Floral organ development gene NsAGL6 as well as plant expression vector and construction method thereof

The technology of a plant expression vector and construction method is applied to the NsAGL6 organ development gene of water lily and the field of plant expression vector and construction thereof, which can solve the problem that the function of the AGL6 gene is not completely determined, and achieve the effect of improving flower type and flowering period.

Inactive Publication Date: 2012-07-04
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

However, the function of the AGL6 gene has not been fully determined so far

Method used

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  • Floral organ development gene NsAGL6 as well as plant expression vector and construction method thereof
  • Floral organ development gene NsAGL6 as well as plant expression vector and construction method thereof
  • Floral organ development gene NsAGL6 as well as plant expression vector and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Construction of plant expression vector pCAMBIA 1301-220-NsAGL6

[0040] 1. Cloning of NsAGL6:

[0041] The water lily cultivar 'Yellow Prince' (Nymphaea sp.cv. 'Yellow Prince') was selected as the material to extract the total RNA of flower buds, and 1 μg of total RNA was reverse-transcribed into cDNA according to the M-MLV reverse transcription kit (TaKaRa), and used RNase digested the cDNA product, referring to the NsAGL6 sequence information of water lily (NCBI accession number: AB495345), analyzed and designed primers to amplify NsAGL6 by Primer Premier 5 software;

[0042] Upstream primer NsAGL6-F: 5′-CGCGGATCCCTTTGAGTGATCATCGCAAGA-3′ (SEQ ID NO.2)

[0043] Downstream primer NsAGL6-R: 5'-TCCCCCGGGATGGTATGGATTACAGGACCC-3' (SEQ ID NO.3)

[0044] Using bud cDNA as a template, carry out PCR reaction, 50 μL reaction system: 10×RCR Bμffer 5.0 μL, NsAGL6-F, NsAGL6-R primers 1.0 μL each (20 μmol L -1 ), dNTP mix 4.0μL (2.5mmol·L -1 ), PrimeSTAR TM HS DNA ...

Embodiment 2

[0047] Example 2 Transformation of Arabidopsis thaliana with plant expression vector pCAMBIA 1301-220-NsAGL6 and observation of flower development characteristics

[0048] 1. Competent preparation and freeze-thaw transformation of Agrobacterium strain EHA105

[0049] Pick a single colony of EHA105 from the YEB (50mg / L rifampicin) plate, inoculate it in 50mL YEB liquid medium containing 50mg / L rifampicin, culture at 200rpm, 28°C until the OD value is 0.5, and then ice-bath the bacteria solution 30min, centrifuge to collect the bacteria, suspend in 2mL pre-cooled 100mM CaCl 2 (20% glycerol) solution, 200 μL / tube aliquoted for use.

[0050] Take 10 μL of pCAMBIA 1301-220-NsAGL6 vector plasmid, add 200 μL of competent cells, bathe in ice for 30 minutes, freeze in liquid nitrogen for 5 minutes, 37°C for 5 minutes, add 800 μL of YEB liquid medium, pre-culture for 4 hours at 28°C and 200 rpm, and spread the bacterial solution on YEB (50mg / L rifampicin + 50mg / L kanamycin) solid medi...

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Abstract

The invention belongs to the field of molecular biology, and discloses a nymphaea floral organ development gene NsAGL6 as well as a plant expression vector and a construction method thereof. The sequence of the nymphaea floral organ development gene NsAGL6 is shown in SEQ ID No.1. The expression vector pCAMB IA 1301-220-NsAGL6 constructed in the invention is obtained by connecting the NsAGL6 geneto the linear pCAMB IA 1301-220 plasmid. The plant expression vector is applied to plant genetic transformation, the NsAGL6 gene is over-expressed under the drive of a CaMV35S starter, the AGL6 protein is synthesized in quantity, and the expression of the downstream gene is regulated so that the nymphaea blooms in advance and the plant flower type is changed.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a water lily flower organ development gene NsAGL6, a plant expression vector and a construction method thereof. Background technique [0002] AGL6 is an important transcription factor involved in the establishment of floral meristem and floral organ formation. At present, the homologous gene of AGL6 has been cloned from corn, petunia, hyacinth, rice and other plants, and its function has been analyzed. The study found that the AGL6 gene of these plants is involved in the regulation of flower development, although the phylogenetic relationship of these plants is not the same. The functions of these genes mostly focus on making plants flower in advance and changing the shape and number of floral organs. AGL6 genes all contain highly conserved MADS domains, and have certain similarities in expression patterns. But so far the function of AGL6 gene has not been fully determined. [...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66
Inventor 陈发棣罗火林陈素梅刘兆磊管志勇
Owner NANJING AGRICULTURAL UNIVERSITY
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