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Malic acid transporter gene for controlling organic acid content in apple pulp and application of transporter gene

A technology of apple pulp and malic acid, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of unconfirmed functional mechanism, etc., achieve the effect of improving apple varieties, accurate results, and improving screening efficiency

Active Publication Date: 2015-04-29
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It has been reported that there are candidate genes that may control the content of malic acid in apple fruit, but their functional mechanism has not been confirmed
And there is no report of utilizing molecular markers to analyze the content of malic acid in natural populations before the present invention's report

Method used

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  • Malic acid transporter gene for controlling organic acid content in apple pulp and application of transporter gene
  • Malic acid transporter gene for controlling organic acid content in apple pulp and application of transporter gene
  • Malic acid transporter gene for controlling organic acid content in apple pulp and application of transporter gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: extraction and determination of malic acid

[0037]After the fruit is ripe, remove the peel and core, and place the pulp at -80°C after quick freezing in liquid nitrogen; take the sample stored at -80°C, grind it into powder with a mortar, take 1g of the ground sample and add 6mL of ddH 2 Mix well with O, and centrifuge at 15,000 rpm for 15 min at 4°C; the supernatant is passed through Waters C 18 Column and 0.22μm Sep-Pak microporous membrane; the filtrate was stored at -80ºC for the determination of sugar and acid.

[0038] The determination of sugars and organic acids uses high-performance liquid chromatography; the chromatographic conditions for the determination of sugars are: Sugar-Pak I, mobile phase double distilled water, the flow rate is 0.8mL / min, and the detector is a differential detector; the chromatographic conditions for the determination of organic acids The conditions are: Shodex KC-811 column, the mobile phase is 0.02mol / L K 2 HPO 4 ...

Embodiment 2

[0039] Example 2: Typing detection of CAPS and SSR molecular markers

[0040] Take 50-100 mg of apple leaves for the extraction of apple genomic DNA, and the extraction of apple genomic DNA uses the plant genomic DNA extraction kit (Tiangen). The molecular marker PCR reaction uses a 10 µL reaction system, as follows: 40 μM dNTPs 0.6 μL, 10×PCR Buffer 1.0 μL, 10 μM Primer Pair 0.4 μL, rTaq 0.1 μL, ddH 2 0 5.4 μL, DNA (20 ng / μL) 2.5 μL; the reaction program is: 95°C, 5 min; [95°C, 30s; 58°C, 30s; 72°C, 45s] 35 cycles; the final extension at 72°C for 10 min .

[0041] Polyacrylamide gel electrophoresis detection: The denatured PCR products were detected on 8% polyacrylamide gel electrophoresis; the electrophoresis condition was 80w constant power, and the electrophoresis buffer was 1×TBE; first, pre-electrophoresis for 30 min until the glass The plate is hot; after preheating, insert a comb to remove air bubbles, load 1 μL of sample, and electrophoresis time is 1-1.5 h.

[004...

Embodiment 3

[0049] Example 3: Yeast expression system detects the acidifying ability of MdAlMT9 to vacuoles

[0050] The CDS region of the MdAlMT9 gene was connected to pRS406 to construct the pRS406-Ma yeast expression vector, and the pRS406 empty vector and pRS406-Ma were transformed into Saccharomyces cerevisiae ( Saccharomyces cerevisiae ); the transformed yeast were cultured on YPDA medium containing 0.02g / mL to OD 600NM =0.6, collect the bacteria, dissolve in 1mL of ddH 2 In O, mechanically break the yeast cell wall, centrifuge at 12 000 rpm for 5 min to get the supernatant, and measure the pH value with a pH meter.

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Abstract

The invention discloses a malic acid transporter gene for controlling organic acid content in apple pulp and application of the transporter gene, and relates to the field of apple molecule genetic breeding. The sequence of the gene is as shown in SEQ ID NO.1 and the sequence of encoded protein is as shown in SEQ ID NO.2. The application is as follows: (1) designing a molecular marker according to the sequence for marker screening in breeding; and (2) detecting and screening related acidity screening phenotypes according to the expression quantity of the gene or the sequence subtype of a transcript, or improving or reducing the expression quantity of the gene by virtue of a transgenic method so as to acquire expected pulp acidity characters. The organic acid transporter gene disclosed by the invention can be used for determining pulp acidity; on the basis of the assembling result of RNA-Seq and the annotation result of a whole genome frame diagram, the obtained gene is more accurate; and the gene can be screened at the early stage of apple breeding, so that breeding time can be saved, and meanwhile screening efficiency can be improved.

Description

technical field [0001] The invention relates to the field of apple molecular genetic breeding, in particular to a method for controlling the organic acid content of apple pulp [0002] The malate transporter gene and its application. Background technique [0003] Apple is the most grown and consumed fruit in the world. It has the characteristics of high heterozygosity, self-incompatibility and long juvenile period. These characteristics, especially the long childhood period, severely restrict the genetic breeding of apples and the cultivation of new varieties. Fruit quality directly determines the commercial value of the fruit and affects the share in the consumer market. The content of components such as acid, sugar and aroma substances in the fruit is the main factor to determine the quality of the fruit. The organic acid content of apples is an important factor affecting the taste quality, and has an important impact on the consumption market share and the quality of f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12Q1/68
Inventor 韩月彭马百全王鲁谷超
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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