR程序：94℃ 预变性10min；94℃变性1min，58℃复性1min，72℃延伸1min，循环35 次；72℃延伸5min；①PCR program: Pre-denaturation at 94°C for 10 minutes; denaturation at 94°C for 1 minute, renaturation at 58°C for 1 minute, extension at 72°C for 1 minute, cycle 35 times; extension at 72°C for 5 minutes;
②PCR扩增产物用1％琼脂糖电泳检测，取阳性克隆的菌液送公司进行测序。测序结果表明所得序列包含一个完整的ORF，长402 bp。全长ORF序列见SEQ ID No.1。②PCR amplification products were detected by 1% agarose electrophoresis, and the bacterial solution of positive clones was sent to the company for sequencing. Sequencing results showed that the obtained sequence contained a complete ORF with a length of 402 bp. See SEQ ID No.1 for the full-length ORF sequence.
实施例2Example 2
ABA、NaCl处理条件下TaDHN1 基因的表达分析Expression analysis ofTaDHN1 gene under ABA and NaCl treatment conditions
2．1 材料处理2.1 Material Handling
小麦材料山融3号的种子正常萌发，1周后去掉胚乳，Hangload培养液继续培养1周。盐胁迫在液体培养基中施加200 mM NaCl，ABA处理施加100 μM ABA，分别在处理后的0、0.5、3、12、24，48小时取幼嫩的叶片和根系用于提取总RNA。The seeds of the
2．2 Trizol法提取小麦Total RNA。 2.2 Trizol method to extract wheat Total RNA. the
1．将组织材料放入液氮预冷的研钵中，在液氮中充分研磨成粉末； 1. Put the tissue material into a liquid nitrogen pre-cooled mortar, and fully grind it into powder in liquid nitrogen;
2．待液氮挥发干，立即转移到2ml的离心管中，每100mg材料约加入1ml的Invitrogen公司的TRIzol提取液，剧烈振荡混匀样品，使样品充分裂解，室温放置5分钟；2. After the liquid nitrogen evaporates to dryness, immediately transfer to a 2ml centrifuge tube, add about 1ml of Invitrogen’s TRIzol extract for every 100mg of material, shake and mix the sample vigorously to fully lyse the sample, and place it at room temperature for 5 minutes;
3．加入0.2ml氯仿，剧烈振荡混匀15秒，室温放置10分钟；3. Add 0.2ml of chloroform, shake vigorously for 15 seconds, and place at room temperature for 10 minutes;
4．4℃，12000rpm离心15分钟；4. Centrifuge at 12,000 rpm for 15 minutes at 4°C;
5．用移液器小心吸出上层水相，加入新的1.5ml的离心管中，加入500μl的异丙醇(1:1体积)，充分混匀，-20℃,沉淀30min； 5. Carefully suck out the upper aqueous phase with a pipette, add it to a new 1.5ml centrifuge tube, add 500μl of isopropanol (1:1 volume), mix well, and settle at -20°C for 30min;
6．4℃，12000rpm离心10min，小心弃去上清液；6. Centrifuge at 12000rpm for 10min at 4°C, carefully discard the supernatant;
7．RNA沉淀用1ml的75%乙醇洗涤。4℃，8000rpm离心10min收集沉淀；7. The RNA pellet was washed with 1 ml of 75% ethanol. Centrifuge at 8000rpm for 10min at 4°C to collect the precipitate;
8．重复用75%乙醇洗涤一次RNA沉淀；8. Wash the RNA pellet with 75% ethanol repeatedly;
9．去上清，RNA沉淀于无菌操作台上晾干约10-15分钟，RNA略显透明，加入适当体积（30-50μl）的RNase-free水充分溶解(可放于-80℃长期保存)；9. Remove the supernatant, dry the RNA pellet on a sterile operating table for about 10-15 minutes, the RNA is slightly transparent, add an appropriate volume (30-50μl) of RNase-free water to fully dissolve (can be stored at -80°C for long-term storage) ;
10．紫外分光光度计及1%Agrose凝胶电泳检测RNA浓度及质量。10. The concentration and quality of RNA were detected by UV spectrophotometer and 1% Agrose gel electrophoresis.
