Plant high-pH saline-alkaline tolerance gene SucHP and application thereof

Abstract

The invention discloses a plant high-pH saline-alkaline tolerance gene SucHP and researches the expression of a high-pH saline and alkaline stress induction gene in a common seepweed herb seedling period by using suppression subtractive hybridization and high-density lattice film technology. The plant high-pH saline-alkaline tolerance gene SucHP can be prepared by the following steps of: firstly separating a gene of a coding tonoplast proton pump from common seepweed herbs; constructing a sense expression vector of the gene; and converting Arabidopsis. A transgenic plant has high high-pH saline-alkaline tolerance and can normally grow under the conditions of 100mM NaHCO3+200mM NaCl and 8.5 of pH. The plant high-pH saline-alkaline tolerance gene SucHP can be used for plant genetic transformation and increases plant saline and alkaline resistance.

Description

technical field

[0001] The invention belongs to the field of biotechnology, in particular to a plant high pH saline-alkali tolerance gene SucHP and its application. Background technique

[0002] Due to the long-term impact of natural factors and human factors, soil salinization is becoming more and more serious around the world. At present, there are about 1 billion hectares of saline-alkali land in the world, while the area of ​​saline-alkali land in my country is 99.13 million hectares (accounting for about 10% of the world's total area). These saline-alkali lands mainly include NaCl, Na 2 SO 4 Saline soil dominated by neutral salt, and NaHCO 3 、Na 2 CO 3 Soda saline-alkali soil dominated by alkaline salts. In my country's ever-expanding inland saline-alkali land, a large part is soda saline-alkali soil. In this type of soil, due to the large amount of carbonate (NaHCO 3 , Na2CO 3 ) hydrolysis, the soil pH is as high as 8.0 or more. Therefore, NaHCO in soda salin...

Examples

Embodiment 1

[0028] Embodiment 1: Cloning of Suaeda salsa tonoplast proton pump gene SucHP

[0029] 1) Extraction of total RNA: Suaeda salsa seeds were collected from Baicheng, Jilin Province. After sterilizing Suaeda salsa seeds with mercuric chloride, they were sown in MS medium, and after 3 weeks, the seedlings were transferred to the hydroponic system, and after 4 weeks of cultivation in the hydroponic system, they were treated with 100mM NaHCO 3 +200mM NaCl for treatment; after 24 hours of treatment, take the whole plant (including roots and leaves), fix it with liquid nitrogen, and store it in a -80°C freezer for later use;

[0030] Suaeda salsa total RNA was extracted using RNAeasy mini kit (promoga company product);

[0031] 2) Separating the above-mentioned Suaeda salsa total RNA treated with saline, using the magnetic bead method (Promega company) to isolate and purify mRNA, utilizing the Library Construction Kit (Clontech) kit for reverse transcription, and establishing the Sua...

Embodiment 2

[0037] Embodiment 2RT-PCR detects the Suaeda salsa of saline-alkali stress

[0038] RT-PCR detection primers:

[0039] Primer 5 (upstream sequence): 5'-CTgCTggAAACACTACCgCT-3'

[0040] Primer 6 (downstream sequence): 5'-CATTgTCCCAAgCACCTCCT-3'

[0041] Primer 7 (upstream sequence): 5'-TACCACATCCAAggAAggCA-3'

[0042] Primer 8 (downstream sequence): 5'-ACCCAAgTCCAACTACgAg-3'

[0043] Primer 5 and primer 6 are the primers of the SucHP gene in the vector, and the amplified fragment is 569bp; primer 7 and primer 8 are the primers of the internal reference gene Actin, and the amplified fragment is 438bp; 3 Treat 0h, 6h, 12h, 24h, 48h. Use the cDNA of Suaeda salsa roots, stems and leaves as templates to detect gene expression; after the PCR reaction is completed, perform 1% agarose gel electrophoresis detection on the PCR products, and the detection results are shown in figure 1 , the results showed that the expression of SucHP gene had a certain salt-alkaline inducibility.

Embodiment 3

[0044] Example 3 Construction of Plant Expression Vector pCB35S-SucHP

[0045] According to the sequence of SEQ ID No.1, design and construct the ATTB1 and ATTB2 adapter primers of the expression vector:

[0046] Primer 1: 5′-AAAAAGCAGGCTATGGGTGTTGTTCTTCTTC-3′

[0047] Primer 2: 5′-AGAAAGCTGGGTTTAGAAAGATCTTGAAGAGCA-3′

[0048] Primer 3: 5′-ggggACAAgTTTgTACAAAAAAgCAggCT-3′

[0049] Primer 4: 5′-ggggACCACTTTgTACAAgAAAgCTgggT-3′

[0050] 1) Using the total cDNA of Suaeda salsa as a template, use primer 1 and primer 2 with ATTB1 and ATTB2 adapters to carry out the first round of PCR amplification. The amplification system is as follows:

