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Strong-salt-tolerant plant gene SseNHX1 as well as encoding protein and application of strong-salt-tolerant plant gene SseNHX1

A salt-tolerant gene and plant technology, applied in the field of plant genetic engineering, can solve the problems of low single gene activity, low salt tolerance, limited production application and promotion, etc., and achieve the effect of improving screening efficiency and salt tolerance

Inactive Publication Date: 2014-08-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual production and application, plant salt-tolerant genetic engineering mostly focuses on the cloning and genetic transformation of a single salt-tolerant gene. Although a single salt-tolerant gene can improve the salt tolerance of plants to a certain extent, however, due to the The defects of low activity and low salt tolerance greatly limit the production, application and promotion of this gene

Method used

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  • Strong-salt-tolerant plant gene SseNHX1 as well as encoding protein and application of strong-salt-tolerant plant gene SseNHX1
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  • Strong-salt-tolerant plant gene SseNHX1 as well as encoding protein and application of strong-salt-tolerant plant gene SseNHX1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1. Suaeda salsa and salicornia Na + / H + Cloning of the antiporter gene

[0031] The total RNA was extracted from the leaves of Suaeda salsa and Salicornia with Trizol reagent, and the first strand of cDNA was synthesized by reverse transcription using the total RNA as a template. Using the synthesized cDNA as a template, the reference pair encodes Na + / H + The cDNA sequences of the wild-type salt-tolerant genes SsNHX1 (derived from Suaeda salsa, its GenBank sequence number: AY261806) and SeNHX1 (derived from Salicornia, its GenBank sequence number: AY131235) of the antiporter were designed with the following primers (5' end Contains BamHI and SalI restriction sites respectively):

[0032] ssF(5'-CGC GGATCC ATGTGGTCACAGTTAAGCTC-3') SEQ ID NO: 3

[0033] ssR(5'-ACGC GTC GAC TTATGTTCTCTGTGACAAAATTAGTGG-3') SEQ ID NO: 4

[0034] seF(5'-CGC GGATCC ATGTTGTCACAATTGAGCTC-3') SEQ ID NO: 5

[0035] seR(5'-ACGC GTC GAC TGTTCTGTCTAGCAAATTGTC-3') SEQ ID N...

Embodiment 2

[0037] Example 2. DNA shuffling of SsNHX1 and SeNHX1 genes

[0038] 1) Preparation of shuffling templates Using the pMD-18T-SsNHX1 and pMD-18T-SeNHX1 plasmids as templates, the two genes were amplified by PCR with TaqPlus DNA polymerase, and purified as templates for DNA family shuffling.

[0039] 2) DNaseI random enzyme digestion ultraviolet absorption method was used to measure the concentration of the purified DNA, and templates with a content of 1.5 μg were mixed respectively. Take the mixed template and add it to 50 μL enzyme digestion reaction system.

[0040] Firstly, DNaseI (No. EN0525Fermentas, 10U / μl) was diluted 100 times with 0.15M NaCl (Sigma) to a concentration of 0.1U / μL.

[0041] DNaseI digestion reaction system:

[0042]

[0043] Prepare enzyme digestion reaction solution according to the above system, mix well, 15°C, 10min, then add 3μL diluted DNaseI, 0.3U, mix well, 15°C, 2min, 90°C, 10min, stop the reaction. The digested product was subjected to 2% a...

Embodiment 3

[0053] Example 3. Construction of intermediate vector pYES2-GFP

[0054] Design the following primers according to the cDNA sequence of the green fluorescent protein GFP gene (GenBank sequence number: AY013825) (the 5' end contains BamHI and SalI restriction sites respectively)

[0055] GFP-F: (5'-CGC GGATCC ATGAGTAAAGGAGAAGAAC-3') SEQ ID NO: 7

[0056] GFP-R: (5'-ACGC GTCGA CTTATTTGTATAGTTCAT-3') SEQ ID NO: 8

[0057] Amplify the GFP gene fragment with high-fidelity Pfu DNA polymerase, connect it to the pGM-T vector, transform it into Escherichia coli TOP10, clone the GFP gene sequence containing the BamHI / SalI restriction site, and confirm it by sequencing Correctness of vector pGM-GFP sequence.

[0058] There is no SalⅠ restriction site on the yeast expression vector pYES2, but there is an EcoRI restriction site. The target gene GFP of the vector pGM-GFP has EcoRI restriction sites on both sides, and there is no EcoRI restriction site in the middle of the GFP gene fra...

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Abstract

The invention discloses a strong-salt-tolerant plant gene SseNHX1 as well as an encoding protein and application of the strong-salt-tolerant plant gene SseNHX1. The strong-salt-tolerant plant gene SseNHX1 is as shown in SEQ ID NO:1; a reverse Na<+> / H<+> transport protein for encoding the strong-salt-tolerant plant gene SseNHX1 is as shown in SEQ ID NO:2; the strong-salt-tolerant plant gene SseNHX1 is applied to the aspect of culturing salt-tolerant plants. According to the invention, a strong-salt-tolerant gene (SseNHX1) obtained after two reverse Na<+> / H<+> transport protein genes (NHX1) with different sources in the same family are recombined is transplanted into tobacco in a genetic transformation way, therefore, after the tobacco is subjected to salt stress treatment, the salt tolerance of the transgenic tobacco can be further improved, and a basis is provided for culturing novel genetic materials with stronger salt tolerance.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a plant strong salt tolerance gene SseNHX1 and its encoded protein and application. Background technique [0002] The problem of soil salinization is one of the most serious environmental problems in the world at present. Various factors including population expansion continue to make human beings develop and utilize large areas of land, and gradually generate new secondary salinization, which accelerates soil salinization. process. At present, about 20% of the cultivated land and 50% of the irrigated land in the world are affected by salinization to varying degrees, and the annual average is about 120,000 hm 2 Secondary salinization of land occurs (Zhu J K, Plant salt tolerance. Trends in plant science. 2001, 6(2):66-71). Therefore, the development and efficient utilization of soil resources is the top priority of protecting human food security. For a long time, in or...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84C12N1/21A01H5/00
Inventor 王罡季静吴广霞高海伶王昱蓉
Owner TIANJIN UNIV
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