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Pyrus fruit sugar transporter gene PbTMT4 and application thereof

A sugar transporter, pear fruit technology, applied in the field of genetic engineering

Inactive Publication Date: 2017-11-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TMT gene family is an important family of sugar transporters on the tonoplast, and it has been reported in species such as Arabidopsis, rice, grape, and apple, but there are few reports on TMT in pears

Method used

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  • Pyrus fruit sugar transporter gene PbTMT4 and application thereof
  • Pyrus fruit sugar transporter gene PbTMT4 and application thereof
  • Pyrus fruit sugar transporter gene PbTMT4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Pear TMT family gene screening and information analysis

[0034] Using the protein sequences of Arabidopsis thaliana AtTMT1 (At1g20840), AtTMT2 (At4g35300), AtTMT3 (At3g51490) as templates in the Arabidopsis thaliana genome database and pear genome database (http: / / peargenome.njau.edu.cn / ) Blastp, to obtain homologous gene sequences. The obtained homologous sequences were clustered with the homologous sequences of each species by using the software MEGA6.0 to construct a phylogenetic tree, and those clustered with Arabidopsis TMT family genes were candidate genes. The conserved domains of all candidate genes were analyzed by the Pfam database (http: / / pfam.xfam.org / ), and sequences inconsistent with the conserved domains of TMT family genes were removed, and the conserved domains of the NCBI conservative structure database CCD (http: / / www. ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) to further verify the conserved domains, and finally obtain and name six pear TM...

Embodiment 2

[0037] Example 2 Analysis of PbTMT gene expression pattern in pear

[0038] Taking 'Yali' as the test material, roots, stems, young leaves, mature leaves, flowers, young fruits (30 days after full flowering), mature fruits (150 days after full flowering) and seeds were collected as samples, total RNA was extracted and reverse transcribed, The resulting first-strand cDNA was used to amplify the PbTMT gene.

[0039] Total RNA was extracted by CTAB method (CTAB extraction buffer included 2% CTAB, 2% PVP K-30, 0.05% spermidine, 10mM Tris HCl (pH=8.0), 25mM EDTA, 2M NaCl), and 1 μg RNA sample was taken, Incubate with 1U DNase I (purchased from Fermentas) at 37°C for 30min, immediately place on ice for 5min, add 1 μL EDTA (25mM) and incubate at 65°C for 30min. For first-strand cDNA synthesis One-Step RT-PCR SuperMix Reverse Transcription Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), operated according to the instructions of the kit.

[0040] Using pear Tubulin...

Embodiment 3

[0051] Example 3 PbTMT gene expression in pears and its relationship with fruit sugar accumulation

[0052] 1. qRT-PCR analysis of pear PbTMT gene during pear fruit development

[0053] Taking 'Yali' as the test material, the fruit samples were collected from 10 days after full flowering, and samples were collected every 20 days until the fruit was ripe. The pulp samples were treated with liquid nitrogen and stored at -80°C.

[0054] The methods for extracting total RNA from pear pulp and synthesizing cDNA, as well as the primers and reaction conditions used for qRT-PCR of each gene of pear PbTMT are the same as in Example 2.

[0055] qRT-PCR analysis of the relative expression of PbTMT gene during pear fruit development, see image 3 a.

[0056] 2. Changes of soluble sugar content during pear fruit development

[0057] The extraction steps of soluble sugar are as follows: Accurately weigh 2.0 g of pulp tissue in a pre-cooled mortar, add liquid nitrogen and fully grind it i...

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Abstract

The invention discloses a pyrus fruit sugar transporter gene PbTMT4 and application thereof. The nucleotide sequence of the pyrus fruit sugar transporter gene PbTMT4 is as shown in SEQ ID NO.1. Tomato plants overexpressing the pyrus gene PbTMT4 bloom and bear fruits in advance obviously, and the sugar contents of fruits are obviously higher than the sugar content of fruits of wild type tomato plants. The pyrus fruit sugar transporter gene PbTMT4 can be applied to constructing transgenic plants blooming in advance and enhancing the sugar contents of plant fruits.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to pear fruit sugar transporter gene PbTMT4 and its application. Background technique [0002] Sugar is the basic substance for fruit growth and development, and it is also an important indicator for evaluating fruit quality. At the same time, sugar, as a signaling molecule, participates in a variety of metabolic and hormone pathways (Ljung et al., 2015), and regulates plant growth and gene expression (Wind et al., 2010). [0003] The vacuole is an important organ for sugar accumulation in plants. Normally, reducing sugars accumulated in vacuoles account for about 90% of free hexoses, and only a small amount of hexoses exists in the cytoplasm (Voitsekhovskaja et al., 2006). The ability of vacuoles to accumulate sugar is mainly regulated by sugar transporters located in the tonoplast membrane. The sugar transporters identified in the model plant Arabidopsis thaliana are mainly tonop...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/08
CPCC07K14/415C12N15/8245C12N15/827
Inventor 张虎平张绍铃程瑞吴巨友程寅胜吴俊王鹏齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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