注：a)用紫外分光光度计检测RNA的产量，在260nm处的吸光度，1OD=40ug/ml。根据在260nm和280nm处的吸光值，检测RNA的纯度,纯RNA的OD260/OD280比值应接近2.0(比值最好在1.9～2.1之间)。 Note: a) Use a UV spectrophotometer to detect the yield of RNA, the absorbance at 260nm, 1OD=40ug/ml. According to the absorbance value at 260nm and 280nm, detect the purity of RNA, and the ratio of OD260 /OD280 of pure RNA should be close to 2.0 (the ratio should preferably be between 1.9 and 2.1).
b)用1%Agrose凝胶电泳检侧RNA的质量及大小。吸取1ul RNA加入3μl的RNase-free水，加1μl上样缓冲液6 5℃变性5分钟。电泳后用EB染色，另取3μl的2kb DNAMarker作为对照。 b) Detect the quality and size of RNA by 1% Agrose gel electrophoresis. Aspirate 1ul of RNA and add 3ul of RNase-free water, add 1ul of loading buffer to denature at 65°C for 5 minutes. After electrophoresis, stain with EB, and take another 3 μl of 2kb DNAMarker as a control. the
2．3第一链cDNA的合成 2.3 Synthesis of first-strand cDNA
采用PrimeScriptTM RT-PCR Kit 进行。反应步骤如下：The PrimeScript™ RT-PCR Kit was used. The reaction steps are as follows:
1．在Microtube 管中配制下列混合液。1. Prepare the following mixes in Microtube tubes.
dNTP Mixture (10 mM) 1μl dNTP Mixture (10 mM) 1μl
Oligo dT Primer (2.5μM) 1μlOligo dT Primer (2.5μM) 1μl
Total RNA 4μlTotal RNA 4μl
RNase free H2O 4μlRNase free H2 O 4 μl
2．在PCR仪上进行变性、退火反应。2. Perform denaturation and annealing reactions on a PCR instrument.
65℃ 5 min 65℃ 5 min
4℃ 1 min4°C 1 min
3．离心数秒钟使RNA/引物等的混合液聚集于Microtube管底部。3. Centrifuge for a few seconds to pool the RNA/primer mixture at the bottom of the Microtube.
4．在上述Microtube管中配制下列反转录反应液 4. Prepare the following reverse transcription reaction solution in the above Microtube tube
上述变性、退火后反应液 10 μlReaction solution after denaturation and annealing 10 μl
5×PrimerScriptTM Buffer 4 μl5×PrimerScript™ Buffer 4 μl
RNase Inhibitor (40U/μl) 0.5 μlRNase Inhibitor (40U/μl) 0.5 μl
PrimScriptTMRTase 0.5 μlPrimScript™ RTase 0.5 μl
Rnase Free dH2O 5 μlRNase Free dH2 O 5 μl
5．在PCR仪上按下列条件进行反转录反应5. Carry out the reverse transcription reaction on the PCR instrument according to the following conditions
42℃ 15-30 min42℃ 15-30 min
95℃ 5 min95℃ 5 min
4℃ 保温4°C insulation
2．4 PCR 反应及电泳2.4 PCR reaction and electrophoresis
1．以cDNA 为模板，进行PCR 反应。引物如下1. Using cDNA as template, carry out PCR reaction. Primers are as follows
TaAct-S： 5’- GTTCCAATCTATGAGGGATACACGC -3’TaAct-S: 5’- GTTCCAATCTATGAGGGATACACGC-3’
TaAct-A: 5’- GAACCTCCACTGAGAACAACATTACC -3’TaAct-A: 5’- GAACCTCCACTGAGAACAACATTACC -3’
D1ORF5: 5’-TCTAGAATGGAGTACCAGGGGCAGCAC-3’（XbaI）D1ORF5: 5'-TCTAGAATGGAGTACCAGGGGCAGCAC-3' (XbaI)
D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’(SacI)D1ORF3: 5'-GAGCTCTCAGTGCTGTCCGCCGGG-3'(SacI)
2．PCR 体系2. PCR system
ddH2O 4.7μlddH2 O 4.7μl
10× buffer 2μl10× buffer 2μl
Primer1（2μM） 1μlPrimer1 (2 μM) 1 μl
Primer2（2μM） 1μlPrimer2 (2μM) 1μl
dNTP（10mM each） 0.2μldNTP (10mM each) 0.2μl
rTaq polymerase（5U/μl） 0.1μlrTaq polymerase (5U/μl) 0.1μl
逆转录cDNA 模板 1μlReverse transcription cDNA template 1μl
Total Volume 10μlTotal Volume 10μl
3．PCR 程序3. PCR program
94℃ 5min；25～30 cycles，94℃ 20s，57℃ 60s，72℃ 45s；72℃ 5min。94°C 5min; 25～30 cycles, 94°C 20s, 57°C 60s, 72°C 45s; 72°C 5min.
