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Eremochloa ophiuroides rooting promoting gene EoSINAT5 as well as plant expression vector and application thereof

A plant expression vector and a technology for promoting rooting, which are applied in the field of EoSINAT5, a rooting-promoting gene, and plant expression vectors thereof, and can solve the problems of weak rooting ability of A.

Active Publication Date: 2020-10-27
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

[0011] The purpose of the present invention is to solve the problem of weak rooting ability of directional improvement of Pseudomonas spp., and provide a new E3 ubiquitin ligase gene EoSINAT5, the nucleotide sequence of which is shown in SEQ ID NO.1

Method used

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  • Eremochloa ophiuroides rooting promoting gene EoSINAT5 as well as plant expression vector and application thereof
  • Eremochloa ophiuroides rooting promoting gene EoSINAT5 as well as plant expression vector and application thereof
  • Eremochloa ophiuroides rooting promoting gene EoSINAT5 as well as plant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Cloning of embodiment 1.EoSINAT5 gene

[0082] Take the genus 'Ganbei' as the material, take 0.15 g of the root, extract the total RNA of the leaves according to the operation method of the Trizol RNA extraction kit (TaKaRa), and reverse according to the M-MLV reverse transcription kit (TaKaRa) According to the sequence information of the gene in the chrysanthemum library, use primer 5 software to design specific primers to amplify EoSINAT5;

[0083] Upstream primer EoSINAT5-F: 5′-ATGGCATCAGTTACTTATAT-3′ (SEQ ID NO.2),

[0084] Downstream primer EoSINAT5-R: 5'-CCGCTCCTTCCAGATCCTCC-3' (SEQ ID NO.3);

[0085] Use the root cDNA as a template to carry out PCR reaction, 50 μL reaction system: 5.0 μL 10×PCR Buffer, 1.0 μL each of EoSINAT5-F and EoSINAT5-R primers (20 μmol L -1 ), dNTP mix 4.0μL (2.5mmol·L -1 ), TaqDNA Polymerase 0.2μL, cDNA template 1μL, ddH 2 O 37.8 μL; reaction program: pre-denaturation at 95°C for 5 min, then melting at 94°C for 45 sec, annealing at 55...

Embodiment 2

[0086] Embodiment 2. Construction of plant expression vector pCAMBIA1305.1-EoSINAT5

[0087] Design primers according to the full-length gene sequence of EoSINAT5 to carry out PCR reaction, use the positive plasmid in the above-mentioned embodiment 1 as a template, introduce homologous recombination arms respectively at the upstream and downstream of the EoSINAT5 gene,

[0088] Upstream primer 1305-EoSINAT5-F:

[0089] 5'-ggtacccggggatcctctagaATGGCATCAGTTACTTATATTGATGATAGC-3' (SEQ ID NO.4), downstream primer 1305-EoSINAT5-R: 5'-tgcctgcaggtcgactctagaCCGCTCCTTCCAGATCCTCC-3' (SEQ ID NO.5);

[0090] With high-fidelity enzyme (PrimeSTAR TM HS DNA Polymerase, TaKaRa) for PCR reaction, 50 μL reaction system: 10×HS PCR Buffer 5.0 μL, 1305-EoSINAT5-F, 1305-EoSINAT5-R primers 1.0 μL each (20 μmol L -1 ), dNTP mix 4.0μL (2.5mmol·L -1 ), PrimeSTAR TM HS DNA Polymerase 0.4 μL, cDNA template 1 μL, ddH 2 O 37.6 μL; reaction program: pre-denaturation at 95°C for 5 min, then melting at ...

Embodiment 3

[0092] Example 3. Transformation of Arabidopsis thaliana by Agrobacterium EHA105-mediated pollen tube infection

[0093] Pick a single colony of EHA105 from a YEB (50 μg / mL rifampicin) plate, inoculate it in 50 mL of YEB liquid medium containing 50 μg / mL rifampicin, cultivate it at 200 rpm, 28°C until the OD value is 0.5, and then bathe the bacteria in ice 30min, centrifuge to collect the bacteria, suspend in 2mL pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200 μL / tube aliquoted for use. Take 10 μL of the pCAMBIA1305.1-EoSINAT5 vector plasmid, add 200 μL of competent cells, bathe in ice for 30 minutes, freeze in liquid nitrogen for 5 minutes, and add 800 μL of YEB liquid medium for 5 minutes at 37°C, pre-culture for 4 hours at 28°C and 200 rpm, and spread the bacterial solution on YEB (50 μg / mL rifampicin + 50 μg / mL kanamycin) solid medium, cultured in the dark at 28°C for 2 days, picked a single clone for detection, and selected positive clones to transform Arabidopsis ...

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Abstract

The invention belongs to the field of plant genetic engineering and transgenic breeding, and relates to an Eremochloa ophiuroides rooting promoting gene EoSINAT5 as well as a plant expression vector and application thereof. The Eremochloa ophiuroides rooting promoting gene EoSINAT5 has nucleotide sequence shown as SEQ ID NO.1. The gene EoSINAT5 is transferred into genome DNA of a target plant andtranscribed. Phenotypic observation and statistical analysis on transgenic plants show that compared with non-transgenic plants, root length and root number of transgenic plants are increased significantly. The exogenous E3 ubiquitin ligase gene EoSINAT5 is transformed and normally transcribed and expressed, so that root growth of target plants is regulated, and new germplasm with high rooting capacity is obtained. The gene has great significance for cultivation of excellent lawn varieties and wide application in production.

Description

technical field [0001] The invention belongs to the fields of plant genetic engineering and transgenic breeding, and relates to EoSINAT5, a rooting-promoting gene of Escherichia coli, a plant expression vector and application thereof. Background technique [0002] Eremochloa ophiuroides (Munro.) Hack.] belongs to the genus Centipede Grass of the Poaceae Panicaceae. It is the only excellent warm-season C4 herb in this genus that can be used as turfgrass. It is also recognized at home and abroad as originating in my country. The best warm-season lawn grass. It has important characteristics such as barren resistance, less pests and diseases, low maintenance level, good ornamental value and high lawn value. It is also called "lazy grass" (Hanna, 1995). Widely used in construction (Liu et al.2003). As a native grass species, False Thrift Grass has the characteristics of resistance to diseases and insect pests, resistance to barrenness and low maintenance, and has good ornamental...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/82C12N1/21C12N15/11A01H5/06A01H6/20
CPCC12N9/93C12N15/8261C12Y603/02019
Inventor 王晶晶刘建秀宗俊勤李丹丹郭爱桂郭海林陈静波李建建
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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