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Pear hexose transport protein gene PbHT1 and application thereof

A transporter and hexose technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of unreported research reports, unclear effect of pear monosaccharide transporter function, etc., and achieve the effect of increasing hexose content

Active Publication Date: 2016-02-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the functional role of pear monosaccharide transporters in the process of fruit sugar accumulation and the impact on fruit quality are not clear, and there are no related research reports.

Method used

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  • Pear hexose transport protein gene PbHT1 and application thereof
  • Pear hexose transport protein gene PbHT1 and application thereof
  • Pear hexose transport protein gene PbHT1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of embodiment 1 pear PbHT1 gene

[0034] Total RNA was extracted from 'Yali' pulp 40-100 days after full flowering and reverse-transcribed, and the obtained first-strand cDNA was used to amplify the PbHT1 gene. Total RNA was extracted by CTAB method (CTAB extraction buffer: 2% CTAB, 2% PVPK-30, 0.05% spermidine, 10mM Tris HCl (pH=8.0), 25mM EDTA, 2M NaCl), and 1 μg RNA sample was treated with 1UDNaseI (purchased After incubating at 37°C for 30 minutes from TaKaRa Company), 1 μL of LEDTA (25 mM) was added and incubated at 65°C for 10 minutes. The first-strand cDNA was synthesized using a TOYOBO reverse transcription kit (purchased from TakaRa, and operated according to the kit instructions. The amplification primers were: forward primer PbHT1-F1: 5'-TCCCAAGAACCCTAACAATGCC-3' (SEQ ID NO.3); Reverse primer PbHT1-R1: 5'-TGGGAACAATAATATTCAGTGCTACC-3' (SEQ ID NO.4). 25μL PCR reaction system includes: 1 × PCR buffer (purchased from TakaRa company), 2.5mM MgCl 2 (purc...

Embodiment 2

[0037] Example 2 Changes in PbHT1 gene expression and sugar content during pear fruit development

[0038] 1. qRT-PCR analysis of pear PbHT1 gene during pear fruit development

[0039] The extraction of pear pulp total RNA, the method for cDNA synthesis are the same as in Example 1. Use pear β-tubulin (AB239681) as an internal control, the nucleotide sequence of the primer is as follows: forward primer TUB-F: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO.5), reverse primer TUB-R: 5'- CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 6). Utilize PrimerPremier5.0 to design gene-specific qRT-PCR primer pair in the open reading frame of PbHT1 gene, the nucleotide sequence of primer is as follows: Forward primer PbHT1-F2: 5'-CCTCTGCGTGGCAATCGTCAT-3' (SEQ ID NO.7), Reverse primer PbHT1-R2: 5'-TTCTCCAGGGTTCCCATCCAC-3' (SEQ ID NO. 8).

[0040] The qRT-PCR uses the SYBRGreen kit (purchased from TaKaRa Company, operated according to the instructions of the kit). The 20 μL qRT-PCR reaction system includes: ...

Embodiment 3

[0044] Example 3 Subcellular Localization of PbHT1 Gene

[0045] The present embodiment utilizes onion epidermis to study the subcellular localization of the PbHT1 gene, and the expression vector used is pCAMBIA1302, which has the GFP gene ( Figure 4 ). Use RT-PCR to amplify the entire ORF of the PbHT1 gene, design amplification primers that amplify the ORF sequence and remove the stop codon, and then add two restriction sites, BglII and SpeI, to the 5' ends of the forward and reverse primers. , that is, the amplification primer with restriction site is obtained: the nucleotide sequence of the forward primer PbHT1-F3 is: 5'-GA AGATC TATGCCTGCTGTTGGTAT-3' (SEQ ID NO.9), the reverse primer PbHT1-R3 nucleotide sequence is: 5'-GG ACTAGT TGGGAACAATAATATTCAGTGCTACC-3' (SEQ ID NO. 10). The underline is the enzyme cutting site, AGATCT is the BglⅡ enzyme cleavage site, ACTAGT It is the SpeI restriction site. First, the amplified product is installed on the pMD19-T vector to ...

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Abstract

The invention discloses a pear hexose transport protein gene PbHT1 and application thereof. The hexose transport protein gene PbHT1 is separated from a pear fruit, the nucleotide sequence of the gene is as indicated in SEQ ID No. 1, and the coded amino acid sequence of the gene is as indicated in SEQ ID No. 2. The gene PbHT1 is transformed into a tomato through a southern buddhism genetic transformation method, a transgenic plant is obtained, and through biological function verification, it is shown that the clonal gene PbHT1 has the effects of postponing the flowering phase and raising the hexose content in the fruit. Through finding of the gene PbHT1, a new gene resource is provided for molecular breeding for improving quality of the plant fruit.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, it relates to a gene PbHT1 encoding a hexose transporter that is isolated and cloned from pear (Pyrusbretschneideri) fruit, and also relates to the application of a pear hexose transporter gene PbHT1 in regulating plant growth and fruit sugar accumulation. Background technique [0002] The sugar produced by the light and action of plant leaves is the most basic nutrient and energy substance for plant growth and development. It is synthesized in plant-derived organs and then transported to various sink organs for storage or absorption. At the same time, sugar also has a variety of biological functions, such as providing osmotic driving force for fruit cell expansion, and as a signal molecule and hormones to form a network, which regulates fruit growth and gene development through complex signal transduction mechanisms. Expression etc. (Leon and Sheen, 2003; Chen Junwei et al...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 张绍铃王利芬黄小三齐笑笑靳丛许林林
Owner NANJING AGRICULTURAL UNIVERSITY
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