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Gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata

A technology of phosphate synthase and Selaginella mats, applied in the field of plant genetic engineering, can solve the problems of limited research and development of TPS gene

Inactive Publication Date: 2009-11-25
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are relatively many studies and utilizations of TPS genes in Escherichia coli and fungi, while research on the development of TPS genes in plants is still very limited.

Method used

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  • Gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata
  • Gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata
  • Gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] This embodiment is the extraction and purification of the total RNA of Selaginella cuspidatum leaves, comprising the following steps:

[0117] (1) Put the young leaves of Selaginella cushion-like into a mortar pre-cooled by liquid nitrogen, immediately add liquid nitrogen, grind the sample as much as possible, and put the sample into 0.1g per tube before the liquid nitrogen volatilizes. Immediately add 1 mL Trizol (Invitrogen) to each tube in a 1.5 mL centrifuge tube pre-cooled with liquid nitrogen, shake vigorously, and place at room temperature for 5 min.

[0118] (2) Centrifuge at 4°C and 12000r / min for 2min, and transfer the supernatant into a new RNase-free centrifuge tube.

[0119] (3) Add 0.1mL 5mol / L NaCl to each tube, mix well, then add 0.3mL chloroform to each tube, shake vigorously for 15s, place at room temperature for 3min, centrifuge at 12000r / min at 4°C for 15min, avoid inhalation as much as possible In the case of the intermediate layer, aspirate the su...

Embodiment 2

[0123] This embodiment is the cloning of the middle fragment of SpTPS1 gene, the method is as follows:

[0124] Using the purified total RNA of Selaginella cuspidatum leaves obtained in the above-mentioned Example 1 as a template, oligod(T)18:5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T) 18 -3' is the reverse transcription primer, according to Takara's PrimeScript TM The Reverse Transcriptase manual was used to synthesize the first strand of cDNA, and the total volume of the amplification system was 20 μL; the first strand of cDNA of the middle fragment was obtained.

[0125]First, by comparing the amino acid sequences of five known trehalose-6-phosphate synthases: Selaginella lepidophylla TPS1 U96736.1; tomato TPS1 (Solanumlycopersicum TPS1E.F151131.1); Arabidopsis TPS1 (Arabidopsis thaliana TPS1NM106505.4); TPS1 (Physcomitrella patens subsp TPS1XM001778453); sugarcane TPS1 (Saccharum hybrid cultivar TPS1 EU761244), the conserved region in the middle position was determined, a...

Embodiment 3

[0161] This embodiment is the cloning of the 3' end of the SpTPS1 gene, the method is as follows:

[0162] According to the sequencing results of the above intermediate fragment, design and synthesize a specific upstream primer:

[0163] S1: 5'-GCCATCAGAGAAAGCGGTTACT-3',

[0164] In addition, design and synthesize a 3′ end universal downstream primer:

[0165] AP2: 5'-TACGTACGGCATGACAGTG-3';

[0166] Using the first strand of the intermediate fragment cDNA obtained in the foregoing Example 2 as a template, PCR amplification was performed using upstream primer S1 and downstream primer AP2;

[0167] The amplification system is:

[0168] TaqPlusPCR Master Mix 10μL,

[0169] cDNA template 2 μL,

[0170] 2× Primer (S1 / AP2) 0.5 μL,

[0171] wxya 2 O 7μ.

[0172] The amplification procedure is:

[0173] 94°C, 3min;

[0174] 94°C, 30s; 55°C, 45s; 72°C, 1min 30s (33 cycles)

[0175] 72°C, 8min; keep warm at 12°C.

[0176] The resulting amplified products were detected by 1%...

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Abstract

The invention discloses a gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata. The sequence has a nucleotide sequence represented by SEQ ID NO.1. The sequence is a new TPS gene sequence and the research objects of the TPS gene are further increased to a plurality of plants such as the elaginella pulvinata from the microorganisms including bacillus coli and fungi, and a few limited plants. In addition, new plant variety with corresponding excellent drought resistance and salt resistance is expected to culture if the TPS gene sequence is guided in the gene of other plants such as maize through a transgenic technology according to the excellent drought resistance and salt resistance shown by the trehalose catalytically synthesized by the TPS gene in selaginella pulvinata plant. The invention further discloses a method for cloning the gene sequence of trehalose-6-phosphate synthetase derived from selaginella pulvinata.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a trehalose-6-phosphate synthase gene sequence derived from Selaginella cuspidatum and a cloning method thereof. Background technique [0002] Selaginella pulvinata Maxim is a plant of the genus Selaginella in the family Selaginaceae, commonly known as "Nine Dead Resurrection Grass". It generally grows in barren mountains and dry rock crevices, and is extremely resistant to drought and cold. When the weather is dry, the twigs curl up and huddle up to retain water in the body. Once it gets rain and the temperature rises, the curled twigs will spread out. Studies have shown that recovery plants such as Selaginella cuspidatum contain a large amount of stress metabolite-trehalose. [0003] Trehalose, also known as yeast sugar, is a non-reducing sugar composed of two glucopyranose molecules linked by α-1,1 glycosidic bonds. Protect biological cells un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12P19/34
Inventor 李晚忱付凤玲林荆牟禹蒋伟
Owner SICHUAN AGRI UNIV
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