根据内参Actin 的扩增情况确定PCR 的循环数，调整cDNA 模板的加入量。 Determine the number of PCR cycles according to the amplification of the internal reference Actin, and adjust the amount of cDNA template added. the
4．1％琼脂糖凝胶电泳。结果见图2中a、b、c、d。 4.1% agarose gel electrophoresis. The results are shown in Figure 2 a, b, c, d. the
实施例3Example 3
35S启动子植物表达载体的构建Construction of 35S Promoter Plant Expression Vector
植物表达载体pCAMBIA-super1300是含有35S 启动子和NPTⅡ基因的双元载体，在其多克隆位点上含有限制性内切酶XbaI和SacI位点。根据基因TaDHN1的cDNA序列，设计包含完整ORF的基因特异性引物，且在引物5′端引入XbaI 、SacI双酶切位点，引物序列为：The plant expression vector pCAMBIA-super1300 is a binary vector containing 35S promoter and NPTⅡ gene, and contains restriction endonucleaseXba I andSac I sites in its multiple cloning site. According to the cDNA sequence of the geneTaDHN1 , gene-specific primers containing the complete ORF were designed, andXba I andSac I double restriction sites were introduced at the 5' end of the primer. The primer sequence is:
D1ORF5: 5’-TCTAGAATGGAGTACCAGGGGCAGCAC-3’（XbaI）D1ORF5: 5'-TCTAGAATGGAGTACCAGGGGCAGCAC-3' (Xba I)
D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’(SacI)D1ORF3: 5'-GAGCTCTCAGTGCTGTCCGCCGGG-3'(Sac I)
用该对引物扩增TaDHN1的cDNA序列。然后分别用限制性内切酶XbaI和SacI双酶切载体pCAMBIA-super1300和目的基因片断。完全酶切的载体在1%琼脂糖凝胶上电泳分离后，经胶回收，然后与双酶切的cDNA 片段连结，构建获得植物表达载体pCAMBIA-super1300/TaDHN1。The cDNA sequence ofTaDHN1 was amplified with this pair of primers. Then the vector pCAMBIA-super1300 and the target gene fragment were cut with restriction endonucleasesXba I andSac I respectively. The fully digested vector was separated by electrophoresis on 1% agarose gel, recovered by gel, and then ligated with the double digested cDNA fragment to construct the plant expression vector pCAMBIA-super1300/TaDHN1.
3．1 质粒pCAMBIA-super1300空载体和目的基因片断XbaI和SacI双酶切 3.1 Plasmid pCAMBIA-super1300 empty vector and target gene fragmentXba I andSac I double enzyme digestion
酶切体系如下：The enzyme digestion system is as follows:
XbaI lμlXba I lμl
SacI 1μlSac I 1μl
pCAMBIA-super1300载体 pCAMBIA-super1300 vector
(或目的基因片段) 5μl(or target gene fragment) 5μl
10×Buffer M 1μl10×Buffer M 1μl
ddH2O To 20μlddH2 O To 20μl
于37℃恒温水浴锅酶切3 小时以上。Enzyme digestion in a constant temperature water bath at 37°C for more than 3 hours.
3．2 酶切产物的电泳与回收 3.2 Electrophoresis and recovery of digested products
双酶切完成后，以1×TAE 为电泳缓冲液，将酶切产物进行0.8％琼脂糖凝胶电泳。在紫外透射仪下用洁净刀片切下pCAMBIA-super1300中载体的大片段和目的基因片段，琼脂糖凝胶回收试剂盒回收目的条带。After the double digestion was completed, the digested product was subjected to 0.8% agarose gel electrophoresis with 1×TAE as the electrophoresis buffer. Cut out the large fragment of the vector and the target gene fragment in pCAMBIA-super1300 with a clean blade under the ultraviolet transilluminator, and recover the target band with the agarose gel recovery kit.
3．3 连接 3.3 Connection
经酶切的pCAMBIA-super1300载体片段和目的基因片段以摩尔比1:4 的比例进行16℃连接过夜。The digested pCAMBIA-super1300 vector fragment and the target gene fragment were ligated overnight at 16°C at a molar ratio of 1:4.
