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Malic acid transporter gene PbrTDT1 from pyrus bretschneideri and application of malic acid transporter gene PbrTDT1

A transporter, malic acid technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of unclear role of TDT transporter, and achieve the effect of improving screening efficiency

Active Publication Date: 2020-12-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Fruit quality is the key to fruit tree production, and the content and composition of organic acids in fruit are important parameters to measure fruit quality, but the role of TDT transporter in the accumulation of organic acids in pear fruit is still unclear

Method used

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  • Malic acid transporter gene PbrTDT1 from pyrus bretschneideri and application of malic acid transporter gene PbrTDT1
  • Malic acid transporter gene PbrTDT1 from pyrus bretschneideri and application of malic acid transporter gene PbrTDT1
  • Malic acid transporter gene PbrTDT1 from pyrus bretschneideri and application of malic acid transporter gene PbrTDT1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning of embodiment 1PbrTDT1 gene

[0036] The pulp of 'Dangshan Suli' which was 90 days after the full flowering stage was used as the test material, RNA was extracted and reverse transcribed, and the obtained first-strand cDNA was used to amplify the full length of the PbrTDT1 gene. Amplified gene PbrTDT1 primer pair is: forward primer PbrTDT1-F1: 5'-ATGGATCATCATCCAGTTTCCGACG-3' (SEQ ID NO: 3); reverse primer PbrTDT1-R1: 5'-TCACTGAACTGCAGGTTCATTTGTCC-3' (SEQ ID NO: 4 ). The 50μl reaction system includes 100ng template DNA, 1.0μM the aforementioned primers, 10mM dNTPs, 5×Q5 reaction buffer (Q5 Reaction Buffer), 1U Q5 high-fidelity polymerase (Q5 High-Fidelity DNA Polymerase) (the above buffer and Q5 High-fidelity polymerases were purchased from NewEngland Biolabs). The PCR reaction program was: 95°C, pre-denaturation for 30 seconds; 95°C, denaturation for 30 seconds, 60°C, annealing for 45 seconds, 72°C, extension for 1 minute, 35 cycles; after the cycle was comple...

Embodiment 2

[0037] Example 2 Analysis of the expression pattern of the PbrTDT1 gene

[0038] 1. Changes of malic acid content during pear fruit development

[0039] To analyze whether the PbrTDT1 gene is related to the metabolism of malic acid in pear pulp. The present invention measures the malic acid content in five periods during the dynamic development of pear fruit. The specific measurement steps are as follows: Accurately weigh 2.0 g of the pulp tissue sample in a pre-cooled mortar, add 8 mL of 80% ethanol, fully grind it into a homogenate, transfer it to a 10 mL centrifuge tube, bathe in 37°C for 30 minutes, and ultrasonically for 15 minutes. Centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant to a 25mL volumetric flask, repeat the operation 3 times, combine the supernatant and make to volume. Take 2mL of the extract, evaporate to dryness with a rotary evaporator (model: RE-3000, Shanghai Rongya Biochemical Instrument Factory), dissolve it in 1mL of sterile distille...

Embodiment 3

[0045] Example 3 Subcellular Localization of PbrTDT1 Gene

[0046] The present invention uses Arabidopsis protoplasts to study the subcellular localization of PbrTDT1. The entire open reading frame of the PbrTDT1 gene was amplified by PCR, and two restriction sites, XbaI and BamHI, were respectively added to the two ends of the amplification primers. The nucleotide sequence of the primer is: forward primer PbrTDT1-F3:5'-GCTCTAGAATGGATCATCATCCAGTTCCGACG-3'(SEQ ID NO:9); reverse primer PbrTDT1-R3:5'-CGGGATCCCTGAACTGCAGGTTCATTTGTCC-3'(SEQ IDNO:10) . The underline is the enzyme cutting site, TCTAGA is the XbaI restriction site, GGATCC It is the restriction site of BamHI restriction enzyme. Firstly, the amplified product was loaded on the pEASY-Blunt Zero vector to obtain a pEASY-Blunt Zero-PbrTDT1 recombinant vector. At the same time, the pEASY-Blunt Zero-PbrTDT1 and 35S-YFP vectors were digested with XbaI and BamHI, and the products were recovered and ligated to obtain the...

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Abstract

The invention discloses a malic acid transporter gene PbrTDT1 from pyrus bretschneideri and application of the malic acid transporter gene PbrTDT1, and belongs to the technical field of plant geneticengineering. The novel malic acid transporter gene PbrTDT1 is obtained by cloning from pyrus bretschneideri fruits, a nucleotide sequence of the malic acid transporter gene PbrTDT1 is shown as SEQ IDNO: 1 in a sequence listing, and a coded amino acid sequence of the malic acid transporter gene PbrTDT1 is shown as SEQ ID NO: 2 in the sequence listing. The gene is transformed into tomatoes throughagrobacterium-mediated genetic transformation to obtain transgenic plants, and biological function verification shows that the malic acid content in fruits of the transgenic tomatoes is obviously increased, which indicates that the cloned PbrTDT1 gene has the function of regulating and controlling the malic acid content of fruit pulp. The TDT gene PbrTDT1 from the pyrus bretschneideri can be applied to fruit acidity character improvement or high-acid transgenic fruit construction, and provides a new gene resource for molecular breeding for improving the quality of plant fruits.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to a pear malate transporter coding gene PbrTDT1 and its application in regulating the malic acid content of fruit pulp. Specifically, it involves cloning a TDT (tonoplast dicarboxylate transporter, tonoplast dicarboxylate transporter) family transport gene PbrTDT1 from 'Dangshansuli' (Pyrus bretschneideriRehd cv. 'Dangshansuli'), and then introducing the gene into tomato to obtain The content of malic acid in the transgenic tomato fruit was significantly increased. Background technique [0002] Organic acids play a key role in plant metabolism, together with metabolites such as sugars and phenols, play an important role in fruit quality. As an important organic acid, malic acid is the main metabolite of many plants and has multiple functions. Such as maintaining osmotic pressure, regulating pH, participating in stomatal opening and closing, and responding to biotic and abiot...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8243
Inventor 张绍铃许林林谢智华齐开杰钱铭郭志华张雨馨
Owner NANJING AGRICULTURAL UNIVERSITY
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