Pear sugar transport protein gene PbSUT2 and application thereof

A technology of sucrose transporter and gene, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve problems such as unseen functional research

Active Publication Date: 2016-06-01
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on the function of plant sucrose transporters mainly focuses on Arabidopsis, tobacco, tomato and other plants, but there are few reports on pear sucrose transporters, although the pear sucrose transporters have been cloned PbSUT1 (Zhang et al., 2013 ) and PpSUT2 (Tang et al., 2014), but there are no related reports on the functional studies of pear sucrose transporter or fruit tree plant sucrose transporter

Method used

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  • Pear sugar transport protein gene PbSUT2 and application thereof
  • Pear sugar transport protein gene PbSUT2 and application thereof
  • Pear sugar transport protein gene PbSUT2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cloning of pear PbSUT2 gene

[0034] Using the pulp of'Ya Pear' 100 days after blooming as the test material, total RNA was extracted and reverse transcribed, and the first strand cDNA obtained was used to amplify the PbSUT2 gene. Use CTAB method (CTAB extraction buffer includes 2% CTAB, 2% PVPK-30, 0.05% spermidine, 10 mM Tris·HCl (pH = 8.0), 25 mM EDTA, 2M NaCl) to extract total RNA, take 1 μg RNA sample, and pass 1UDNaseI (purchase After incubating at 37°C for 30 min from TaKaRa company, add 1μLEDTA (25mM) and incubate at 65°C for 10 min. The TOYOBO reverse transcription kit (purchased from TakaRa Company, according to the kit instructions for the synthesis of the first strand cDNA. The amplification primers are: forward primer PbSUT2-F1: 5'-CCATGCCAGCTCCAGAAG-3' (corresponding to SEQIDNO.3) ; Reverse primer PbSUT2-R1: 5'-ACCTCATGTGACAGCTCTGG-3' (corresponding to SEQIDNO.4). 25μL PCR reaction system includes: 1×PCR buffer (purchased from TakaRa), 2.5mMMgCl 2 ...

Embodiment 2

[0037] Example 2 Changes in PbSUT2 gene expression and sugar content during pear fruit development

[0038] 1. qRT-PCR analysis of PbSUT2 gene in pear fruit development

[0039] The methods of extracting total RNA from pear pulp and cDNA synthesis are the same as in Example 1. Using pear tubulin (AB239681) as an internal control, the nucleotide sequence of the primer is as follows:

[0040] Forward primer TUB-F: 5’-TGGGCTTTGCTCCTCTTAC-3’,

[0041] Reverse primer TUB-R: 5'-CCTTCGTGCTCATCTTACC-3'.

[0042] Use PrimerPremier5.0 to design gene-specific qRT-PCR primer pairs in the open reading frame of the PbSUT2 gene. The nucleotide sequences of the primers are as follows:

[0043] Forward primer PbSUT2-F2: 5’-CCTCCAGATGGCATTGTGATAGC-3’,

[0044] Reverse primer PbSUT2-R2: 5'-GCGGGATTACTATTGCCAGATT-3'.

[0045] qRT-PCR uses SYBRGreen kit (purchased from TaKaRa company, according to the kit instructions). The 20μL qRT-PCR reaction system includes: 10μL 2×SYBRPremixExTaq, 0.4μL forward primer, ...

Embodiment 3

[0049] Example 3 Subcellular localization of PbSUT2 gene

[0050] In this example, the onion epidermis was used to study the subcellular location of the PbSUT2 gene. The expression vector used was pCAMBIA1302, and the expression vector had the GFP gene ( Figure 4 ). The entire ORF of the PbSUT2 gene was amplified by RT-PCR, the ORF amplification primers were the PbSUT2-F1 and PbSUT2-R1 primers in Example 1, and the 5'ends of the ORF amplification primers PbSUT2-F1 and PbSUT2-R1 were respectively Adding the two restriction sites of BglII and SpeI, the amplification primer with restriction sites is obtained: the nucleotide sequence of the forward primer PbSUT2-F3 is: 5'-GA AGATCT CCATGCCAGCTCCAGAAG-3’, reverse primer PbSUT2-R3 nucleotide sequence: 5’-GG ACTAGT ACCTCATGTGACAGCTCTGG-3’. The underline is the restriction site, AGATCT Is the BglII restriction site, ACTAGT SpeI restriction site. First install the amplified product on the pMD19-T vector to obtain the recombinant vec...

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Abstract

The invention discloses a pear sugar transport protein gene PbSUT2 and application thereof. A nucleotide sequence of the pear sugar transport protein gene separated from a pear fruit is shown as SEQ ID No. 1, and a coded amino acid sequence of the pear sugar transport protein gene is shown as SEQ ID No. 2. The gene PbSUT2 is transformed into a tomato through an agrobacterium mediated genetic transformation method; the biological function verification of an obtained transgenic plant indicates that the cloned gene PbSUT2 has the functions of promoting plant early flowering and increasing the content of sucrose in the fruit. The discovery of the gene PbSUT2 provides new genetic resources for promoting the molecular breeding of plant fruit quality.

Description

Technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, it relates to a gene PbSUT2 encoding sucrose transporter isolated and cloned from Pyrusbretschneideri fruit, and also relates to the application of a pear sucrose transporter gene PbSUT2 in regulating plant growth and fruit quality. Background technique [0002] Pear is one of the most important fruits in the world and the third largest cultivated fruit tree in my country. Sugar is the precursor for the synthesis of other quality characteristic components of fruit and flavor substances such as organic acids, anthocyanins and aromatic substances. It is a key substance linking the primary and secondary metabolism of plants; at the same time, sugar also has a variety of biological functions For example, it provides osmotic driving force for the expansion of fruit cells, as well as signal molecules and hormones and other signals to form a network, which regulates fruit growth and ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/84A01H5/00
Inventor 张绍铃王利芬黄小三齐笑笑许林林谢智华张虎平
Owner NANJING AGRICULTURAL UNIVERSITY
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