3．4 转化 3.4 Transformation
连接产物热激法转化大肠杆菌DH5α感受态细胞，转化菌在含Kan 50μg/ml 的LB 固体平板上37℃培养16 小时左右。The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method, and the transformed bacteria were cultured on LB solid
3．5 重组子的鉴定 3.5 Identification of recombinants
质粒酶切鉴定。提取阳性克隆质粒，对质粒进行XbaI和SacI双酶切，酶切体系同3．1。酶切产物经0.8％琼脂糖凝胶电泳后，检测到已切开的合适大小的目的基因条带和载体条带，证明载体构建正确（图3）。Identification of plasmid restriction enzymes. Extract the positive cloned plasmid, carry outXba I andSac I double enzyme digestion on the plasmid, the enzyme digestion system is the same as 3.1. After the digested product was subjected to 0.8% agarose gel electrophoresis, the excised target gene band and vector band of appropriate size were detected, proving that the vector was constructed correctly (Figure 3).
实施例4Example 4
农杆菌感受态的制备与转化Preparation and Transformation of Competent Agrobacterium
4．1 农杆菌GV3101感受态的制备4.1 Preparation of Competent Agrobacterium GV3101
1．从YEP 平板(含50μg/ml 利福平)上挑取根癌农杆菌单菌落，接种于含50μg/ml1. Pick a single colony of Agrobacterium tumefaciens from the YEP plate (containing 50 μg/ml rifampicin) and inoculate it on a plate containing 50 μg/ml
利福平的YEP 液体培养基中，200rpm/min，28℃培养过夜；In the YEP liquid medium of rifampicin, 200rpm/min, culture overnight at 28°C;
2．取2ml 过夜培养液接种于50ml 含相同抗生素的YEP 液体培养基中在相同条件2. Take 2ml of overnight culture solution and inoculate it in 50ml of YEP liquid medium containing the same antibiotics under the same conditions
下培养至OD600 达0.5；Under culture until OD600 reaches 0.5;
3．菌液冰浴30min，4℃，5000rpm 离心10min，收集菌体；3. Bacterial solution in ice bath for 30min, centrifuged at 5000rpm at 4°C for 10min to collect the bacterial cells;
4．将菌体重悬于冰浴的10ml 0.15mol/L 的NaCl 中，离心收集菌体；4. Resuspend the bacteria in 10ml of 0.15mol/L NaCl in an ice bath, and collect the bacteria by centrifugation;
5．再悬浮于1ml 20mmol/L 冰预冷的CaCl2 溶液中，以200μl/管将菌液分装在1.5ml5. Resuspend in 1ml 20mmol/L ice-cooled CaCl2 solution, and divide the bacterial solution into 1.5ml with 200μl/tube
Eppendorf 管中，置液氮中速冻1min，-70℃保存备用。In Eppendorf tubes, freeze in liquid nitrogen for 1 min, and store at -70°C for later use.
4．2 冻融法转化根癌农杆菌GV3101 4.2 Transformation of Agrobacterium tumefaciens GV3101 by freeze-thaw method
1．在室温下融化农杆菌感受态细胞，加入1μg 表达载体质粒DNA，混匀后冰浴1. Thaw Agrobacterium competent cells at room temperature, add 1 μg expression vector plasmid DNA, mix well and ice bath
30min；30min;
2．置液氮速冻1min，迅速移至37℃保温3min；2. Quickly freeze in liquid nitrogen for 1 minute, then quickly move to 37°C for 3 minutes;
3．加入无抗生素的YEP 800μl，28℃震荡培养3hr；3. Add 800 μl of YEP without antibiotics, shake and incubate at 28°C for 3 hours;
4．7000rpm 离心30s 收集菌体，涂于含50μg/ml 利福平、50μg/ml Kan 的YEP 平板上，28℃倒置暗培养2-3 天。4. Centrifuge at 7000rpm for 30s to collect the bacteria, spread on the YEP plate containing 50μg/ml rifampicin and 50μg/ml Kan, and incubate in the dark at 28°C for 2-3 days.
4．3 菌体PCR 鉴定，鉴定结果如图4。 4.3 PCR identification of bacteria, the identification results are shown in Figure 4. the
菌体PCR所用引物同实施例3。方法及程序同2.4。 The primers used for phage PCR were the same as those in Example 3. The method and procedure are the same as 2.4. the
实施例5Example 5
转基因功能验证——拟南芥转化、筛选及表型分析Functional verification of transgenes - Arabidopsis transformation, screening and phenotypic analysis
5．1 拟南芥的种植5.1 Planting of Arabidopsis
将野生型拟南芥种子用7.5%次氯酸钠溶液（包括7.5%次氯酸钠和0.01% Triton-X 100）消毒15分钟，然后用无菌水漂洗5-6次，点播于MS平板上，于4°C 春化2-3天。然后移植到营养钵中（营养土与蛭石按等比例混合），23°C 培养，16/8 h 光周期，光强30-40μmolm-2 s-1；待植株开花后，剪去其主枝顶端，促进侧枝发展。在剪枝后的4-6 天内，进行农杆菌转化。The wild-type Arabidopsis seeds were sterilized with 7.5% sodium hypochlorite solution (including 7.5% sodium hypochlorite and 0.01% Triton-X 100) for 15 minutes, then rinsed with sterile water 5-6 times, sowed on MS plates at 4°C Vernalization takes 2-3 days. Then transplant it into a nutrient pot (the nutrient soil and vermiculite are mixed in equal proportions), cultivate at 23°C, with a photoperiod of 16/8 h, and a light intensity of 30-40 μmolm-2 s-1; branches to promote the development of lateral branches. Within 4-6 days after pruning, perform Agrobacterium transformation.
