A Novel Salt Tolerance Gene zmubp in Zoysia rugosa and Its Expression Vector and Application

A technology of Zoysia grove and salt tolerance gene, applied in the field of molecular biology, can solve the problem that salt tolerance function has not been reported, and achieve the effect of improving salt tolerance

Inactive Publication Date: 2019-04-16
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

The research group applied the FOX capture system to the halophyte Zoysia sativa, and found a phenotype of Arabidopsis with significantly enhanced salt tolerance, and obtained a candidate gene for salt tolerance of Zoysia sativa through sequencing verification BYZGR , through bioinformatics analysis, BYZGR It encodes a protein containing U-box domain, which has 70.52% and 32.5% homology with OsUBP39 (XP_015651212) in rice and AtUBP38 (AT5G65200) in Arabidopsis, respectively. However, the salt tolerance function of OsUBP39 and AtUBP38 has not yet to report

Method used

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  • A Novel Salt Tolerance Gene zmubp in Zoysia rugosa and Its Expression Vector and Application
  • A Novel Salt Tolerance Gene zmubp in Zoysia rugosa and Its Expression Vector and Application
  • A Novel Salt Tolerance Gene zmubp in Zoysia rugosa and Its Expression Vector and Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Zoysia folium BYZGR clone of ( figure 1 ).

[0029] 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, take 1 µg of total RNA according to the M-MLV reverse transcription kit (TaKaRa), reverse transcribe into cDNA, and digest the cDNA product with RNase , design primers to amplify BYZGR ;

[0030] Upstream primer ZmUBP-F: 5′-ATGCCAAAGGACAGGAGC-3′ (SEQ ID NO.2);

[0031] Downstream primer ZmUBP-R: 5′-TTGTGCAGGGTCACCGTC-3′ (SEQ ID NO.3).

[0032] Using the extracted leaf cDNA as a template, carry out PCR reaction, 20 μL reaction system: 2×LA RCR Mix 10 μL, ZmUBP-F, ZmUBP-R primers 1.0 μL ea...

Embodiment 2

[0033] Example 2. Plant expression vector pEarleyGate103- BYZGR build ( figure 1 , figure 2 ).

[0034] Design primers for PCR reaction in target gene BYZGR 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 BYZGR fragment with Bam H I and not Ⅰ The double-digested pENTR1A connection was transformed into Escherichia coli, 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;

[0035] Upstream primer ZmUBP-BamHI-F: 5′-GGATCCGGATGCCAAAGGACAGGAGC-3′ (SEQ ID NO.4);

[0036]Downstream primer ZmUBP-NotI-R: 5'-GCGGCCGCGATTGTGCAGGGTC...

Embodiment 3

[0040] Example 3 Plant expression vector pEarleyGate103- BYZGR Genetic transformation of Arabidopsis thaliana and identification of its salt tolerance ( image 3 , Figure 4 ).

[0041] 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, aliquot 200 µL / tube for use. Take 5 µL of pEarleyGate103- BYZGR 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 mediu...

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Abstract

The invention belongs to the field of molecular biology, and discloses a salt-tolerant gene ZmPDI in a halophyte zoysia matrella as well as a plant expression vector and application thereof. The sequence of the salt-tolerant gene ZmPDI in the zoysia matrella is SEQ ID NO. 1. The plant expression vector is obtained by carrying out double enzyme digestion on ZmPDI through BamHI and EcoRV, then inserting the ZmPDI into a gateway entry vector pENTR1A, and finally subjecting the ZmPDI and a pEarleyGate103 expression vector plasmid to recombination reaction. The ZmPDI in the zoysia matrella, which is provided by the invention, is a novel salt-tolerant gene; the gene can be used for improving the salt tolerance of a plant, and can be applied to the creation of novel salt-tolerant germplasm and the improvement of a plant variety.

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 BYZGR And its plant expression vector and application. Background technique [0002] Soil salinization has become a key stress factor affecting plant growth and yield, and the cultivation and utilization of salt-tolerant plants is the most favorable way to solve plant growth under salt stress. With the continuous improvement of molecular biology technology, it has become a hot research direction to improve plant stress resistance through genetic engineering methods combined with molecular design concepts. Therefore, mining and utilizing salt-tolerant genes is very important. [0003] The FOX capture system obtains a transgenic Arabidopsis library containing different cDNA insertions by overexpressing the full-length cDNA library of the target plant in the model plant Arabidopsis thaliana. According to the significant mutation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
CPCC07K14/415C12N15/66C12N15/8205C12N15/8273
Inventor 陈煜宗俊勤陈静波刘建秀李建建汪毅
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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