CN1425675A - Cotton Na+/H+ reverse transport protein gene and its cloning method and use - Google Patents
Cotton Na+/H+ reverse transport protein gene and its cloning method and use Download PDFInfo
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- CN1425675A CN1425675A CN 02151530 CN02151530A CN1425675A CN 1425675 A CN1425675 A CN 1425675A CN 02151530 CN02151530 CN 02151530 CN 02151530 A CN02151530 A CN 02151530A CN 1425675 A CN1425675 A CN 1425675A
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Abstract
本发明涉及棉花中Na+/H+反向转运蛋白GhNHX1基因的克隆、重组及耐盐性功能分析，属于分子生物学和生物技术领域。从0.4mol·L－1NaCl处理的棉花叶片中提取总RNA，反转录成cDNA。利用兼并引物，进行常规聚合酶链式反应(PCR)，得到中间片段，然后进行3′和5′末端快速扩增获得全长cDNA。将该基因转入盐敏感酵母突变体，能在一定程度上恢复其抗盐能力。进一步利用该基因转化烟草，获得的转基因植株能在200mmol·L－1的盐浓度下正常生长。因此，该基因是一个重要的抗盐基因，可以用于转化盐敏感植物来提高其耐盐能力，对于盐敏感植物在盐碱地上的推广种植，具有非常重要的经济效益和社会效益。
The invention relates to the cloning, recombination and salt-tolerance function analysis of the Na + /H + antiporter GhNHX1 gene in cotton, belonging to the fields of molecular biology and biotechnology. Total RNA was extracted from cotton leaves treated with 0.4mol·L -1 NaCl and reverse transcribed into cDNA. Using degenerate primers, conventional polymerase chain reaction (PCR) was carried out to obtain the middle fragment, and then the 3' and 5' ends were rapidly amplified to obtain the full-length cDNA. Transferring the gene into the salt-sensitive yeast mutant can restore its salt-resistant ability to a certain extent. Further using the gene to transform tobacco, the obtained transgenic plants can grow normally under the salt concentration of 200mmol·L -1 . Therefore, this gene is an important salt-resistant gene, which can be used to transform salt-sensitive plants to improve their salt-tolerant ability, and has very important economic and social benefits for the popularization and planting of salt-sensitive plants on saline-alkali land.
Description
(一)技术领域(1) Technical field
本发明涉及棉花中Na+/H+反向转运蛋白基因GhNHX1的克隆、重组及耐盐性功能的分析和应用，属于分子生物学和生物技术领域。The invention relates to the analysis and application of cloning, recombination and salt tolerance function of Na + /H + antiporter gene GhNHX1 in cotton, and belongs to the field of molecular biology and biotechnology.
(二)背景技术(2) Background technology
高浓度的盐引起植物体内的离子失衡和高渗胁迫，导致植物发育不良，生长缓慢特别是降低农作物产量。严重的盐胁迫或渗透胁迫会引起植物体内的第二胁迫——氧化胁迫的发生，甚至导致植物死亡。因此，盐胁迫受到人们的广泛关注，提高作物的耐盐性亦愈来愈受到人们的重视。在高盐下，植物通过产生胁迫蛋白和可溶性渗透调节物质来减轻盐胁迫造成的毒害。早期的耐盐基因工程主要集中在清除自由基和增加渗透调节物质两个方面，转基因植株的耐盐能力有所提高，但效果不太明显。最近，定位在质膜和液泡膜上的Na+/H+反向转运蛋白基因从许多植物中分离出来。研究结果表明质膜上的Na+/H+反向转运蛋白负责Na+的外排，从而保持植物细胞内低Na+水平。而液泡膜上的Na+/H+反向转运蛋白则负责Na+的液泡区域化，维持细胞内的离子均衡。这大大促进了耐盐基因工程方面的研究工作并取得突破性进展。Blumwald等利用基因工程手段在番茄和油菜中过量表达Na+反向转运蛋白基因，得到了世界上第一批真正意义上的耐盐植株，参见于张虹霞等人于2001年《自然生物技术)》中的转基因抗盐番茄植株叶中积累盐而果实中不积累盐，19：765-768(Hong-Xia Zhang，Eduardo Bhmmwald，2001，Naturebiotechnology.19：765-768)。High concentrations of salt cause ion imbalance and hyperosmotic stress in plants, resulting in poor plant development, slow growth and especially reduced crop yields. Severe salt stress or osmotic stress will cause the second stress in plants - oxidative stress, and even cause plant death. Therefore, people pay more and more attention to salt stress, and people pay more and more attention to improving the salt tolerance of crops. Under high salinity, plants mitigate the toxicity caused by salt stress by producing stress proteins and soluble osmoregulatory substances. Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants was improved, but the effect was not obvious. Recently, Na + /H + antiporter genes localized on the plasma membrane and tonoplast membrane were isolated from many plants. The findings suggest that Na + /H + antiporters on the plasma membrane are responsible for Na + efflux, thereby maintaining low Na + levels in plant cells. The Na + /H + antiporter on the tonoplast membrane is responsible for the localization of Na + vacuoles and maintains the ion balance in the cell. This has greatly promoted the research work on salt-tolerant genetic engineering and made breakthroughs. Blumwald et al. used genetic engineering to overexpress the Na + antiporter gene in tomato and rapeseed, and obtained the first batch of truly salt-tolerant plants in the world. See Yu Zhang Hongxia and others in 2001 "Nature Biotechnology)" Transgenic salt-tolerant tomato plants accumulate salt in leaves but not in fruits, 19: 765-768 (Hong-Xia Zhang, Eduardo Bhmmwald, 2001, Naturebiotechnology. 19: 765-768).
由于Na+/H+反向转运蛋白基因在耐盐过程中所起的作用，本发明人从棉花中分离编码液泡型Na+/H+反向转运蛋白的基因。根据其它植物中发表的Na+/H+反向转运蛋白的氨基酸序列，设计引物，利用反转录-聚合酶链式反应，从棉花中分离出编码液泡型Na+/H+反向转运蛋白的基因。将该基因转入盐敏感酵母突变体，能在一定程度上恢复其抗盐能力。进一步构建正义表达载体，转化烟草，转基因植株具有较高的耐盐性，耐盐性可达200mmolL-1。该基因可用于植物的遗传转化，提高植物的耐盐能力。Due to the role of the Na + /H + antiporter gene in the process of salt tolerance, the inventors isolated a gene encoding a vacuolar Na + /H + antiporter from cotton. According to the amino acid sequence of Na + /H + antiporter published in other plants, primers were designed, and the coding vacuolar Na + /H + antiporter was isolated from cotton by reverse transcription-polymerase chain reaction gene. Transferring the gene into the salt-sensitive yeast mutant can restore its salt-resistant ability to a certain extent. The sense expression vector was further constructed, and tobacco was transformed. The transgenic plants had higher salt tolerance, and the salt tolerance could reach 200mmolL -1 . The gene can be used for genetic transformation of plants to improve the salt-tolerant ability of plants.
(三)发明内容(3) Contents of the invention
本发明首次从棉花中分离出编码液泡型Na+/H+反向转运蛋白的全长cDNA，连接到表达载体上，利用农杆菌侵染法转化烟草，获得的转基因植株耐盐性高达200mmolL-1。The present invention isolates the full-length cDNA encoding the vacuolar Na + /H + antiporter from cotton for the first time, connects it to the expression vector, and transforms tobacco using the Agrobacterium infection method, and the salt tolerance of the obtained transgenic plants is as high as 200mmolL - 1 .