5．2 拟南芥转化 5.2 Arabidopsis transformation
把200 ml 菌液倒入浅盘中。将修剪好的拟南芥倒扣并使所有花序浸入悬浮菌液中，轻轻搅动沾花30 sec-1 min。取出花盆侧放于托盘中，用保鲜袋包裹以保湿。将托盘置暗处培养24 h。然后取出营养钵并直立放置，恢复光照，继续培养植株至成熟。Pour 200 ml of bacterial solution into a shallow dish. Turn the pruned Arabidopsis upside down and immerse all the inflorescences in the suspension solution, and gently stir to soak the flowers for 30 sec-1 min. Take out the flowerpot and put it on the side of the tray, wrap it in a fresh-keeping bag to keep it moist. Incubate the trays in the dark for 24 h. Then take out the nutrient bowl and place it upright, restore the light, and continue to cultivate the plants until they mature.
5．3 阳性植株的筛选：T0 代种子用7.5%次氯酸钠溶液（包括7.5%次氯酸钠和0.01% Triton-X100）消毒后，点播在MS选择培养板(30 mg/L 潮霉素)上。于4℃下春化2-3天。移入培养室中培养。大约10天左右，挑选潮霉素抗性植株（长出真叶1-2 对，根伸长至培养基中）并移栽到营养钵中。培养直至种子成熟。同样方法筛选T1 代种子得到T2 代植株。并在T2 代植物中挑选抗性比为3:1 的单插入独立株系，并获得纯合T3代株系进行转基因拟南芥的分子检测和表型鉴定。 5.3 Screening of positive plants: After the seeds of the T0 generation were sterilized with 7.5% sodium hypochlorite solution (including 7.5% sodium hypochlorite and 0.01% Triton-X100), they were sown on MS selective culture plates (30 mg/L hygromycin). Vernalize at 4°C for 2-3 days. Transfer to culture room for cultivation. About 10 days or so, pick hygromycin-resistant plants (1-2 pairs of true leaves grow, roots extend into the medium) and transplant them into nutrient pots. Cultivate until the seeds mature. The same method was used to screen T1 generation seeds to obtain T2 generation plants. A single-insertion independent line with a resistance ratio of 3:1 was selected from the T2 generation plants, and a homozygous T3 generation line was obtained for molecular detection and phenotypic identification of transgenic Arabidopsis. the
5．4 转基因拟南芥的PCR鉴定 5.4 PCR identification of transgenic Arabidopsis
1．拟南芥基因组DNA的提取 1. Extraction of Arabidopsis Genomic DNA
（1）取100mg 左右的新鲜叶片，放入1.5ml 离心管中，液氮速冻，研磨器中磨碎，加入600μl 预热至65℃的2×CTAB 提取缓冲液，混匀置于65℃水浴中放置90min；(1) Take about 100mg of fresh leaves, put them into a 1.5ml centrifuge tube, freeze them quickly in liquid nitrogen, grind them in a grinder, add 600μl of 2×CTAB extraction buffer preheated to 65°C, mix well and place in a 65°C water bath placed in the middle for 90min;
（2）混合物冷至室温后加入等体积的酚/氯仿/异戊醇，混匀，4℃，12000rpm 离心10min；(2) After cooling the mixture to room temperature, add an equal volume of phenol/chloroform/isoamyl alcohol, mix well, and centrifuge at 12,000 rpm for 10 min at 4°C;
（3）取上清，加入等体积的氯仿/异戊醇，混匀，4℃，12000rpm 离心10min；(3) Take the supernatant, add an equal volume of chloroform/isoamyl alcohol, mix well, and centrifuge at 12000rpm for 10min at 4°C;
（4）取上清，加入1/10 体积的3mol/L NaAc（pH5.3）和0.7 倍体积的异丙醇，仔细混匀，室温放置15min，沉淀DNA；(4) Take the supernatant, add 1/10 volume of 3mol/L NaAc (pH5.3) and 0.7 times the volume of isopropanol, mix carefully, and place at room temperature for 15min to precipitate DNA;
（5）用一玻璃钩将DNA 钩出，并转移至一装有800μl 70%乙醇的干净Eppendorf 管中，室温放置数分钟洗涤沉淀，6000g 离心5min；(5) Hook up the DNA with a glass hook, and transfer it to a clean Eppendorf tube filled with 800μl 70% ethanol, leave it at room temperature for a few minutes to wash the precipitate, and centrifuge at 6000g for 5min;
（6）尽量去尽上清，空气干燥数分钟，溶于适量TE 缓冲液中。(6) Remove the supernatant as much as possible, air dry for several minutes, and dissolve in an appropriate amount of TE buffer.
