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Novel plant salt-resistant gene ZmDUF1644, and expression vector and application thereof

A plant expression vector, zmduf1644 technology, applied in the field of molecular biology, can solve the problems of low efficiency and long cycle, and achieve the effect of improving salt tolerance and plant 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

Conventional breeding methods such as hybrid radiation have made great contributions to stress-resistant new varieties. However, due to the long cycle and low efficiency, they are not enough to meet the current needs of stress-resistant breeding. In order to speed up the process of stress-resistant breeding, fast and efficient genetic engineering technology is gradually Attracted by the society, the development of excellent stress-resistance genes is very important; existing studies have shown that molecular breeding of high-efficiency stress-resistance regulatory genes has a positive effect on improving plant stress resistance
[0003] Studies have found that there is a kind of DUF1644 gene family in plants, and the encoded protein includes a relatively conserved 160 amino acid sequence, which contains 9-10 cysteine ​​sites, but the plant biological functions it participates in are rarely reported ; recently found a salt stress-induced expression from rice OsSIDP366 Gene (encoding DUF1644 family protein), its overexpression significantly improves rice salt tolerance (Guo.et al., 2015)

Method used

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  • Novel plant salt-resistant gene ZmDUF1644, and expression vector and application thereof
  • Novel plant salt-resistant gene ZmDUF1644, and expression vector and application thereof
  • Novel plant salt-resistant gene ZmDUF1644, and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Zoysia folium ZmDUF1644 clone.

[0027] 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 ZmDUF1644 ;

[0028] Upstream primer ZmDUF1644-F: 5′-ATGCCAAAGGACAGGAGC-3′ (SEQ ID NO.2);

[0029] Downstream primer ZmDUF1644-R: 5′-TTGTGCAGGGTCACCGTC-3′ (SEQ ID NO.3).

[0030] Using the extracted leaf cDNA as a template, carry out PCR reaction, 20 µL reaction system: 10 µL of 2×LA RCR Mix, 1.0 µL each of ZmDUF1644-F and ...

Embodiment 2

[0031] Example 2. Plant expression vector pEarleyGate103- ZmDUF1644 build.

[0032] Design primers for PCR reaction in target gene ZmDUF1644 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 ZmDUF1644 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;

[0033] Upstream primer ZmDUF1644-BamHI-F: 5′-GGATCCGGATGCCAAAGGACAGGAGC-3′ (SEQ ID NO.4);

[0034] Downstream primer ZmDUF1644-NotI-R: 5'-GCGGCCGCGATTGTGCAGGGTCACCGTC-3'...

Embodiment 3

[0038] Example 3 Plant expression vector pEarleyGate103- ZmDUF1644 Genetic transformation of Arabidopsis thaliana and identification of its salt tolerance. 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- ZmDUF1644 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 d...

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Abstract

The invention belongs to the field of molecular biology and discloses a salt-resistant gene ZmDUF1644 of a halophyte zoysia matrella, and an expression vector and an application thereof. The sequence of a novel salt-resistant gene ZmDUF1644 in zoysia matrella is as shown in SEQ ID NO.1. The plant expression vector is obtained by performing a recombination reaction with pEarleyGate103 expression vector plasmids after inserting ZmDUF1644 subjected to double enzyme digestion of BamHI and NotI into a gateway entry vector pENTR1A. The ZmDUF1644 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 ZmDUF1644 And its plant expression vector and application. Background technique [0002] Salt stress significantly inhibits plant growth and development, so the cultivation and utilization of salt-resistant germplasm is very important. Conventional breeding methods such as hybrid radiation have made great contributions to stress-resistant new varieties. However, due to the long cycle and low efficiency, they are not enough to meet the current needs of stress-resistant breeding. In order to speed up the process of stress-resistant breeding, fast and efficient genetic engineering technology is gradually With the attention of the society, the development of excellent stress-resistant genes is very important; existing studies have shown that molecular breeding of high-efficiency stress-resistant regulatory genes has a positive ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
CPCC07K14/415C12N15/66C12N15/8205C12N15/8273
Inventor 宗俊勤陈煜刘建秀陈静波张兵李丹丹
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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