从棉花叶片中提取总RNA，然后将2微克总RNA反转录成cDNA。根据其它植物中的Na+/H+反向转运蛋白中保守的氨基酸序列，设计一对兼并引物：Total RNA was extracted from cotton leaves, and then 2 μg of total RNA was reverse transcribed into cDNA. According to the conserved amino acid sequences in Na + /H + antiporters in other plants, design a pair of degenerate primers:
正向引物：5′-CC(GATC)CC(GATC)AT(TAC)AT(TAC)TT(TC)AA(TC)GC-3′Forward primer: 5′-CC(GATC)CC(GATC)AT(TAC)AT(TAC)TT(TC)AA(TC)GC-3′
反向引物：5′-GT(GATC)AC(GATC)TT(AG)TGCCA(GATC)GT(AG)TA-3′Reverse primer: 5′-GT(GATC)AC(GATC)TT(AG)TGCCA(GATC)GT(AG)TA-3′
进行常规聚合酶链式反应(PCR)，取2μl PCR产物连接到pGEM-T easy载体上，转化DH5α感受态细胞，涂平板，筛选阳性克隆，挑白斑摇菌，提取质粒，并进行酶切鉴定，之后进行序列测定。然后进行3′和5′末端快速扩增获得全长cDNA。具体的PCR试剂与条件为：Carry out conventional polymerase chain reaction (PCR), take 2μl of PCR product and connect it to the pGEM-T easy vector, transform DH5α competent cells, smear the plate, screen positive clones, pick the white spot shake, extract the plasmid, and carry out enzyme digestion identification , followed by sequence determination. The full-length cDNA was then obtained by rapid amplification of the 3' and 5' ends. The specific PCR reagents and conditions are:
10×反应缓冲液 5μl10×
25mM MgCl2 4μl25mM MgCl2 4μl
10mM脱氧核苷酸混合物(dNTP) 1μl10mM deoxynucleotide mixture (dNTP) 1μl
正向引物(10μM) 2μlForward primer (10μM) 2μl
反向引物(10μM) 2μlReverse primer (10μM) 2μl
模板cDNA 1μl
Taq DNA聚合酶 0.5μlTaq DNA polymerase 0.5μl
总体积 50μlTotal volume 50μl
PCR反应条件为：94℃ 5分钟，然后进入下列循环：94℃ 1分钟，55℃ 1分钟，72℃ 1分钟，共30个循环，最后72(C延伸5分钟。The PCR reaction conditions were: 94°C for 5 minutes, and then entered the following cycle: 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, a total of 30 cycles, and finally 72°C for 5 minutes.
结果扩增到一个614bp的DNA片段。取2μl PCR产物连接到pGEM-T easy载体(Promega公司产品)，转化DH5α细胞(常用载体宿主细胞)，平板过夜培养。挑取白色菌斑，提取质粒DNA，用于序列测定。As a result, a 614bp DNA fragment was amplified. Take 2 μl of the PCR product and connect it to the pGEM-T easy vector (promega company product), transform DH5α cells (commonly used vector host cells), and culture the plate overnight. Pick white plaques and extract plasmid DNA for sequence determination.
经过3′和5′末端快速扩增获得全长的cDNA为2485bp，包括起始密码子前的上游序列和多聚A尾巴。开放阅读框部分为1629bp，由此推得具543个氨基酸的一段序列，将此氨基酸序列在国际基因库中进行比较，表明与已发表的野滨藜、水稻和拟南芥的Na+/H+反向转运蛋白的氨基酸同源性分别为77.30％、72.74％和76.61％，表明经过上述克隆步骤得到了编码Na+/H+反向转运蛋白的基因。该基因序列如下：序列表(1)SEQ ID NO 1的信息(a)序列特征The full-length cDNA was 2485bp after rapid amplification at the 3' and 5' ends, including the upstream sequence before the start codon and the poly A tail. The open reading frame part is 1629bp, and a sequence of 543 amino acids is deduced from it. The amino acid sequence is compared in the international gene bank, which shows that it is different from the published Na+/H+ reactions of Abaminus, rice and Arabidopsis. The amino acid homology to the transporter was 77.30%, 72.74% and 76.61%, respectively, indicating that the gene encoding the Na+/H+ antiporter was obtained through the above cloning steps. The gene sequence is as follows: Sequence Listing (1) Information of SEQ ID NO 1 (a) Sequence Features
*长度：2485碱基对 * Length: 2485 bp
*类型：核酸 * Type: nucleic acid
*链型：双链 * Chain type: double chain
*拓扑结构：线性(b)分子类型：cDNA(c)假设：否(d)反义：否(e)最初来源：棉花(f)序列描述：SEQ IN N0.1 * Topology: Linear (b) Molecular Type: cDNA (c) Hypothesis: No (d) Antisense: No (e) Original Source: Cotton (f) Sequence Description: SEQ IN N0.1
ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60
TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120
CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180CACGTGGGTAAGTCCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTTGTTTTCT 180
CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTTGGTTCAAT 240
AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300
ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360
TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAATTTTCGAAGATTTACATTGTT 420TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAAATTTTCGAAGATTTACATTGTT 420
GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480
TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540
TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600
TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660
TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720
CAAGTGGGGGTAAAAGCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTCTTTATCTATC 780CAAGTGGGGGTAAAAGTCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTTCTTTATCTATC 780
TTCTGCCCCCTATTATATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840TTCTGCCCCCCTATTATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840
ACTTTATCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900ACTTTATTCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900
CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960
TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020
ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080
CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140
GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTATTTTTAGCAAGCACTATGCTGG 1200GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTTATTTTTAGCAAGCACTATGCTGG 1200
GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260
CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320
AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380
ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440
TGTCATTTGTTGCTGAGACTTTTCTCTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500TGTCATTTGTTGCTGAGACTTTTCTCTTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500
AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560
TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620
TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680
CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740
GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800
TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860
CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920
TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAACA 1880TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAAACA 1880
GCATTCGTGCACTTCTTACAACTCCAGCACACACTGTTCATTACTATTGGCGAAAGTTTG 2040GCATTCGTGCACTTCTTACAACTCCAGCACCACTGTTCATTACTATTGGCGAAAGTTTG 2040
ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100
CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160
CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220
CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280
CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340
TCTTTCCATTTGACATTTACGAATATTTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400TCTTTCCATTTGACATTTACGAATATTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400
CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460
TATTTTTATTAAAAAAAAAAAAAAA 2485TATTTTTATTAAAAAAAAAAAAAAA 2485
(2)SEQ IN NO.2的信息(2) Information of SEQ IN NO.2
(a)序列特征(a) Sequential features
*长度：543氨基酸 * Length: 543 amino acids
*类型：氨基酸 * Type: amino acid
*链型：单链 * Chain type: single chain
*拓扑结构：线性 * Topology: Linear
(b)分子类型：蛋白质(b) Molecule type: protein
(c)序列描述MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 543根据上述序列，设计构建表达载体的引物：(c)序列描述MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 543根据上述序列，设计构建表达载体的引物：
正向引物：5′-ATGGTGGCTCCGCAGTTAGCT-3′Forward primer: 5′-ATGGTGGCTCCGCAGTTAGCT-3′
反向引物：5′-ACCTCATTGCCATTGAGGCAG-3′Reverse primer: 5′-ACCTCATTGCCATTGAGGCAG-3′
以叶的总RNA反转录的cDNA为模板，进行PCR扩增出含全长编码框的序列，先连接到pMD18-T载体上，进行测序鉴定。然后用XbaI和SalI切下该片段，插入到表达载体PBI121的35S启动子的下游。将构建好的表达载体转入农杆菌EHAl05。Using the reverse-transcribed cDNA of the total leaf RNA as a template, the sequence containing the full-length coding frame was amplified by PCR, which was first connected to the pMD18-T vector for sequencing identification. Then the fragment was excised with XbaI and SalI, and inserted into the downstream of the 35S promoter of the expression vector PBI121. The constructed expression vector was transformed into Agrobacterium EHA105.