2．PCR 扩增 2. PCR amplification
以上面提取的拟南芥基因组DNA 为模板，用基因特异性引物（同实施例3）进行PCR扩增。Using the Arabidopsis genomic DNA extracted above as a template, PCR amplification was carried out with gene-specific primers (same as in Example 3).
PCR 体系同2.4。PCR 反应程序为： 94℃ 预变性5min；94℃变性45sec，58℃复性45sec，72℃延伸1min，循环35 次；72℃延伸7min。PCR扩增产物经琼脂糖凝胶电泳后检测到在转基因拟南芥植株中扩增出一400bp左右的目的条带，在转空载体的植株中未见扩增条带。 The PCR system is the same as 2.4. The PCR reaction program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 sec, 58°C refolding for 45 sec, 72°C extension for 1 min, 35 cycles; 72°C extension for 7 min. After the PCR amplification product was subjected to agarose gel electrophoresis, it was detected that a target band of about 400 bp was amplified in the transgenic Arabidopsis plants, and no amplified band was seen in the plants transformed with the empty vector. the
5．5 转基因拟南芥的表型鉴定 5.5 Phenotype identification of transgenic Arabidopsis
1．拟南芥的种植 1. Planting of Arabidopsis
T3代单拷贝纯合拟南芥株系的种子用7.5%次氯酸钠溶液（包括7.5%次氯酸钠和0.01% Triton-X 100）消毒15分钟，然后用无菌水漂洗5-6次，点播于MS平板上，于4°C 春化2-3天，然后移植到营养钵中（营养土与蛭石按等比例混合），23°C 培养，16/8 h 光周期，光强30-40μmolm-2 s-1。Seeds of T3 single-copy homozygous Arabidopsis lines were sterilized with 7.5% sodium hypochlorite solution (including 7.5% sodium hypochlorite and 0.01% Triton-X 100) for 15 minutes, then rinsed with sterile water 5-6 times, and seeded on MS plates above, vernalized at 4°C for 2-3 days, then transplanted into nutrient pots (mixing nutrient soil and vermiculite in equal proportions), cultured at 23°C, 16/8 h photoperiod, light intensity 30-40μmolm-2 s-1.
2．NaCl 和干旱胁迫处理 2. NaCl and drought stress treatments
NaCl处理：将萌发2天的拟南芥幼苗（对照及转基因株系）小心移植含有50 mM、100 mM NaCl的MS培养皿中，竖直培养一周观察表型。结果表明在在正常MS培养基上，Col-0野生型拟南芥和转TaDHN1基因的拟南芥表型差异不大，而在含有50 mM 或100 mM NaCl的MS培养基上，转TaDHN1基因的拟南芥植株的根系明显长于野生型Col-0，T测验表明二者根长差异达到了显著性水平（图5-A）。NaCl treatment: Arabidopsis thaliana seedlings (control and transgenic lines) germinated for 2 days were carefully transplanted into MS petri dishes containing 50 mM and 100 mM NaCl, and cultured vertically for one week to observe the phenotype. The results showed that on normal MS medium, the phenotypes of Col-0 wild-type Arabidopsis and Arabidopsis transgenic withTaDHN1 gene were not significantly different, while on MS medium containing 50 mM or 100 mM NaCl, transgenicTaDHN1 gene The root system of the Arabidopsis plants was significantly longer than that of the wild-type Col-0, and the T test showed that the difference in root length between the two reached a significant level (Fig. 5-A).
