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Sugarcane saccharose translocator ShSUT2 genes and application

A sucrose transporter and gene-like technology, which is applied to the sugarcane ShSUT2-like sucrose transporter and its encoding gene and application fields, can solve the problem of unclear molecular genetic mechanism, restriction on breeding of high-sugar varieties, and increase in the content of enzymatically synthesized sugars, etc. question

Inactive Publication Date: 2014-04-16
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The selection of high-sugar varieties through conventional breeding has been limited. Scientists at home and abroad have also devoted themselves to the molecular breeding research of increasing the sugar content of sugarcane, such as the key enzymes of sugarcane endogenous sucrose and glucose metabolism (vacuole acid invertase, neutral invertase , sucrose phosphate synthase, aldolase, UDP glucose hydrolase) gene modification, the total sucrose content of transgenic sugarcane plants did not increase; in addition, the exogenous sucrose isomerase gene, artichoke fructosyltransferase gene, etc. were introduced into sugarcane, The content of sucrose decreased, the content of enzyme-synthesized sugars increased, but the total sugar content did not increase
Sucrose accumulation in sugarcane is a dynamic process, which is affected by many factors, such as internal factors (photosynthesis efficiency, key enzyme activities of sucrose metabolism, output capacity of photosynthetic compounds, hormones, etc.) , pests, lodging, etc.), the modification of a single gene in sugarcane has not fundamentally increased the sugar content of sugarcane, and it is urgent to further study the molecular genetic mechanism of sugarcane sucrose content and yield traits, and find the key control points for the regulation of sucrose accumulation. Make a qualitative change in the genetic improvement of sugarcane
[0004] At present, the molecular genetic mechanism of sugarcane sucrose accumulation is still unclear, and there are few endogenous regulatory genes with clear functions reported

Method used

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  • Sugarcane saccharose translocator ShSUT2 genes and application
  • Sugarcane saccharose translocator ShSUT2 genes and application
  • Sugarcane saccharose translocator ShSUT2 genes and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Cloning and analysis of the full-length coding region of the ShSUT2 gene

[0040] 1. Extraction of total RNA

[0041] Take 0.1 g of fresh sugarcane stems with 7-9 nodes (counting from the growth point downward), 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.

[0042] 2. Synthesis of the first strand of cDNA

[0043] 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...

Embodiment 2

[0068] Example 2. Bioinformatics analysis of ShSUT2 and its encoded protein

[0069] The full-length cDNA sequence of ShSUT2 obtained in Example 1 was subjected to bioinformatics analysis using DNAMAN and OMIGA software. The full-length sequence of ShSUT2A was 1797 bp, encoding a protein consisting of 598 amino acid residues. The full-length sequence of ShSUT2B is 1779bp, encoding a protein consisting of 592 amino acid residues. The structure diagram of ShSUT2 is shown in Figure 5 shown. The domain of ShSUT2 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

[0070] Example 3. Functional analysis of ShSUT2 in yeast mutants

[0071] The ShSUT2 gene amplified in Example 1 was amplified by primers containing SpeI and XhoI restriction sites (primer sequence F: 5'-GGACTAGT AGCCTGAGCCCCAGATCTCACT-3', R: 5'-GGATGGGCGGCTCTTACATACACT-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 DH5a competent cells, positive clones were screened for PCR identification of bacterial liquid, and the plasmids of 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 digested with the sam...

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Abstract

The invention discloses two saccharose translocator ShSUT2 genes derived from sugarcane and application. According to the saccharose translocator genes ShSUT2A and ShSUT2B, amino acid sequences are respectively shown in SEQ ID NO 2 and 4, and base sequences of encoding genes ShSuT2A and ShSUT2B are respectively shown in SEQ ID NO 1 and 3. Absorbing function deficient mutant by yeast saccharose shows that the proteins encoded by ShSUT2A and ShSUT2B have saccharose transport activity. The ShSUT2A and ShSUT2B encoding genes are responsible for absorbing saccharose of apoplast in sugarcane plant and participate in transportation, distribution and accumulated physiological processes of sugarcane plant saccharose. The genes can provide new ways for improving transportation and distribution efficiency of sugarcane saccharose, promoting growth of the plant and improving sugarcane yields and sugar content.

Description

technical field [0001] The invention relates to a plant sucrose transporter encoding gene and its application, in particular to a sugarcane ShSUT2-like sucrose transporter and its encoding gene and application. Background technique [0002] Sugarcane is an important sugar and energy crop. Sugar cane produces 70% of the world's sucrose, accounting for 92% of my country's sugar production; sugar cane is also used to produce biofuel alcohol and power generation. At present, the way to increase the yield of sugarcane is, on the one hand, by expanding the planting area, and on the other hand, through the selection and promotion of high-quality sugarcane high-yield new varieties or high-fiber energy sugarcane new varieties. Conventional breeding is still the main means of cultivating fine varieties of sugarcane. However, due to factors such as narrow genetic background of sugarcane germplasm resources, different flowering periods, genetic rules of breeding target-related traits,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N1/21C12N15/81C12N1/19C12N15/11A01H5/00C12R1/865
Inventor 赵婷婷冯翠莲王俊刚张树珍杨本鹏王文治曾军
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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