转化烟草，在含卡那霉素的LB固体培养基上筛选，将筛选出的抗性苗在100mmolL-1、200mmolL-1和30mmolL-1氯化钠的培养基上培养。结果表明，与野生型相比，转基因植株仍能正常生长，具较高的耐盐能力。Tobacco was transformed, screened on LB solid medium containing kanamycin, and the screened resistant shoots were cultured on 100mmolL -1 , 200mmolL -1 and 30mmolL -1 sodium chloride medium. The results showed that, compared with the wild type, the transgenic plants could still grow normally and had higher salt tolerance.
转化酵母盐敏感突变株，在选择培养基上筛选转化子，将筛选出的阳性转化子在喊含1600mM氯化钠的选择培养基上培养。结果表明GhNHX1对酵母盐敏感突变株有补偿作用，转6hNHX1的酵母盐敏感突变株的耐盐能力大大增强。Yeast salt-sensitive mutants were transformed, transformants were screened on selective media, and the positive transformants screened were cultured on selective media containing 1600 mM sodium chloride. The results showed that GhNHX1 had a compensatory effect on yeast salt-sensitive mutant strains, and the salt-tolerant ability of yeast salt-sensitive mutant strains transfected with 6hNHX1 was greatly enhanced.
根据上述技术，从棉花中分离出编码液泡型Na+/H+反向转运蛋白的基因GhNHX1，该基因过量表达能导致转基因烟草具较高的耐盐性。让该基因在棉花中表达，将会提高棉花的耐盐性；如果与特异启动子连接，对其表达进行时空和胁迫诱导进行调控，将更具有针对性，具有非常重要的经济效益和社会效益.According to the above technique, the gene GhNHX1 encoding vacuolar Na + /H + antiporter was isolated from cotton, and the overexpression of this gene can lead to higher salt tolerance in transgenic tobacco. Expressing the gene in cotton will improve the salt tolerance of cotton; if it is connected to a specific promoter, its expression can be regulated by time and space and stress induction, which will be more targeted and have very important economic and social benefits .
(四)附图说明图1是野生型和转GhNHX1基因植株的光合速率的测定结果柱状图横坐标表示NaCl浓度梯度，纵坐标表示光合速率值；W代表野生型，T代表转基因植株，mM表示mmol/L-1。图2是野生型和转基因植株的叶绿素含量的测定结果柱状图横坐标表示NaCl浓度梯度，纵坐标表示叶绿素含量；W代表野生型，T代表转基因植株，mM表示mmol/L-1。图3是GhNHX1基因对酵母盐敏感突变体的补偿作用图横坐标表示NaC1浓度梯度，纵坐标表示600nm处的光吸收值；control代表野生型酵母菌株K601，R100代表盐敏感酵母突变株，R100/pYES2代表转pYES2(空载体)的酵母菌株，R100/pYES2 GhNHX1代表转GhNHX1基因的酵母菌株，mM表示mmol/L-1。(4) Description of drawings Fig. 1 is the measurement result of the photosynthetic rate of wild-type and transgenic GhNHX1 gene plant. mmol/L -1 . Fig. 2 is the histogram of the measurement results of chlorophyll content of wild-type and transgenic plants . Fig. 3 is a graph showing the compensation effect of the GhNHX1 gene on yeast salt-sensitive mutants. pYES2 represents the yeast strain transformed with pYES2 (empty vector), R100/pYES2 GhNHX1 represents the yeast strain transformed with GhNHX1 gene, and mM represents mmol/L -1 .
(五)具体发明实施方式实施例1：棉花Na+/H+反向转运蛋白基因的克隆方法(5) Specific Invention Embodiment Example 1: Cloning method of cotton Na + /H + antiporter gene
1.总RNA的提取：采用RNAeasy mini kit(promoga公司产品)提取总RNA。1. Extraction of total RNA: Total RNA was extracted using RNAeasy mini kit (product of Promoga).
2.cDNA第一条链的合成：取2微克总RNA，加入5×反应缓冲液4μl，10mmolL-1脱氧核糖核酸(dNTP)2μl，核糖核酸酶抑制剂(40-200u/μ1)0.5μl，引物T26(10pmol/μ1)1μl，反转录酶(10u/μ1)2μl，42℃反应60分钟，85℃ 10分钟终止反应。2. Synthesis of the first strand of cDNA: Take 2 micrograms of total RNA, add 4 μl of 5× reaction buffer, 2 μl of 10 mmolL -1 deoxyribonucleic acid (dNTP), 0.5 μl of ribonuclease inhibitor (40-200u/μ1), Primer T26 (10pmol/μl) 1μl, reverse transcriptase (10u/μl) 2μl, react at 42°C for 60 minutes, and stop the reaction at 85°C for 10 minutes.
3.PCR反应：聚合酶链式反应(PCR)试剂与条件为：3. PCR reaction: polymerase chain reaction (PCR) reagents and conditions are:
首先将下列试剂混在一起：First mix the following reagents together:
10×反应缓冲液 5μl10×
10mM脱氧核苷酸混合物(dNTP) 1μl10mM deoxynucleotide mixture (dNTP) 1μl
正向引物(10μmolL-1) 2μlForward primer (10μmolL -1 ) 2μl
反向引物(10μmolL-1) 2μlReverse primer (10μmolL -1 ) 2μl
模板cDNA 1μl
TaqDNA聚合酶 0.5μlTaqDNA polymerase 0.5μl
总体积 50μlTotal volume 50μl
PCR反应条件为：94℃ 5分钟，然后进入下列循环：94℃ 1分钟，55℃ 1分钟，72℃ 1.5分钟，共30个循环，最后72℃延伸5分钟。The PCR reaction conditions are: 94°C for 5 minutes, and then enter the following cycle: 94°C for 1 minute, 55°C for 1 minute, 72°C for 1.5 minutes, a total of 30 cycles, and finally 72°C for 5 minutes.
4.基因克隆：取2μl PCR与pGEM-T easy载体进行连接，操作步骤按Promega公司产品pGEM-T easy Vector system说明书进行。然后连接产物转化大肠杆菌DH5α菌株，在表面涂有5-溴-4-氯-3-吲哚-β-D-半乳糖苷)和X-gal的含氨苄青霉素(100微克/毫升)的LB平板上生长过夜。挑取白色菌落，在LB液体培养基中培养过夜。4. Gene cloning: Take 2 μl of PCR and connect it with the pGEM-T easy vector, and the operation steps are carried out according to the instructions of the pGEM-T easy Vector system of Promega company. The ligation product was then transformed into E. coli DH5α strain, coated with 5-bromo-4-chloro-3-indole-β-D-galactoside) and X-gal on LB containing ampicillin (100 μg/ml) Grow overnight on plates. Pick white colonies and culture them overnight in LB liquid medium.
5.质粒DNA的提取：碱法提取质粒DNA。5. Extraction of plasmid DNA: extraction of plasmid DNA by alkaline method.
6.序列测定：本工作在上海生工生物工程技术服务有限公司进行。6. Sequence determination: This work was carried out at Shanghai Sangon Bioengineering Technology Service Co., Ltd.