渗透胁迫处理：将萌发2天的拟南芥幼苗（对照及转基因株系）小心移植含有50 mM、100 mM、150 mM 甘露醇的MS培养皿中，竖直培养一周观察表型。结果表明在在正常MS培养基上，Col-0野生型拟南芥和转TaDHN1基因的拟南芥表型差异不大，而在含有50 mM 100 mM或150 mM 甘露醇的MS培养基上，转TaDHN1基因的拟南芥植株的根系明显长于野生型，T测验表明二者根长差异达到了显著性水平（图5-B）。 Osmotic stress treatment: 2-day-germinated Arabidopsis seedlings (control and transgenic lines) were carefully transplanted into MS petri dishes containing 50 mM, 100 mM, and 150 mM mannitol, and cultured vertically for one week to observe the phenotype. The results showed that on normal MS medium, the phenotypes of Col-0 wild-type Arabidopsis andTaDHN1 transgenic Arabidopsis had little difference, while on MS medium containing 50
干旱处理：将萌发一周的拟南芥幼苗（对照及转基因株系）移植培养土中，培养20天后开始控水（不浇水）干旱处理。干旱处理15天后观察表型。结果表明干旱处理15天后，野生型拟南芥植株萎蔫，一些株系的叶子变得干枯(图5-C)；而转了TaDHN1外源基因的拟南芥植株长势旺盛，能正常结出荚果(图5 C TaDHN1-8和TaDHN1-20株系)。 Drought treatment: Arabidopsis thaliana seedlings (control and transgenic lines) germinated for one week were transplanted into the culture soil, and after 20 days of cultivation, water control (no watering) drought treatment was started. Phenotypes were observed after 15 days of drought treatment. The results showed that after 15 days of drought treatment, the wild-type Arabidopsis plants wilted, and the leaves of some lines became dry (Fig. 5-C); while the Arabidopsis plants transfected withthe TaDHN1 exogenous gene grew vigorously and produced pods normally (Fig. 5C TaDHN1-8 and TaDHN1-20 strains).
序列表sequence listing
SQ ID No.1SQ ID No.1
<110>济南大学 <110> Jinan University
<120>小麦耐盐、抗旱基因TaDHN1及其应用<120> Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<141>2013-10-22<141>2013-10-22
<160> 1<160> 1
<210> 1<210> 1
<211>402<211>402
<212>cDNA<212> cDNA
<213>小麦<213> Wheat
<221>小麦耐盐、抗旱基因TaDHN1及其应用<221>Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<222>（1）…（402）<222>(1)...(402)
<400>1<400>1
1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT
61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG
121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC
181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC
241 AAGGAGAAGC TCCCCGGCGG AGCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG241 AAGGAGAAGC TCCCCGGCGG AGCCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG
301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC
361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA
SQ ID No.2SQ ID No.2
<110>济南大学 <110> Jinan University
<120>小麦耐盐、抗旱基因TaDHN1及其应用<120> Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<141>2013-10-22<141>2013-10-22
<160> 1<160> 1
<210> 1<210> 1
<211>133<211>133
<212>AA<212>AA
<213>小麦<213> Wheat
<221>小麦耐盐、抗旱基因TaDHN1及其应用<221>Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<222>（1）…（133）<222>(1)...(133)
<400>1<400>1
1 MEYQGQHGHA TDKVEEYGQP 1 MEYQGQHGHA TDKVEEYGQP
21 VAGHGGFTGR PTGTHGAQLQ 21 VAGHGGFTGR PTGTHGAQLQ
41 ATRDDHKTDG VLRRSGSSSS 41 ATRDDHKTDG VLRRSGSSSS
61 SSSEDDGVGG RRKKGMKEKI 61 SSSEDDGVGG RRKKGMKEKI
81 KEKLPGGAHK DATAGQQHTA 81 KEKLPGGAHK DATAGQQHTA
101 VAGEYAGTHG TEATGEKKGV 101 VAGEYAGTHG TEATGEKKGV
121 MDKIKEKLPG GQH*121 MDKIKEKLPG GQH*
序列表sequence listing
the
SQ ID No.1SQ ID No.1
<110>济南大学<110> Jinan University
<120>小麦耐盐、抗旱基因TaDHN1及其应用<120> Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<141>2013-10-22<141>2013-10-22
<160> 1<160> 1
<210> 1<210> 1
<211>402<211>402
<212>cDNA<212> cDNA
<213>小麦<213> Wheat
<221>小麦耐盐、抗旱基因TaDHN1及其应用<221>Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<222>（1）…（402）<222>(1)...(402)
<400>1<400>1
the
1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT
61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG
121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC
181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC
241 AAGGAGAAGC TCCCCGGCGG AGCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG241 AAGGAGAAGC TCCCCGGCGG AGCCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG
301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC
361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA
the
the
the
the
the
the
SQ ID No.2SQ ID No.2
<110>济南大学<110> Jinan University
<120>小麦耐盐、抗旱基因TaDHN1及其应用<120> Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<141>2013-10-22<141>2013-10-22
<160> 1<160> 1
<210> 1<210> 1
<211>133<211>133
<212>AA<212>AA
<213>小麦<213> Wheat
<221>小麦耐盐、抗旱基因TaDHN1及其应用<221>Wheat Salt Tolerance and Drought Resistance Gene TaDHN1 and Its Application
<222>（1）…（133）<222>(1)...(133)
<400>1<400>1
the
1 MEYQGQHGHA TDKVEEYGQP1 MEYQGQHGHA TDKVEEYGQP
21 VAGHGGFTGR PTGTHGAQLQ21 VAGHGGFTGR PTGTHGAQLQ
41 ATRDDHKTDG VLRRSGSSSS41 ATRDDHKTDG VLRRSGSSSS
61 SSSEDDGVGG RRKKGMKEKI61 SSSEDDGVGG RRKKGMKEKI
81 KEKLPGGAHK DATAGQQHTA81 KEKLPGGAHK DATAGQQHTA
101 VAGEYAGTHG TEATGEKKGV101 VAGEYAGTHG TEATGEKKGV
121 MDKIKEKLPG GQH*121 MDKIKEKLPG GQH*
Claims (8)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086635A (en)* | 2014-04-10 | 2014-10-08 | 内蒙古农业大学 | New drought resistant gene CkDHN1 in Caragana korshinskii Kom. |
| CN105063062A (en)* | 2015-08-10 | 2015-11-18 | 济南大学 | Wheat salt-resistant drought-resistant gene TaDHN3, and expression vector and applications thereof |
| CN111187778A (en)* | 2020-02-10 | 2020-05-22 | 济南大学 | Wheat salt-tolerant gene TaFLZ2 and application thereof |
| CN119752991A (en)* | 2024-12-18 | 2025-04-04 | 西北农林科技大学 | Application of Dehydrin Genes in Plant Drought Resistance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703465A (en)* | 2012-05-04 | 2012-10-03 | 济南大学 | Salt-tolerant drought-tolerant wheat gene TaWRKY79 and application thereof |
| CN102703466A (en)* | 2012-05-04 | 2012-10-03 | 济南大学 | Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof |
| CN103319582A (en)* | 2012-03-19 | 2013-09-25 | 中国农业科学院作物科学研究所 | Plant stress tolerance-associated protein TaNF-YA 1, coding genes thereof and applications |
- 2013
- 2013-12-11CNCN201310686299.6Apatent/CN103667315A/enactivePending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103319582A (en)* | 2012-03-19 | 2013-09-25 | 中国农业科学院作物科学研究所 | Plant stress tolerance-associated protein TaNF-YA 1, coding genes thereof and applications |
| CN102703465A (en)* | 2012-05-04 | 2012-10-03 | 济南大学 | Salt-tolerant drought-tolerant wheat gene TaWRKY79 and application thereof |
| CN102703466A (en)* | 2012-05-04 | 2012-10-03 | 济南大学 | Wheat salt-tolerant and drought-resistant gene TaWRKY80 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| NCBI: "GENBANK ACCESSION EMT30992.1", 《GENBANK》* |
| 张宁 等: "小麦脱水素基因TaDHN-1的特征及其对非生物胁迫相应", 《中国农业科学》* |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086635A (en)* | 2014-04-10 | 2014-10-08 | 内蒙古农业大学 | New drought resistant gene CkDHN1 in Caragana korshinskii Kom. |
| CN104086635B (en)* | 2014-04-10 | 2017-11-28 | 内蒙古农业大学 | A new anti-drought gene CkDHN1 in Caragana korshinskii |
| CN105063062A (en)* | 2015-08-10 | 2015-11-18 | 济南大学 | Wheat salt-resistant drought-resistant gene TaDHN3, and expression vector and applications thereof |
| CN111187778A (en)* | 2020-02-10 | 2020-05-22 | 济南大学 | Wheat salt-tolerant gene TaFLZ2 and application thereof |
| CN111187778B (en)* | 2020-02-10 | 2021-08-24 | 济南大学 | Wheat salt tolerance gene TaFLZ2 and its application |
| CN119752991A (en)* | 2024-12-18 | 2025-04-04 | 西北农林科技大学 | Application of Dehydrin Genes in Plant Drought Resistance |
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