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Novel salt-resistant gene ZmGnTl in zoysia matrella and expression vector and application thereof

A technology of Zoysia ditchleaf and salt tolerance gene, applied in the field of molecular biology, to achieve the effect of improving salt tolerance

Inactive Publication Date: 2017-05-31
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the properties of its catalyzed substrates and sequence relatedness, plants include 91 GT families (except GT1-GT94, GT36, GT46 and GT86), and each family contains multiple members; it has been reported that two urine Glycoside diphosphate-glucuronosyltransferase gene ( UGT85A5 and UGT87A2 ) overexpression significantly improved the salt tolerance of Arabidopsis and tobacco, while the salt tolerance functions of other glycosyltransferases have not been reported

Method used

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  • Novel salt-resistant gene ZmGnTl in zoysia matrella and expression vector and application thereof
  • Novel salt-resistant gene ZmGnTl in zoysia matrella and expression vector and application thereof
  • Novel salt-resistant gene ZmGnTl in zoysia matrella and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Zoysia folium wxya clone.

[0032] Choose Zoysia ditch leaves ( Zoysia matrella ) as materials, select healthy turf pieces, place them in a cup filled with quartz sand, and culture them in 1 / 2 Hongland nutrient solution for 20 days, then transfer them to 1 / 2 Hongland nutrient solution containing 300mM NaCl for 7 days, take 0.1g of young leaves, according to the instructions of the Trizol RNA Extraction Kit (TaKaRa), extract the total RNA of the leaves, follow the M-MLV Reverse Transcription Kit (TaKaRa) to take 1 µg of total RNA and reverse transcribe into cDNA, and digest the cDNA with RNase products, designed primers to amplify wxya ;

[0033] Upstream primer ZmGnTL-F: 5′-ATGACGTCACCGGCGCCG-3′ (SEQ ID NO.2);

[0034] Downstream primer ZmGnTL-R: 5′-GTCACGTAGGATGACCGA-3′ (SEQ ID NO.3).

[0035] The extracted leaf cDNA was used as a template for PCR reaction, 20 µL reaction system: 10 µL of 2×LA RCR Mix, 1.0 µL each of ZmGnTL-F and ZmGnTL-R primers (10 µm...

Embodiment 2

[0036] Example 2. Plant expression vector pEarleyGate103- wxya build.

[0037] Design primers for PCR reaction in target gene wxya Respectively introduce enzyme cutting sites upstream and downstream Bam H I and not Ⅰ. The PCR product was connected to the pMD19-T Simple vector, transformed into TOP10 competent cells, and the positive plasmid was extracted. Bam H I and not Ⅰ double digested wxya fragment with Bam HI and not Ⅰ The double-digested pENTR1A connection was transformed into Escherichia coli, and the extracted positive plasmid was Pvu ⅠAfter linearization by single enzyme digestion, carry out LR recombination reaction (Invitrogen) with pEarleyGate103 vector plasmid, transform, extract positive plasmid, electrophoresis detection and sequencing verification as SEQ ID NO.1;

[0038] Upstream primer ZmGnTL-BamH -F: 5'-GGATCCGGATGACGTCACCGGCGCCG-3' (SEQ ID NO. 4);

[0039] Downstream primer ZmGnTL-NotI-R: 5′-GCGGCCGCGAGTCACGTAGGATGACCGA-3′ (SEQ ID NO.5). ...

Embodiment 3

[0043] Example 3 Plant expression vector pEarleyGate103- wxya Genetic transformation of Arabidopsis thaliana and identification of its salt tolerance.

[0044] ①Competent preparation of Agrobacterium strain EHA105 and transformation by freeze-thaw method: Pick a single colony of EHA105 from a YEB (50 µg / mL rifampicin) plate and inoculate it in 50 mL of YEB liquid medium containing 50 µg / mL rifampicin medium, 220 rpm, 28°C to OD value 0.6, then ice-bathed the bacteria solution for 30 min, centrifuged to collect the bacteria, suspended in 2 mL of pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200 µL / tube for use; take 5 µL pEarleyGate103- wxya Add 100 µL of competent cells to the vector plasmid, ice bath for 30 min, freeze in liquid nitrogen for 5 min, 37°C for 5 min, add 800 µL of YEB liquid medium, pre-culture for 3 h at 28°C and 200 rpm, and smear the bacterial solution on YEB ( 50 µg / mL rifampicin + 50 µg / mL kanamycin) solid medium, cultured in the dark at 28°C for 2 day...

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Abstract

The invention belongs to the field of molecular biology and discloses a salt-resistant gene ZmGnTL of a halophyte zoysia matrella and an expression vector and an application thereof. The sequence of a novel salt-resistant gene ZmGnTl in zoysia matrella is SEQ ID NO.1. The plant expression vector is obtained by performing a recombination reaction with pEarleyGate103 expression vector plasmids after inserting ZmGnTL after double enzyme digestion of BamHI and NotI into a gateway entry vector pENTR1A. The ZmGnTL of zoysia matrella provided by the invention is the novel salt-resistant gene which can improve the salt resistance of the plant and can be applied to creating salt-resistant novel germplasms and improving plant varieties.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a new salt-tolerant gene in the halophyte Zoysia folium wxya And its plant expression vector and application. Background technique [0002] Soil salinization is an important stress factor affecting plant growth and development. Salt stress significantly inhibits plant growth and development, and the cultivation and utilization of salt-resistant germplasm is very critical. In order to speed up the process of plant salt-resistant breeding, rapid and efficient genetic engineering technology has been heated With support and attention, mining excellent salt-tolerant genes has become an important prerequisite for salt-tolerant breeding. [0003] Glycosyltransferases (Glycosyltransferases, GT) are essential enzymes in protein glycosylation (oligosaccharides are covalently bound to specific amino acid residues on proteins in the form of glycosides), and have become an important part of gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82C12N15/66A01H5/00
CPCC12N9/1051C12N15/66C12N15/8273
Inventor 陈煜宗俊勤刘建秀陈静波汪毅李丹丹
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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