7.3′和5′序列的分离：按Clontech公司的SMART RACE cDNA Amplification Kit说明书进行。7. Separation of 3' and 5' sequences: according to the instructions of SMART RACE cDNA Amplification Kit of Clontech Company.
8.同源检索：利用BLAST软件将分离出的序列与基因银行中的序列进行比较。实施例2：棉花Na+/H+反向转运蛋白基因GhNHXl，如下序列：8. Homology search: use BLAST software to compare the isolated sequence with the sequence in the gene bank. Embodiment 2: Cotton Na + /H + antiporter gene GhNHX1, following sequence:
(1)SEQ ID NO 1的信息(1) Information on
(a)序列特征(a) Sequential features
*长度：2485碱基对 * Length: 2485 bp
*类型：核酸 * Type: nucleic acid
*链型：双链 * Chain type: double chain
*拓扑结构：线性 * Topology: Linear
(b)分子类型：cDNA(b) Molecular type: cDNA
(c)假设：否(c) Assumption: No
(d)反义：否(d) Antisense: no
(e)最初来源：棉花(e) Original source: Cotton
(f)序列描述：SEQ IN NO.1(f) Sequence description: SEQ IN NO.1
ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60
TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120
CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180CACGTGGGTAAGTCCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTTGTTTTCT 180
CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTTGGTTCAAT 240
AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300
ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360
TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAATTTTCGAAGATTTACATTGTT 420TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAAATTTTCGAAGATTTACATTGTT 420
GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480
TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540
TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600
TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660
TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720
CAAGTGGGGGTAAAAGCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTCTTTATCTATC 780CAAGTGGGGGTAAAAGTCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTTCTTTATCTATC 780
TTCTGCCCCCTATTATATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840TTCTGCCCCCCTATTATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840
ACTTTATCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900ACTTTATTCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900
CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960
TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020
ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080
CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140
GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTATTTTTAGCAAGCACTATGCTGG 1200GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTTATTTTTAGCAAGCACTATGCTGG 1200
GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260
CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320
AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380
ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440
TGTCATTTGTTGCTGAGACTTTTCTCTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500TGTCATTTGTTGCTGAGACTTTTCTCTTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500
AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560
TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620
TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680
CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740
GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800
TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860
CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920
TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAACA 1880TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAAACA 1880
GCATTCGTGCACTTCTTACAACTCCAGCACACACTGTTCATTACTATTGGCGAAAGTTTG 2040GCATTCGTGCACTTCTTACAACTCCAGCACCACTGTTCATTACTATTGGCGAAAGTTTG 2040
ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100
CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160
CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220
CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280
CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340
TCTTTCCATTTGACATTTACGAATATTTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400TCTTTCCATTTGACATTTACGAATATTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400
CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460
TATTTTTATTAAAAAAAAAAAAAAA 2485(3)SEQ IN NO.2的信息(a)序列特征TATTTTTATTAAAAAAAAAAAAAAA 2485(3) Information of SEQ IN NO.2 (a) Sequence feature
*长度：543氨基酸 * Length: 543 amino acids
*类型：氨基酸 * Type: amino acid
*链型：单链 * Chain type: single chain
*拓扑结构：线性 * Topology: Linear
(b)分子类型：蛋白质(b) Molecule type: protein
(c)序列描述MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 543实施例3：表达载体的构建(c)序列描述MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 543实施例3：表达载体的构建
1.根据分离出的棉花Na+/H+反向转运蛋白基因GhNHXl的核苷酸序列，设计引物：1. according to the nucleotide sequence of the isolated cotton Na + /H + antiporter gene GhNHX1, design primers:
正向引物：5′-ATGGTGGCTCCGCAGTTAGCT-3′Forward primer: 5′-ATGGTGGCTCCGCAGTTAGCT-3′
反向引物：5′-ACCTCATTGCCATTGAGGCAG-3’Reverse primer: 5'-ACCTCATTGCCATTGAGGCAG-3'
以叶的总RNA反转录的cDNA为模板，进行聚合酶链式反应。Polymerase chain reaction was carried out using the cDNA reverse transcribed from the total leaf RNA as a template.
2.取2μl PCR与pMD18-T载体进行连接，操作步骤按Promega公司产品pMD18-T Vectorsystem说明书进行。然后转化大肠杆菌DH5α菌株，在表面涂5-溴-4-氯-3-吲哚-β-D-半乳糖苷和X-gal的含氨苄青霉素(100微克/毫升)的LB平板上生长过夜。挑取白色菌落，在LB液体培养基中培养过夜。碱法提取质粒DNA，进行序列测定。2. Take 2 μl of PCR and connect it with the pMD18-T vector, and the operation steps are carried out according to the instructions of the pMD18-T Vectorsystem product of Promega Company. Then transform E. coli DH5α strain and grow overnight on LB plates coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal containing ampicillin (100 μg/ml) . Pick white colonies and culture them overnight in LB liquid medium. Plasmid DNA was extracted by alkaline method and sequenced.
3.用XαbI和SαlI两个限制性内切酶将该基因从pMD18-T载体上切下，与相同酶酶切的PBI121连接。连接产物转化DH5α细胞，然后在含氨苄青霉素的LB固体平板上培养，对菌落进行PCR鉴定和质粒DNA的酶切分析。3. Cut the gene from the pMD18-T vector with two restriction enzymes XαbI and SαllI, and connect it with PBI121 digested with the same enzymes. The ligation product was transformed into DH5α cells, and then cultured on LB solid plates containing ampicillin, and the colonies were identified by PCR and digested with plasmid DNA.
4.将构建好的表达载体转化农杆菌EHAl05。实施例4：基因耐盐性分析4. Transform the constructed expression vector into Agrobacterium EHA105. Example 4: Gene Salt Tolerance Analysis
1.种植烟草。1. To grow tobacco.
2.挑取鉴定好的农杆菌单克隆于含50毫克/升卡那霉素的LB液体培养基中，28℃振荡培养。2. Pick the identified single clone of Agrobacterium in LB liquid medium containing 50 mg/L kanamycin, and culture it with shaking at 28°C.
3.3000rpm离心5分钟，农杆菌沉淀用10X MS培养基悬浮。3. Centrifuge at 3000rpm for 5 minutes, and suspend the Agrobacterium pellet with 10X MS medium.
4.将烟草叶片切成小块，放入上述悬浮液浸泡10分钟。4. Cut the tobacco leaves into small pieces and soak them in the above suspension for 10 minutes.
5.侵染后的叶片切块置于分化培养基(1×MS盐，3％蔗糖，pH5.8，4.5g/L卡拉胶，)上共培养2天，然后转入选择培养基(1×MS盐，3％蔗糖，pH5.8，4.5g/L卡拉胶，卡那霉素100毫克/升，头孢青霉素250毫克/升)筛选，得到抗性植株。5. The leaf cuttings after infection were placed on the differentiation medium (1×MS salt, 3% sucrose, pH5.8, 4.5g/L carrageenan,) and co-cultured for 2 days, then transferred to the selection medium (1 ×MS salt, 3% sucrose, pH5.8, 4.5g/L carrageenan, 100 mg/L kanamycin, 250 mg/L cephalosporin) screening to obtain resistant plants.
