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New salt tolerance gene ZmPDI in zoysia matrella and plant expression vector and application of new salt tolerance gene ZmPDI

A plant expression vector, the technology of Zoysia dulcis, applied in the field of molecular biology to achieve the effect of improving salt tolerance

Inactive Publication Date: 2017-03-22
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

However, there is no report on the regulation of plant salt tolerance by PDI

Method used

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  • New salt tolerance gene ZmPDI in zoysia matrella and plant expression vector and application of new salt tolerance gene ZmPDI
  • New salt tolerance gene ZmPDI in zoysia matrella and plant expression vector and application of new salt tolerance gene ZmPDI
  • New salt tolerance gene ZmPDI in zoysia matrella and plant expression vector and application of new salt tolerance gene ZmPDI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Zoysia folium ZPDI clone of ( figure 1 ).

[0030] 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 ZPDI ;

[0031] Upstream primer ZmPDI-F: 5′-ATGGCGATCCACTCCAGGGT-3′ (SEQ ID NO.2);

[0032] Downstream primer ZmPDI-R: 5′-GAGCTCATCCTTGACGGCCT-3′ (SEQ ID NO.3).

[0033] Using the extracted leaf cDNA as a template, carry out PCR reaction, 20 µL reaction system: 10 µL of 2×RCR Mix, 1.0 µL each of ZmPDI-F and ZmP...

Embodiment 2

[0034] Example 2 Plant expression vector pEarleyGate103- ZPDI build ( figure 2 ).

[0035] Design primers for PCR reaction in target gene ZPDI Respectively introduce enzyme cutting sites upstream and downstream Bam H I and EcoR V. 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 EcoR Ⅴ double digested ZPDI fragment with Bam H I and EcoR Ⅴ The double-digested pENTR1A was ligated, transformed, and the extracted positive plasmid was Nsi 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;

[0036] Upstream primer ZmPDI- Bam HⅠ-F: 5′-GGATCCGGATGGCGATCCACTCCAGGGT-3′ (SEQ ID NO.4);

[0037] Downstream primer ZmPDI- EcoR V-R: 5'-GATATCTGAGCTCATCCTTGACGGCCT-3' (SEQ ID NO. 5). ...

Embodiment 3

[0041] Example 3 Plant expression vector pEarleyGate103- ZPDI Genetic transformation of Arabidopsis thaliana and identification of its salt tolerance.

[0042] 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- ZPDI 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 tolerance gene ZmPDI in zoysia matrella of the halophyte and a plant expression vector and application of the new salt tolerance gene ZmPDI. The sequence of the salt tolerance gene ZmPDI of the zoysia matrella is SEQ ID NO.1. The plant expression vector is obtained in the manner that BamHI and EcoRV are inserted in a gateway entry vector pENTR1A after double enzyme digestion of the ZmPDI and then are subjected to a recombination reaction with pEarleyGate 103 expression vector plasmids. The zoysia matrella ZmPDI is the new salt tolerance gene, can improve salt tolerance of plants and can be applied to creating of new salt tolerance germplasm and improvement of plant breeds.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a salt-tolerant gene ZmPDI of a halophyte Zoysia scutellaria and its plant expression vector and application. Background technique [0002] Salt stress is an important environmental factor affecting plant growth, yield and quality. Therefore, the breeding of new plant stress-resistant varieties and the study of stress-resistant mechanisms have become hot spots in recent years; conventional breeding techniques such as hybridization and radiation mutagenesis are not enough to meet the current requirements of stress-resistant breeding due to their long cycle and low efficiency; In the process of reverse breeding, fast and efficient genetic engineering technology has gradually attracted the attention of the society, and the development of excellent stress-resistant genes is very important; existing studies have shown that molecular breeding of high-efficiency stress-resistant regulator...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
CPCC07K14/415C12N15/66C12N15/8205C12N15/8273
Inventor 陈煜宗俊勤刘建秀陈静波郭海林李丹丹张兵汪毅李建建
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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