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Sugar cane sucrose transport protein ShSUT3 and application of coding gene thereof

A technology of sucrose transporter and gene, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2014-06-25
广西康田农业科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, only one sucrose transporter gene ShSUT1 has been reported in sugarcane, and the encoded protein is involved in the reabsorption of sucrose in the phloem. Other types of sucrose transporters have not been reported.

Method used

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  • Sugar cane sucrose transport protein ShSUT3 and application of coding gene thereof
  • Sugar cane sucrose transport protein ShSUT3 and application of coding gene thereof
  • Sugar cane sucrose transport protein ShSUT3 and application of coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning and analysis of the full-length coding region of the ShSUT3 gene

[0037] 1. Extraction of total RNA

[0038] Take 0.1 g of fresh sugarcane stems with 5-7 nodes (counting downward from the growth point), cut them into flakes, and grind them into powder in liquid nitrogen; add 1 mL Trizol (Gibco, Japan), and extract total RNA according to the kit instructions. Carry out 1.2% agarose gel electrophoresis detection, the result is as follows figure 1 shown. The extracted RNA has two obvious electrophoresis bands, which are 28S RNA and 18S RNA from top to bottom, indicating that the total RNA with higher purity and integrity has been obtained.

[0039] 2. Synthesis of the first strand of cDNA

[0040] Mix 5 μg of sugarcane total RNA with 1 μL (10 pmol / L) of reverse transcription primer (oligo-dT linker primer), heat at 70 °C for 5 min, place it on ice immediately, and then add 5× buffer, 2.5 mmol / L dNTP mixture, Ribonuclease Inhibitor, M-MLV reverse tran...

Embodiment 2

[0064] Example 2. Bioinformatics analysis of ShSUT3 and its encoded protein

[0065] Utilize DNAMAN and OMIGA software to carry out bioinformatics analysis to the full-length cDNA sequence of ShSUT3 obtained in embodiment 1, ShSUT3 this sequence full-length 1605bp, wherein from the 1st position of 5' end to 1605th position is a complete ORF, coded by 534 A protein composed of amino acid residues. The schematic diagram of the structure of ShSUT3 is shown in Figure 5 shown. The domain of ShSUT3 was analyzed with the online Blast tool, and the results showed that the protein belonged to the GPH sucrose superfamily, indicating that the protein was a member of the SUT family. The hydrophobicity of the protein was predicted by the membrane protein online prediction tool TopPred2 ( Figure 6 ) and is a 12-transmembrane protein ( Figure 7 ), conforming to the structure of the sucrose transporter family, indicating that it belongs to the sucrose transporter family.

Embodiment 3

[0066] Example 3. Functional analysis of ShSUT3 in yeast mutants

[0067] The ShSUT3 gene amplified in Example 1 was amplified by primers containing SpeI and XhoI restriction sites (primer sequence F: 5'-GGACTAGT ATTTGCCTGTCCCTATCGTCCTCT-3', R: 5'-GGGAATTCCGGCAGACACCAAGAATCAGACTC-3'), and the amplified The amplified target fragment was recovered and purified, connected to the pMD-19T simple (TaKaRa Code: D104A) vector, the ligated product was transformed into Escherichia coli JM109 competent cells, and the positive clones were screened for bacterial liquid PCR identification, and the plasmids of the positive clones were extracted Perform sequencing. Sequencing results showed that the amplified fragment had the nucleotide sequence of the coding region in sequence 2. The plasmids in the clones with correct sequencing results were extracted, digested with SpeI and XhoI, and inserted between the SpeI and XhoI restriction sites of the yeast expression vector PDR196 that had been d...

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Abstract

The invention discloses a sugar cane sucrose transport protein ShSUT3 and an application of a coding gene thereof. The sugar cane sucrose transport protein ShSUT3 has one of the following amino acid residue sequences: (1) SEQ ID No. 1 amino acid residue sequences contained in a sequence table; (2) the sugar cane sucrose transport protein enables the SEQ ID No. 1 amino acid residue sequences contained in the sequence table to be substituted and / or lacked and / or added through one or more of amino acid residues, has sucrose transport function and is derived from the SEQ ID No. 1. The sugar cane sucrose transport protein ShSUT3 disclosed by the invention has sucrose transport activity, is a cell membrane sucrose transport protein, participates in the transmembrane transport of sucrose and is a key protein which participates in the sucrose accumulating process of a sugar cane. According to the invention, the utilization efficiency and yield of the sucrose can be purposefully enhanced by utilizing the sucrose transport protein gene ShSUT3 disclosed by the invention through gene engineering.

Description

technical field [0001] The invention relates to a sucrose transporter and its encoding gene and application, in particular to a sucrose transporter ShSUT3 derived from sugarcane and its encoding gene and application. Background technique [0002] Sucrose is one of the main end products of photosynthesis in higher plants, and it is also the main form of carbohydrate transportation and storage. At the same time, sucrose is also an important signaling molecule, coordinating the relationship between plant source and sink and responding to adversity such as cold damage and drought. After sucrose is synthesized in the source leaf, it is loaded into the phloem through the symplast or apoplast pathway, transported to the sink organ for long-distance transport and unloaded, and then used for the growth and development of sink cells or for storage. Sucrose export capacity affects plant photosynthetic efficiency and crop yield. [0003] Phloem apoplast loading of sucrose or plant cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 王俊刚赵婷婷张树珍杨本鹏冯翠莲王文治伍苏然
Owner 广西康田农业科技股份有限公司
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