6.将抗性植株移入花盆，每两天浇含10mmolL-1，100mmolL-1，200mmolL-1和300mmolL-1氯化钠的1/2Hoagland营养液，20天后测定野生型和转基因植株的光合速率和叶绿素含量。实施例5：棉花Na+/H+反向转运蛋白基因GhNHX1对酵母盐敏感突变株的补偿作用6. Move the resistant plants into flowerpots, pour 1/2 Hoagland nutrient solution containing 10mmolL -1 , 100mmolL -1 , 200mmolL -1 and 300mmolL -1 sodium chloride every two days, measure the photosynthesis of wild type and transgenic plants after 20 days rate and chlorophyll content. Example 5: Compensation of cotton Na + /H + antiporter gene GhNHX1 on yeast salt-sensitive mutants
1.将克隆到的全长cDNA亚克隆到pMD18-T载体中，构建成pMD18-T-GhNHX1。1. Subcloning the cloned full-length cDNA into pMD18-T vector to construct pMD18-T-GhNHX1.
2.将pMD18-T-GhNHX1和酵母表达载体pYES2经BamHI和HindIII酶切，回收目的条带，在连接酶作用下，构建成pYES2-GhNHX1表达载体。2. Digest pMD18-T-GhNHX1 and yeast expression vector pYES2 with BamHI and HindIII, recover the target band, and construct pYES2-GhNHX1 expression vector under the action of ligase.
3.挑取YPD平板上的酵母菌株K601(野生型)和R100(盐敏感突变株)单菌落与5mlYPD培养液中，30℃振荡培养过夜，再稀释10倍，振荡培养6小时，3000rpm离心5分钟收集菌体，用1.5mlTE重悬菌体。3. Pick a single colony of yeast strains K601 (wild type) and R100 (salt-sensitive mutant strain) on the YPD plate and put them in 5ml of YPD culture medium, shake and culture at 30°C overnight, then dilute 10 times, shake and culture for 6 hours, and centrifuge at 3000rpm for 5 Collect the cells in 1 minute and resuspend the cells with 1.5ml TE.
4.分别取0.1ml上述菌液、3μg pYES2-GhNHX1质粒和0.6mlTE-liOAC-PEG溶液于一1.5ml离心管中，30℃振荡培养30分钟，42℃热击15分钟，3000rpm离心20秒收集菌体，重悬于0.5ml重蒸水中，取200μl涂布于选择培养基上，30℃培养2-4天。4. Take 0.1ml of the above bacterial solution, 3μg of pYES2-GhNHX1 plasmid and 0.6ml of TE-liOAC-PEG solution in a 1.5ml centrifuge tube, culture with shaking at 30°C for 30 minutes, heat shock at 42°C for 15 minutes, and centrifuge at 3000rpm for 20 seconds to collect Bacteria were resuspended in 0.5ml redistilled water, 200μl was taken and spread on the selection medium, and cultured at 30°C for 2-4 days.
5.转化子接种到选择培养液中，30℃振荡培养48小时，测定600nm处的光吸收。5. The transformants were inoculated into the selection medium, cultured with shaking at 30° C. for 48 hours, and the light absorption at 600 nm was measured.
钠离子反向运转蛋白基因序列表-wordSodium ion antiporter gene sequence list-word
SEQUENCE LISTING<110>山东农业大学<120>棉花Na+/H+反向转运蛋白基因的克隆和应用<130>1<160>2<170>PatentIn version 3.1<210>1<211>2485<212>DNA<213>Gossypium hirsutum<220>1<221>CDS<222>(513)..(2141)<223><220><221>5’UTR<222>(1)..(512)<223><220><221>3’UTR<222>(2142)..(2485)<223><400>1acgcggggca acacagtctt gattttgatc gtttttcgct cccatcgaaa gcgaagattt 60taagctgaaa aaagaagaga ggaaaattgt ggcaatttgt tggtgagaaa gtcgaagatt 120cacgtgggta agctccataa acagtgaaac attggatttt cttttttgtt tttgttttct 180caagctctct cttcgaattt actcgtctct ttgaaactgt ccgttttttt ttggttcaat 240aaaatcgcaa attatttgct aatttagaga agaaaattga acggagctga aacaaggatg 300atttgttgct gcatgatgtt gattctccaa aacgattcga gtgcttaagg attttaagat 360tagaaagttc ttgaaatgga cagttcagag gcataaaaat tttcgaagat ttacattgtt 420gaaggagagc ttaaatctga agccttggac tacaactgtt tcagttagaa ggaattggtg 480tttaataaaa tttgatttaa aaagaggtca at atg gtg gct ccg cag tta gct 533SEQUENCE LISTING<110>Shandong Agricultural University<120>Cloning and application of cotton Na+/H+ antiporter gene<130>1<160>2<170>PatentIn version 3.1<210>1<211>2485<212>DNA <213>Gossypium hirsutum<220>1<221>CDS<222>(513)..(2141)<223><220><221>5'UTR<222>(1)..(512)<223> <220><221>3'UTR<222>(2142)..(2485)<223><400>1acgcggggca acacagtctt gattttgatc gtttttcgct cccatcgaaa gcgaagattt 60taagctgaaa aaagaagaga ggaaaattgt ggcaatttgt tggtgagaaa gtcgaagatt 120cacgtgggta agctccataa acagtgaaac attggatttt cttttttgtt tttgttttct 180caagctctct cttcgaattt actcgtctct ttgaaactgt ccgttttttt ttggttcaat 240aaaatcgcaa attatttgct aatttagaga agaaaattga acggagctga aacaaggatg 300atttgttgct gcatgatgtt gattctccaa aacgattcga gtgcttaagg attttaagat 360tagaaagttc ttgaaatgga cagttcagag gcataaaaat tttcgaagat ttacattgtt 420gaaggagagc ttaaatctga agccttggac tacaactgtt tcagttagaa ggaattggtg 480tttaataaaa tttgatttaa aaagaggtca at atg gtg gct ccg cag tta gct 533
Met Val Ala Pro Gln Leu Ala Met Val Ala Pro Gln Leu Ala
1 5gct gtc ttt act aag ttg cag aca cta tct act tca gac cat gcg tct 581Ala Val Phe Thr Lys Leu Gln Thr Leu Ser Thr Ser Asp His Ala Ser1 5GCT GTC TTT AAG TTG CAG CAG ACA CTA TCT TCT TCA GAC Cat GCG TCT 581ALA Val PHR LYS Leu Gln Thr Leu Sering
10 15 20gtg gtc tcc atg aac ata ttt gta gcg ctt ctt tgt gct tgc att gtg 629Val Val Ser Met Asn Ile Phe Val Ala Leu Leu Cys Ala Cys Ile Val10 15 20GTG GTC TCC AAC ATA TTA TTT GCG CTT CTT CTT GCT GCT GCT GTG 629val Val Serite Ale Phe Val Ala Leu Leu Cys Ile Val
25 30 35att ggt cat ctt ttg gag gag aat aga tgg atg aac gaa rca att act 677Ile Gly His Leu Leu Glu Glu Asn Arg Trp Met Ash Glu Ser Ile Thr40 45 50 55gcc ctt atc att ggt gtt ttt act ggg gtc att att ttg ttg aca agt 725Ala Leu Ile Ile Gly Val Phe Thr Gly Val Ile Ile Leu Leu Thr Ser25 35t, GAATG, TTG ATT, ILG, IleTTT, Ilete Ah, Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Leu aca agt 725Ala Leu Ile Ile Gly Val Phe Thr Gly Val Ile Ile Leu Leu Thr Ser
60 65 70ggg ggt aaa agc tct cat ctt tta gtc ttc agt gaa gat ctg ttc ttt 773Gly Gly Lys Ser Ser His Leu Leu Val Phe Set Glu Asp Leu Phe Phe60 65 70GGG GGT AAA AGC TCT Cat CTTA GTC TTC AGT GAA GAA GAA GAA GAT CTG TTC TTT 773GLY GLY LYS Leu Val Phe Set Glu AS
