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Sorbitol dehydrogenase gene derived from haloarcula marismortui and applications thereof

A technology of sorbitol dehydrogenase and Dead Sea salt, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as SDH activity decline

Active Publication Date: 2014-12-03
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have shown that Arabidopsis SDH mutants have a slight increase in sorbitol content and a decrease in SDH activity after drought stress

Method used

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  • Sorbitol dehydrogenase gene derived from haloarcula marismortui and applications thereof
  • Sorbitol dehydrogenase gene derived from haloarcula marismortui and applications thereof
  • Sorbitol dehydrogenase gene derived from haloarcula marismortui and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 The artificial synthesis of sorbitol dehydrogenase gene HmSDHI

[0034] According to the PTDS (PCR-based two-step DNA synthesis, PTDS) method, the sorbitol dehydrogenase gene derived from Halobacterium dead sea was cloned and chemically synthesized. On the basis of keeping the amino acid sequence of the sorbitol dehydrogenase gene unchanged, Primers were designed to synthesize the sorbitol dehydrogenase gene (HmSDHI) of the present invention. The designed primers are as follows:

[0035] 1.HMSDHI-1:Tm=54,60mer

[0036] GGA,TCC,ATG,AGA,CCA,TCT,GCT,TCT,GTT,GGT,CAG,AGA,CCA,GAA,GAG,AGA,CTC,AAT,CCA,CAG

[0037] 2. HMSDHI-2:Tm=54,60mer

[0038] TGA,CAC,CAA,TGA,CTT,TCA,TGC,CTG,GAA,GAG,AGT,GAG,AGC,ACT,GTG,GAT,TGA,GTC,TCT,CTT

[0039] 3.HMSDHI-3:Tm=54,60mer

[0040] CAT,GAA,AGT,CAT,TGG,TGT,CAC,CAG,AGA,TGA,TGA,TGG,TCC,ACA,ACT,CTT,GGA,GAG,AGA,GAG

[0041] 4.HMSDHI-4:Tm=54,60mer

[0042] GAG,GGT,TCT,GAC,AAG,GGC,TTC,ACC,TGG,ATC,TGG,AGA,TGG,TCT,CTC,TCT,CTC,CAA,GAG...

Embodiment 2

[0095] Example 2 Construction of HMSDHI Agrobacterium Binary Vector

[0096] The ORF of the above-mentioned artificially synthesized HMSDHI gene was amplified by PCR, and the head and the tail were respectively added to Bam HI and Sac I cutting sites, and the DNA fragments recovered were connected with the corresponding enzyme-cut vectors after digestion with Bam HI and Sac I, and identified by enzyme digestion And the correct binary vector pYM6854 was obtained after sequencing.

Embodiment 3

[0097] Embodiment 3 Transformation of Agrobacterium by electric shock method

[0098] 1) Prepare Agrobacterium GV3101 competent, the method refers to MicroPulser TMElectroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Corporation) (Raineri et al., 1990).

[0099] 2) Take 50 μL of GV3101 competent cells, add 1 μL of DNA, and transfer to a 0.2 cm electric shock cup for transformation (400Ω, 2.5KV, 25 μf). Add 1 mL of LB medium containing 1% mannitol to recover the culture for 2 hours (28°C, 250rpm). Take 10 μL and 100 μL respectively and smear LB plates (rifampicin 50 μg / mL, gentamicin 50 μg / mL, chloramphenicol 100 μg / mL).

[0100] 3) Pick several clones from the bacteria grown on the LB plate in step 2, extract the Agrobacterium plasmid by alkaline method, identify by enzyme digestion, and detect by PCR.

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Abstract

The invention relates to a sorbitol dehydrogenase gene derived from haloarcula marismortui and applications thereof. The sorbitol dehydrogenase gene is synthesized by a manual method. The nucleotide sequence of the gene is shown as SEQ ID NO 1. The coded amino acid sequence is shown as SEQ ID NO2. Results of arabidopsis thaliana transformation by utilization of the sorbitol dehydrogenase gene by adopting an agrobacterium floral-dip method show that: the sorbitol dehydrogenase gene can successfully improve the salt tolerance performance of transgenic arabidopsis thaliana, so that the sorbitol dehydrogenase gene can be widely applied in plants under high-salinity stress.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, specifically relates to a sorbitol dehydrogenase gene derived from Halobacterium dead sea and its application, in particular to the application of the gene in plant salt tolerance. Background technique [0002] Sorbitol is a sugar alcohol widely distributed in some Rosaceae woody plants. It is the major photosynthetic product, transport sugar and storage substance of these Rosaceae woody plants, serving the role of sucrose in other plants. Sorbitol is also a small molecule osmotic substance, which can change the osmotic potential of cells, thereby improving the ability of plants to resist cold, drought, salt, and boron deficiency. [0003] Sorbitol dehydrogenase (SDH) and sorbitol oxidase (SOX) are key enzymes involved in sorbitol metabolism, in which SDH catalyzes the conversion of sorbitol into fructose and glucose, and SOX catalyzes the oxidation of sorbitol into glucose. [0004] In...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/82A01H5/00
Inventor 高建杰姚泉洪彭日荷付晓燕田永生赵伟韩红娟王波王丽娟韩静
Owner SHANGHAI ACAD OF AGRI SCI
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