[0051] The amplification system and procedures are:

[0052] 2×Taq Mix 12.5μl

[0053] Primer 1 1.0 μl

[0054] Primer 2 1.0 μl

[0055] Template DNA 1.0μl

[0056] ddH2O 9.5μl

[0057] The PCR reaction conditions are:

[0058] After the PCR reaction, take 5 μl of the product and mix it in 2 μl of 6×Loading Buffer, and perform electro...

technical field

[0001] The invention belongs to the field of biotechnology, in particular to a plant high pH saline-alkali tolerance gene SucHP and its application. Background technique

[0002] Due to the long-term impact of natural factors and human factors, soil salinization is becoming more and more serious around the world. At present, there are about 1 billion hectares of saline-alkali land in the world, while the area of ​​saline-alkali land in my country is 99.13 million hectares (accounting for about 10% of the world's total area). These saline-alkali lands mainly include NaCl, Na 2 SO 4 Saline soil dominated by neutral salt, and NaHCO 3 、Na 2 CO 3 Soda saline-alkali soil dominated by alkaline salts. In my country's ever-expanding inland saline-alkali land, a large part is soda saline-alkali soil. In this type of soil, due to the large amount of carbonate (NaHCO 3 , Na2CO 3 ) hydrolysis, the soil pH is as high as 8.0 or more. Therefore, NaHCO in soda salin...

Examples

Embodiment 1

[0028] Embodiment 1: Cloning of Suaeda salsa tonoplast proton pump gene SucHP

[0029] 1) Extraction of total RNA: Suaeda salsa seeds were collected from Baicheng, Jilin Province. After sterilizing Suaeda salsa seeds with mercuric chloride, they were sown in MS medium, and after 3 weeks, the seedlings were transferred to the hydroponic system, and after 4 weeks of cultivation in the hydroponic system, they were treated with 100mM NaHCO 3 +200mM NaCl for treatment; after 24 hours of treatment, take the whole plant (including roots and leaves), fix it with liquid nitrogen, and store it in a -80°C freezer for later use;

[0030] Suaeda salsa total RNA was extracted using RNAeasy mini kit (promoga company product);

[0031] 2) Separating the above-mentioned Suaeda salsa total RNA treated with saline, using the magnetic bead method (Promega company) to isolate and purify mRNA, utilizing the Library Construction Kit (Clontech) kit for reverse transcription, and establishing the Sua...

Embodiment 2

[0037] Embodiment 2RT-PCR detects the Suaeda salsa of saline-alkali stress

[0038] RT-PCR detection primers:

[0039] Primer 5 (upstream sequence): 5'-CTgCTggAAACACTACCgCT-3'

[0040] Primer 6 (downstream sequence): 5'-CATTgTCCCAAgCACCTCCT-3'

[0041] Primer 7 (upstream sequence): 5'-TACCACATCCAAggAAggCA-3'

[0042] Primer 8 (downstream sequence): 5'-ACCCAAgTCCAACTACgAg-3'

[0043] Primer 5 and primer 6 are the primers of the SucHP gene in the vector, and the amplified fragment is 569bp; primer 7 and primer 8 are the primers of the internal reference gene Actin, and the amplified fragment is 438bp; 3 Treat 0h, 6h, 12h, 24h, 48h. Use the cDNA of Suaeda salsa roots, stems and leaves as templates to detect gene expression; after the PCR reaction is completed, perform 1% agarose gel electrophoresis detection on the PCR products, and the detection results are shown in figure 1 , the results showed that the expression of SucHP gene had a certain salt-alkaline inducibility.

Embodiment 3

[0044] Example 3 Construction of Plant Expression Vector pCB35S-SucHP

[0045] According to the sequence of SEQ ID No.1, design and construct the ATTB1 and ATTB2 adapter primers of the expression vector:

[0046] Primer 1: 5′-AAAAAGCAGGCTATGGGTGTTGTTCTTCTTC-3′

[0047] Primer 2: 5′-AGAAAGCTGGGTTTAGAAAGATCTTGAAGAGCA-3′

[0048] Primer 3: 5′-ggggACAAgTTTgTACAAAAAAgCAggCT-3′

[0049] Primer 4: 5′-ggggACCACTTTgTACAAgAAAgCTgggT-3′

[0050] 1) Using the total cDNA of Suaeda salsa as a template, use primer 1 and primer 2 with ATTB1 and ATTB2 adapters to carry out the first round of PCR amplification. The amplification system is as follows:

[0051] The amplification system and procedures are:

[0052] 2×Taq Mix 12.5μl

[0053] Primer 1 1.0 μl

[0054] Primer 2 1.0 μl

[0055] Template DNA 1.0μl

[0056] ddH2O 9.5μl

[0057] The PCR reaction conditions are:

[0058] After the PCR reaction, take 5 μl of the product and mix it in 2 μl of 6×Loading Buffer, and perform electro...

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