75 80 85atc tat ctt ctg ccc cct att ata ttc aat gct ggg ttt cag gtg aaa 821Ile Tyr Leu Leu Pro Pro Ile Ile Phe Ash Ala Gly Phe Gln Val Lys75 80 85ATC TAT CTT CTG CCC CCT ATA TTC AAT GCT GGG TTT CAG GTG AAA 821ile Tyr Leu Pro Pro Ile Phe Ala Gln Val Val Val Lys
90 95 100aag aag caa ttt ttc cgt aac ttt atc acc atc atg ctg ttt ggg gct 869Lys Lys Gln Phe Phe Arg Asn Phe Ile Thr Ile Met Leu Phe Gly Ala90 95 100AAG AAG CGT TTC CGT AAC TTT ACC ATC ATC ATG CTT GGG GCT 869LS LYS GLN PHE PHE ARG Asn PHR ILE MET Leu Phe Gly Gly Gly Ala
105 110 115gtt ggt aca cta ata tct tgt aca att atc tct tta ggt gta att aac 917Val Gly Thr Leu Ile Ser Cys Thr Ile Ile Ser Leu Gly Val Ile Asn120 125 130 135ttc ttc aag gaa atg gac att ggc tcc tta gac att gga gat ttt cta 965Phe Phe Lys Glu Met Asp Ile Gly Ser Leu Asp lle Gly Asp Phe Leu105 110 115gtt ggt aca cta ata tct tgt aca att atc tct tta ggt gta att aac 917Val Gly Thr Leu Ile Ser Cys Thr Ile Ile Ser Leu Gly Val Ile Asn120 125 130 135ttc ttc aag gaa atg gac att ggc tcc tta gac att gga gat ttt cta 965Phe Phe Lys Glu Met Asp Ile Gly Ser Leu Asp lle Gly Asp Phe Leu
140 145 150gca att ggt gca ata ttt gct gcg aca gat tct gtt tgc aca ctg cag 1013Ala Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser Val Cys Thr Leu Gln140 145 150GCA AT GCA ATA TTT GCG ACA GCG ACA GAT TCT GTT GTT TGC ACA CTG CAG 1013ALA ILE PHE ALA ALA ALA ASP Ser Val Cys THR Leu Gln
155 160 165gtg ctt aat cag gat gag act cca tta ctc tac agt ttg gtt ttc gga 1061Val Leu Asn Gln Asp Glu Thr Pro Leu Leu Tyr Ser Leu Val Phe Gly155 160 165GTG CTT AAT CAG GAG GAG ACT CCA TTA CTC TAC AGT TTG GGA 1061val Leu Asn Gln ASP GLU Leu Leu Tyr Seru Val Phe Gly Gly Gly
170 175 180gag ggt gtt gta aat gat gca aca tct gtg gtg ctt ttc aat gca atc 1109Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Leu Phe Asn Ala Ile170 175 180gag GTT GTA AAT GCA ACA TCA TCT GTG GTG CTC AAT GCA ATC 1109GLU GLY Val Val Val Val Val Val Leu PHE Asn Ala Ile
185 190 195cag agt ttt gac ctc gtt aat acc agt cct aga att ctt ctg gag ttt 1157Gln Ser Phe Asp Leu Val Asn Thr Ser Pro Arg Ile Leu Leu Glu Phe200 205 210 215att ggc agc ttt ttg tat tta ttt tta gca agc act atg ctg gga gtg 1205Ile Gly Ser Phe Leu Tyr Leu Phe Leu Ala Ser Thr Met Leu Gly Val185 190 195cag agt ttt gac ctc gtt aat acc agt cct aga att ctt ctg gag ttt 1157Gln Ser Phe Asp Leu Val Asn Thr Ser Pro Arg Ile Leu Leu Glu Phe200 205 210 215att ggc agc ttt ttg tat tta ttt tta gca agc act atg ctg gga gtg 1205Ile Gly Ser Phe Leu Tyr Leu Phe Leu Ala Ser Thr Met Leu Gly Val
220 225 230att gtt ggg ttg gtt agt gct tac atc atc aaa aag ttg tac ttt gga 1253220 225 230att gtt ggg ttg gtt agt gct tac atc atc aaa aag ttg tac ttt gga 1253
钠离子反向运转蛋白基因序列表-wordIle Val Gly Leu Val Ser Ala Tyr Ile Ile Lys Lys Leu Tyr Phe GlySodium ion anti-transport protein gene sequence list-wordIle Val Gly Leu Val Ser Ala Tyr Ile Ile Lys Lys Leu Tyr Phe Gly
235 240 245agg cac tca aca gat cgt gaa ttt gct ctt atg atg ctt atg gca tac 1301Arg His Ser Thr Asp Arg Glu Phe Ala Leu Met Met Leu Met Ala Tyr235 240 245AGG CAA ACA GAT CGT GAA TTT GCT CTT ATG ATG CTT ATG GCA TAC 1301ARG HIS Serg Glu PHE Ala Leu Met Leu Met Ala Tyr
250 255 260ctt tcg tat atc atg gct gaa ctg ttc tat ttg agt ggc att ctt aca 1349Leu Ser Tyr Ile Met Ala Glu Leu Phe Tyr Leu Ser Gly Ile Leu Thr250 255 260CTT TAT ATC ATC ATG GCT GAA CTG TAT TTG AGT GGC ATT CTT ACA 1349leu Serle Met Ala Glu Phe Tyr Leu Serle Leu Thr
265 270 275gta ttc ttt tgt ggg att gtg atg tca cat tat acc tgg cac aat gta 1397Val Phe Phe Cys Gly Ile Val Met Ser His Tyr Thr Trp His Ash Val280 285 290 295act gag agt tca aga gta act aca aag cat gcc ttt gct acc ttg tca 1445Thr Glu Ser Ser Arg Val Thr Thr Lys His Ala Phe Ala Thr Leu Ser265 270 275gta ttc ttt tgt ggg att gtg atg tca cat tat acc tgg cac aat gta 1397Val Phe Phe Cys Gly Ile Val Met Ser His Tyr Thr Trp His Ash Val280 285 290 295act gag agt tca aga gta act aca aag cat gcc ttt gct acc ttg tca 1445Thr Glu Ser Ser Arg Val Thr Thr Lys His Ala Phe Ala Thr Leu Ser
300 305 310ttt gtt gct gag act ttt ctc ttt ctt tat gtc ggg atg gat gct ttg 1493Phe Val Ala Glu Thr Phe Leu Phe Leu Tyr Val Gly Met Asp Ala Leu300 305 310ttt GCT GCT GAG Act TTT CTT CTT CTT CTT GGG ATG GT GCT GCT GCT GCT 1493PHE VAL Ala Glu Thr Phe Leu Tyr Val Gly Met Ala Leu
315 320 325gac atg gag aag tgg aga ttt gtc agt gat agc cct gga acg tca gtt 1541Asp Met Glu gys Trp Arg Phe Val Ser Asp Ser Pro Gly Thr Ser Val315 320 325GAC Atg Aag AAG TGG AGA TTT GTC AGC AGC CCT GGA ACG TCA GTT 1541asp Met Gys TRP ARG PHE VAL Ser Pro Gly Thr Ser Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val
330 335 340gct gtt agt gct gtg ctg atg ggt ctt gtt atg gtt gga aga gcg gct 1589Ala Val Ser Ala Val Leu Met Gly Leu Val Met Val Gly Arg Ala Ala330 335 340GCT GCT GCT GCT GTG ATG GGT GGT GGA AGA GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG GCG
345 350 355ttt gtg ttt ccc ctg tca ttt tta tcc aac ttg gca aag aaa tca act 1637Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Ala Lys Lys Ser Thr360 365 370 375agt gag aaa atc agc ttc agg gaa caa att ata ata tgg tgg gct ggg 1685Ser Glu Lys Ile Ser Phe Arg Glu Gln Ile Ile Ile Trp Trp Ala Gly345 350 355ttt gtg ttt ccc ctg tca ttt tta tcc aac ttg gca aag aaa tca act 1637Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Ala Lys Lys Ser Thr360 365 370 375agt gag aaa atc agc ttc agg gaa caa att ata ata tgg tgg gct ggg 1685Ser Glu Lys Ile Ser Phe Arg Glu Gln Ile Ile Ile Trp Trp Ala Gly
