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Pear Sucrose Transporter Gene pbsut2 and Its Application

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

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

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

Method used

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  • Pear Sucrose Transporter Gene pbsut2 and Its Application
  • Pear Sucrose Transporter Gene pbsut2 and Its Application
  • Pear Sucrose Transporter Gene pbsut2 and Its Application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Cloning of embodiment 1 pear PbSUT2 gene

[0034] Total RNA was extracted from 'Yali' pulp 100 days after full flowering and reverse-transcribed, and the obtained first-strand cDNA was used to amplify the PbSUT2 gene. Extract total RNA by CTAB method (CTAB extraction buffer includes 2% CTAB, 2% PVP K-30, 0.05% spermidine, 10mM Tris HCl (pH=8.0), 25mM EDTA, 2M NaCl), and take 1 μg RNA sample After being incubated with 1 U DNaseI (purchased from TaKaRa Company) at 37° C. for 30 minutes, 1 μL of EDTA (25 mM) was added and incubated at 65° C. for 10 minutes. The first-strand cDNA was synthesized with a TOYOBO reverse transcription kit (purchased from TakaRa, and operated according to the kit instructions. Amplification primers were: forward primer PbSUT2-F1: 5'-CCATGCCAGCTCCAGAAG-3' (corresponding to SEQ ID NO. 3); Reverse primer PbSUT2-R1: 5'-ACCTCATGTGACAGCTCTGG-3' (corresponding to SEQ ID NO.4). The 25 μL PCR reaction system includes: 1×PCR buffer (purchased from TakaRa...

Embodiment 2

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

[0038] 1. qRT-PCR analysis of pear PbSUT2 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. 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 Primer Premier 5.0 to design gene-specific qRT-PCR primer pairs within 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 used SYBR Green kit (purchased from TaKaRa Company, operated according to kit instructions). The 20 μL qRT-PCR reaction system includes: 10 μL 2×...

Embodiment 3

[0049] Example 3 Subcellular Localization of PbSUT2 Gene

[0050] The present embodiment utilizes the onion epidermis to study the subcellular localization of the PbSUT2 gene, and the expression vector used is pCAMBIA1302, which has the GFP gene ( Figure 4 ). Utilize RT-PCR to amplify the whole ORF of the PbSUT2 gene, the ORF amplification primers are the PbSUT2-F1 and PbSUT2-R1 primers in Example 1, and the 5' ends of the ORF amplification primers PbSUT2-F1 and PbSUT2-R1 are respectively Add BglⅡ and SpeI two restriction sites to get amplification primers with restriction sites: the nucleotide sequence of the forward primer PbSUT2-F3 is: 5'-GA AGATCT CCATGCCAGCTCCAGAAG-3', reverse primer PbSUT2-R3 nucleotide sequence is: 5'-GG ACTAGT ACCTCATGTGACAGCTCTGG-3'. 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 obtain the recombinant...

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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 a sucrose transporter that is isolated and cloned from a pear (Pyrus bretschneideri) 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 important fruits in the world, and it is also the third largest cultivated fruit tree in my country. Sugar is the precursor for the synthesis of other fruit quality characteristic components and flavor substances such as organic acids, anthocyanins and aromatic substances, and is a key substance connecting the primary metabolism and secondary metabolism of plants; at the same time, sugar also has a variety of biological functions , such as providing osmotic driving force for the cell expansion of the fruit, and as a signal molecule and hormone to form a network, it regulates fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H5/08A01H6/82
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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