Cotton Na+/H+ reverse transport protein gene and its cloning method and use

Abstract

The present invention relates to the cloning, recombination and salt tolerance analysis of cotton Na+ / H+ reverse transport protein GhNHX1 gene, and belongs to the field of molecular biology and biological technology. Total RNA is extracted from cotton leaf treated with 0.4 mol / L NaCl solution and inversely transcribed into cDNA. Facultative primer is used for conventional PCR to obtain intermediate segment, and whole length cDNA is obtained through fast amplification in 3' and 5' ends. The gene is transformed to salt-sensitive yeast mutant to restore its salt tolerant capacity to some degree; and the gene is further transformed to tobacco to obtain transgenic plant capabl eof growing normally in salt concentration of 200 mmol / L. Therefore, the gene is one important salt tolerant gene and may be used in raising the salt tolerant capacity of plant for plantation in saline land.

Description

(1) Technical field

[0001] The present invention relates to Na in cotton + / H + The invention relates to cloning, recombination and salt tolerance function analysis and application of antiporter gene GhNHX1, belonging to the field of molecular biology and biotechnology. (2) Background technology

[0002] High concentrations of salt cause ion imbalance and hyperosmotic stress in plants, resulting in stunted plant development, slow growth, and especially reduced crop yields. Severe salt stress or osmotic stress can cause the second stress in plants, oxidative stress, and even lead to plant death. Therefore, salt stress has received extensive attention, and more and more attention has been paid to improving the salt tolerance of crops. Under high salinity, plants alleviate the toxicity caused by salt stress by producing stress proteins and soluble osmotic regulators. The early salt tolerance genetic engineering mainly focused on scavenging free radicals and increasing osmot...

Examples

Embodiment 2

[0097] 8. Homology search: The isolated sequences were compared with the sequences in the gene bank using BLAST software. Example 2: Cotton Na + / H + Antiporter gene GhNHX1, the following sequence:

[0098] (1) Information of SEQ ID NO 1

[0099] (a) Sequence features

[0100] * Length: 2485 base pairs

[0101] * Type: Nucleic Acid

[0102] * Chain type: double chain

[0103] * Topology: Linear

[0104] (b) Molecular type: cDNA

[0105] (c) Assumption: No

[0106] (d) Antonym: No

[0107] (e) Original Source: Cotton

[0108] (f) Sequence description: SEQ IN NO.1

[0109] ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60

[0110] TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120

[0111] CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180

[0112] CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240

[0113] AAAATCGCAAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300

[0114] ATTTGTTGCTGCATGATGTTGAT...

Embodiment 3

[0156] (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

[0157] 1. According to the isolated cotton Na + / H + Nucleotide sequence of antiporter gene GhNHX1, designed primers:

[0158] Forward primer: 5'-ATGGTGGCTCCGCAGTTAGCT-3'

[0159] Reverse primer: 5'-ACCTCATTGCCATTGAGGCAG-3'

[0160] The polymerase chain reaction was performed using the reverse transcribed cDNA from the total RNA of the leaf as a template.

[0161] 2...

Embodiment 4

[0163] 4. Transform the constructed expression vector into Agrobacterium EHA105. Example 4: Analysis of genetic salt tolerance

[0164] 1. To grow tobacco.

[0165] 2. Pick the identified Agrobacterium monoclonal in LB liquid medium containing 50 mg / L kanamycin, and culture with shaking at 28°C.

[0166] 3. Centrifuge at 3000rpm for 5 minutes, and suspend the Agrobacterium pellet with 10X MS medium.

[0167] 4. Cut the tobacco leaves into small pieces and soak in the above suspension for 10 minutes.

[0168] 5. The infected leaf cuttings were placed on differentiation medium (1×MS salt, 3% sucrose, pH5.8, 4.5g / L carrageenan) for 2 days, and then transferred to selection medium (1 ×MS salt, 3% sucrose, pH 5.8, 4.5 g / L carrageenan, kanamycin 100 mg / L, cephalosporin 250 mg / L) screening to obtain resistant plants.

[0169] 6. Move the resistant plants into flower pots and water with 10mmolL every two days -1 , 100mmolL -1 , 200mmolL -1 and 300mmolL -1 1 / 2 Hoagland nutrient...

(1) Technical field

[0001] The present invention relates to Na in cotton + / H + The invention relates to cloning, recombination and salt tolerance function analysis and application of antiporter gene GhNHX1, belonging to the field of molecular biology and biotechnology. (2) Background technology

[0002] High concentrations of salt cause ion imbalance and hyperosmotic stress in plants, resulting in stunted plant development, slow growth, and especially reduced crop yields. Severe salt stress or osmotic stress can cause the second stress in plants, oxidative stress, and even lead to plant death. Therefore, salt stress has received extensive attention, and more and more attention has been paid to improving the salt tolerance of crops. Under high salinity, plants alleviate the toxicity caused by salt stress by producing stress proteins and soluble osmotic regulators. The early salt tolerance genetic engineering mainly focused on scavenging free radicals and increasing osmot...

Examples

Embodiment 2

[0097] 8. Homology search: The isolated sequences were compared with the sequences in the gene bank using BLAST software. Example 2: Cotton Na + / H + Antiporter gene GhNHX1, the following sequence:

[0098] (1) Information of SEQ ID NO 1

[0099] (a) Sequence features

[0100] * Length: 2485 base pairs

[0101] * Type: Nucleic Acid

[0102] * Chain type: double chain

[0103] * Topology: Linear

[0104] (b) Molecular type: cDNA

[0105] (c) Assumption: No

[0106] (d) Antonym: No

[0107] (e) Original Source: Cotton

[0108] (f) Sequence description: SEQ IN NO.1

[0109] ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60

[0110] TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120

[0111] CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180

[0112] CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240

[0113] AAAATCGCAAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300

[0114] ATTTGTTGCTGCATGATGTTGAT...

Embodiment 3

[0156] (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

[0157] 1. According to the isolated cotton Na + / H + Nucleotide sequence of antiporter gene GhNHX1, designed primers:

[0158] Forward primer: 5'-ATGGTGGCTCCGCAGTTAGCT-3'

[0159] Reverse primer: 5'-ACCTCATTGCCATTGAGGCAG-3'

[0160] The polymerase chain reaction was performed using the reverse transcribed cDNA from the total RNA of the leaf as a template.

[0161] 2...

Embodiment 4

[0163] 4. Transform the constructed expression vector into Agrobacterium EHA105. Example 4: Analysis of genetic salt tolerance

[0164] 1. To grow tobacco.

[0165] 2. Pick the identified Agrobacterium monoclonal in LB liquid medium containing 50 mg / L kanamycin, and culture with shaking at 28°C.

[0166] 3. Centrifuge at 3000rpm for 5 minutes, and suspend the Agrobacterium pellet with 10X MS medium.

[0167] 4. Cut the tobacco leaves into small pieces and soak in the above suspension for 10 minutes.

[0168] 5. The infected leaf cuttings were placed on differentiation medium (1×MS salt, 3% sucrose, pH5.8, 4.5g / L carrageenan) for 2 days, and then transferred to selection medium (1 ×MS salt, 3% sucrose, pH 5.8, 4.5 g / L carrageenan, kanamycin 100 mg / L, cephalosporin 250 mg / L) screening to obtain resistant plants.

[0169] 6. Move the resistant plants into flower pots and water with 10mmolL every two days -1 , 100mmolL -1 , 200mmolL -1 and 300mmolL -1 1 / 2 Hoagland nutrient...

Images