380 385 390ctc atg aga ggc gct gta tct atg gca ctt gca tat aat cag ttt aca 1733Leu Met Arg Gly Ala Val Ser Met Ala Leu Ala Tyr Ash Gln Phe Thr380 385 390CTC Atg Aga GGC GCT GCT GCA CTT GCA TAT AAT CAG TAT CAG TTT ARG GLY ALA VAL ALA Leu Ala Tyr Ash Gln Phe Thr
395 400 405agg ggg ggc cat act cag ttg cga gga aat gca att atg att aca agc 1781Arg Gly Gly His Thr Gln Leu Arg Gly Ash Ala Ile Met Ile Thr Ser395 4005AGG GGC Cat Act CGA GGA AAT GCA ATT ATT AGC 1781arg GLY GLY GLN Leu ARG GLY ALA ILE MET ILE THR Ser Ser
410 415 420acc ata acc att gtt cta ttc agc act gtg gtt ttt ggt tta atg act 1829Thr Ile Thr Ile Val Leu Phe Ser Thr Val Val Phe Gly Leu Met Thr410 415 420ACC ATA ACC ATT GTT CTA TTC AGC AGC AGC GTG GTT GGT GGT TG ACT 1829thr Ile Val Leu Phe Ser Val Phe Gly Leu Met Thr
425 430 435aaa cct cta ata agg ttc ttg ctg cct cat ccc aaa cca aca gcc agc 1877Lys Pro Leu Ile Arg Phe Leu Leu Pro His Pro Lys Pro Thr Ala Set440 445 450 455atg ctc tca gac caa tcc act cca aaa tca atg gag gca cca ttt ctc 1925Met Leu Ser Asp Gln Ser Thr Pro Lys Ser Met Glu Ala Pro Phe Leu425 430 435aaa cct cta ata agg ttc ttg ctg cct cat ccc aaa cca aca gcc agc 1877Lys Pro Leu Ile Arg Phe Leu Leu Pro His Pro Lys Pro Thr Ala Set440 445 450 455atg ctc tca gac caa tcc act cca aaa tca atg gag gca cca ttt ctc 1925Met Leu Ser Asp Gln Ser Thr Pro Lys Ser Met Glu Ala Pro Phe Leu
460 465 470gga agc ggc cag gac tct ttt gat gat agt tta att gga gtt cat cga 1973Gly Ser Gly Gln Asp Ser Phe Asp Asp Ser Leu Ile Gly Val His Arg460 465 470GGA AGC GGC CAG GAC GAC TCT GAT GAT GAT GAT GGA GGA GTT CAT CGA 1973gly Serge Gln Asp ASP ASER Leu Ile Gly Val Val Val Val Val Val Leg Vgal Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val valg
475 480 485cca aac agc att cgt gca ctt ctt aca act cca gca cac act gtt cat 2021Pro Asn Ser Ile Arg Ala Leu Leu Thr Thr Pro Ala His Thr Val His475 485cca Aac AGC AGC AGC ATT CGT GCA CTT CTT ACT CCA CAC Act CAT GTT CAT 2021Pro Asn Serle ARG Ala Leu Thr His ThR Val His His
490 495 500tac tat tgg cga aag ttt gat aat gcc ttc atg cgc cct atg ttt ggt 2069Tyr Tyr Trp Arg Lys Phe Asp Ash Ala Phe Met Arg Pro Met Phe Gly490 495 500TAC TAT TGG CGA AAG TTT GCC TTC ATC ATG CGC CCT ATT GGT 2069tyr Tyr Tys PHE Ala Phe Met ARG Pro Met Phe Gly Gly Gly Gly Gly Gly Gly Gly
505 510 515ggc cgg ggt ttt gtg ccc ttc gtt cct ggc tcc cca aca gaa agg agt 2117Gly Arg Gly Phe Val Pro Phe Val Pro Gly Ser Pro Thr Glu Arg Ser520 525 530 535gaa cct aat ctg cct caa tgg caa tgaggtggtt gaacaagatc tctacaaaaa 2171Glu Pro Asn Leu Pro Gln Trp Gln505 510 515ggc cgg ggt ttt gtg ccc ttc gtt cct ggc tcc cca aca gaa agg agt 2117Gly Arg Gly Phe Val Pro Phe Val Pro Gly Ser Pro Thr Glu Arg Ser520 525 530 535gaa cct aat ctg cct caa tgg caa tgaggtggtt gaacaagatc tctacaaaaa 2171Glu Pro Asn Leu Pro Gln Trp Gln
540tgtacatgta atataacaat gcagtcggtt gcaaaaaaca tgcttctggc gagaagccag 2231tgcggtatgc tttgtatgtt tcatgtatag gctatatttt gttggttttc aagtttcctc 2291aagaggttct tgtttattct ccccgaaact accttcgcac ctgatgctat ctttccattt 2351gacatttacg aatatttatg atctgggtga agcttagggg taggtgtgcc attctatttt 2411gtacgtatac gagtatttat tttgtgttta tatcagtgtg tttagttttt atttttatta 2471aaaaaaaaaa aaaa 2485<210>2<211>543<212>PRT<213>Gossypium hirsutum<400>2Met Val Ala Pro Gln Leu Ala Ala Val Phe Thr Lys Leu Gln Thr Leu1 5 10 15Ser Thr Ser Asp His Ala Ser Val Val Ser Met Asn Ile Phe Val Ala540tgtacatgta atataacaat gcagtcggtt gcaaaaaaca tgcttctggc gagaagccag 2231tgcggtatgc tttgtatgtt tcatgtatag gctatatttt gttggttttc aagtttcctc 2291aagaggttct tgtttattct ccccgaaact accttcgcac ctgatgctat ctttccattt 2351gacatttacg aatatttatg atctgggtga agcttagggg taggtgtgcc attctatttt 2411gtacgtatac gagtatttat tttgtgttta tatcagtgtg tttagttttt atttttatta 2471aaaaaaaaaa aaaa 2485<210>2<211>543<212>PRT<213>Gossypium hirsutum <400> 2MET VAL Ala Pro Gln Leu Ala Ala Val Phe Thr Lys Leu
20 25 3020 25 30
钠离子反向运转蛋白基因序列表-wordLeu Leu Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn ArgSodium ion antitransportation protein gene sequence list-wordLeu Leu Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn Arg
35 40 45Trp Met Asn Glu Ser Ile Thr Ala Leu Ile Ile Gly Val Phe Thr Gly35 40 45Trp Met Asn Glu Ser Ile Thr Ala Leu Ile Ile Gly Val Phe Thr Gly
50 55 60Val Ile Ile Leu Leu Thr Ser Gly Gly Lys Ser Ser His Leu Leu Val65 70 75 80Phe Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe50 55 60VAL ILE Leu Leu THR Ser Gly Gly Lys Sering
85 90 95Asn Ala Gly Phe Gln ValLys Lys Lys Gln Phe Phe Arg Asn Phe Ile85 90 95Asn Ala Gly Phe Gln ValLys Lys Lys Gln Phe Phe Arg Asn Phe Ile
100 105 110Thr Ile Met Leu Phe Gly Ala Val Gly Thr Leu Ile Ser Cys Thr Ile100 105 110Thr Ile Met Leu Phe Gly Ala Val Gly Thr Leu Ile Ser Cys Thr Ile
115 120 125Ile Ser Leu Gly Val Ile Asn Phe Phe Lys Glu Met Asp Ile Gly Ser115 120 125Ile Ser Leu Gly Val Ile Asn Phe Phe Lys Glu Met Asp Ile Gly Ser
130 135 140Leu Asp Ile Gly Asp Phe Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr145 150 155 160Asp Ser Val Cys Thr Leu Gln Val Leu Asn Gln Asp Glu Thr Pro Leu130 135 140leu ASP ILE GLY ASP PHE Leu Ala Ile Gly Ala Ile PHE Ala Ala Ala THR145 150 155 160s THR Leu Gln Val Leu Asn GLU Thr Pro Leu
165 170 175Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser165 170 175Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser
180 185 190Val Val Leu Phe Asn Ala Ile Gln Ser Phe Asp Leu Val Asn Thr Ser180 185 190Val Val Leu Phe Asn Ala Ile Gln Ser Phe Asp Leu Val Asn Thr Ser
195 200 205Pro Arg Ile Leu Leu Glu Phe Ile Gly Ser Phe Leu Tyr Leu Phe Leu195 200 205Pro Arg Ile Leu Leu Glu Phe Ile Gly Ser Phe Leu Tyr Leu Phe Leu
210 215 220Ala Ser Thr Met Leu Gly Val Ile Val Gly Leu Val Ser Ala Tyr Ile225 230 235 240Ile Lys Lys Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Phe Ala210 215 220ALA Ser THR MET Leu Gly Val Ile Val Gly Leu Val
245 250 255Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Ile Met Ala Glu Leu Phe245 250 255Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Ile Met Ala Glu Leu Phe
260 265 270Tyr Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser260 265 270Tyr Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser
275 280 285His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Lys275 280 285 His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Lys
290 295 300His Ala Phe Ala Thr Leu Ser Phe Val Ala Glu Thr Phe Leu Phe Leu305 310 315 320Tyr Val Gly Met Asp Ala Leu Asp Met Glu Lys Trp Arg Phe Val Ser290 295 300His Ala Phe Ala Thr Leu Ser, Val Ala Glu Thr Phe Leu 305 310 320tyr Val Gly Met Ala Leu ASP MET GLU LYS TRG PHE Val Serg PHE Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Serg Phe Val Ser Serg Phe Val Ser
325 330 335Asp Ser Pro Gly Thr Ser Val Ala Val Ser Ala Val Leu Met Gly Leu325 330 335Asp Ser Pro Gly Thr Ser Val Ala Val Ser Ala Val Leu Met Gly Leu
340 345 350Val Met Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser340 345 350Val Met Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser
355 360 365Asn Leu Ala Lys Lys Ser Thr Ser Glu Lys Ile Ser Phe Arg Glu Gln355 360 365Asn Leu Ala Lys Lys Ser Thr Ser Glu Lys Ile Ser Phe Arg Glu Gln
370 375 380Ile Ile Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Met Ala385 390 395 400Leu Ala Tyr Asn Gln Phe Thr Arg Gly Gly His Thr Gln Leu Arg Gly370 375 380ile ILE TRP TRP TRP Ala Gly Leu Met ARG GLY Ala Val Serite Ala385 395 400leu Ala Tyr Asn Phe Thr ARG GLY GLN Leu ARG GLY GLY
405 410 415Asn Ala Ile Met Ile Thr Ser Thr Ile Thr Ile Val Leu Phe Ser Thr405 410 415Asn Ala Ile Met Ile Thr Ser Thr Ile Thr Ile Val Leu Phe Ser Thr
420 425 430Val Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Phe Leu Leu Pro420 425 430Val Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Phe Leu Leu Pro
435 440 445His Pro Lys Pro Thr Ala Ser Met Leu Ser Asp Gln Ser Thr Pro Lys435 440 445His Pro Lys Pro Thr Ala Ser Met Leu Ser Asp Gln Ser Thr Pro Lys
450 455 460Ser Met Glu Ala Pro Phe Leu Gly Ser Gly Gln Asp Ser Phe Asp Asp465 470 475 480Ser Leu Ile Gly Val His Arg Pro Asn Ser Ile Arg Ala Leu Leu Thr450 455 460r Met Glu Ala Pro PHE Leu GLY Serg Gln Asr PHE ASP ASP465 475 480r Leu Ile Gly Val His ARG ALA Leu Leu Thr Thr Thr
485 490 495Thr Pro Ala His Thr Val His Tyr Tyr Trp Arg Lys Phe Asp Asn Ala485 490 495Thr Pro Ala His Thr Val His Tyr Tyr Trp Arg Lys Phe Asp Asn Ala
500 505 510Phe Met Arg Pro Met Phe Gly Gly Arg Gly Phe Val Pro Phe Val Pro500 505 510Phe Met Arg Pro Met Phe Gly Gly Arg Gly Phe Val Pro Phe Val Pro
515 520 525Gly Ser Pro Thr Glu Arg Ser Glu Pro Asn Leu Pro Gln Trp Gln515 520 525Gly Ser Pro Thr Glu Arg Ser Pro Thr Glu Pro Asn Leu Pro Gln Trp Gln
530 535 540530 535 540
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| CNB021515301A CN1190492C (en) | 2002-12-31 | 2002-12-31 | Cotton Na+/H+ reverse transport protein gene and its cloning method and use |
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|---|---|---|---|
| CNB021515301A CN1190492C (en) | 2002-12-31 | 2002-12-31 | Cotton Na+/H+ reverse transport protein gene and its cloning method and use |
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| CN1425675A true CN1425675A (en) | 2003-06-25 |
| CN1190492C CN1190492C (en) | 2005-02-23 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355896C (en) * | 2004-07-30 | 2007-12-19 | 中国科学院上海生命科学研究院 | Method for breeding halophyte of double transgene |
| CN106319082A (en) * | 2016-11-09 | 2017-01-11 | 中国农业科学院棉花研究所 | Method for identifying cotton seedling-stage salt tolerance |
| CN106868039A (en) * | 2017-03-02 | 2017-06-20 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in genetically modified plants are cultivated |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
-
2002
- 2002-12-31 CN CNB021515301A patent/CN1190492C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355896C (en) * | 2004-07-30 | 2007-12-19 | 中国科学院上海生命科学研究院 | Method for breeding halophyte of double transgene |
| CN106319082A (en) * | 2016-11-09 | 2017-01-11 | 中国农业科学院棉花研究所 | Method for identifying cotton seedling-stage salt tolerance |
| CN106868039A (en) * | 2017-03-02 | 2017-06-20 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in genetically modified plants are cultivated |
| CN106868039B (en) * | 2017-03-02 | 2019-11-29 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in cultivation genetically modified plants |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1190492C (en) | 2005-02-23 |
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