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Proteins related to stachyose synthesis and their coding genes and applications

A technology of stachyose and protein, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of improving the quality of cucumber

Inactive Publication Date: 2012-02-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the physiological and biochemical characteristics of cucumber and the changes of related indicators under various stress conditions have been extensively studied, there are still relatively few studies on the metabolism of stachyose and the cloning and regulation of related genes in cucumber. Research on this aspect has important theoretical and practical significance in regulating the sugar metabolism in cucumbers, improving the quality of cucumbers, and enhancing the health effects on the human body.

Method used

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  • Proteins related to stachyose synthesis and their coding genes and applications
  • Proteins related to stachyose synthesis and their coding genes and applications
  • Proteins related to stachyose synthesis and their coding genes and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Acquisition of protein STS related to stachyose synthesis and its encoding gene

[0034] 1. Obtaining the middle fragment of STS gene

[0035] Sow the seeds of cucumber variety Guonong No. 25 in a mixed substrate with peat, vermiculite and field soil (volume ratio 1:1:1). When the plants grow to 5-6 leaves, select young From the young leaves, extract the total RNA of the leaves, and reverse transcribe it into cDNA. Using this cDNA as a template, primers 5'-ACGGTCATTCGTTTCTGTCC-3' and 5'-GCACTTGAACCACCACCATT-3' and primers 5'-CAAAAGCCTATTATAAGGGTCTGACCAACTCTC-3' and 5'-CCA(A / C)CCNGCNCCTTG(A / G)CAGT were designed respectively -3', the intermediate fragments 1 and 2 of the stachyose synthase gene were cloned by RT-PCR. The reaction mixture is as follows:

[0036] wxya 2 o

34μl

10×PCR buffer

5μl

dNTP Mixture (25mM)

4μl

Taq enzyme (5U / μl)

1μl

Upstream primer (10uM)

2μl

Downstream primer (10uM)

...

Embodiment 2

[0055] Embodiment 2, the expression analysis of STS gene in the different tissues of cucumber

[0056] At noon on a sunny day, the mature leaves, tender stems, young fruits and roots of cucumber Guonong No. 25, which had grown to the early melon stage, were selected to extract their total RNA, and the integrity of the RNA was detected by 1% agarose gel electrophoresis. Using the total RNA of the above-mentioned mature leaves, tender stems, young fruits and roots as templates, the second-strand cDNA was obtained by reverse transcription. Using the second-strand cDNA as a template, 5'-GAAGGAAGTTTCAGGTCTGCCAT-3' and 5'-CACATCAACTTTCACTCCTGTAATCC -3' was used as a primer for PCR amplification, and 18s rRNA was used as an internal control for expression analysis. The result is as figure 1 shown. Among them, A is the electrophoresis image of total RNA in different cucumber tissues, B is the RT-PCR electrophoresis image of STS gene in different cucumber tissues, C is the electropho...

Embodiment 3

[0057] Embodiment 3, the construction of STS antisense expression vector and its functional identification

[0058] 1. Construction of STS antisense expression vector

[0059] Using the recombinant expression vector pBS-STS constructed in Example 1 containing the full-length sequence of the STS gene as a template, use primers 5'-ATGGCACCTCCAAATGAC-3' and 5'-CAATCCAAT GAAGATCAAAACT-3' containing SacI and SmaI restriction sites to amplify A fragment containing the ORF of the STS gene was added, and the fragment was recovered and connected to the pMD18-T vector, and the recombinant expression vector was named pMD18-STS. The recombinant expression vector pMD18-STS was sequenced, and the sequencing results showed that the recombinant expression vector contained the complete coding region of STS, and its 5' end and 3' end contained SacI and SmaI restriction sites respectively.

[0060] pMD18-STS was double-digested with SacI and SmaI, and the digested STS fragment was recovered and...

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Abstract

The invention discloses a protein related to stachyose synthesis, its coding gene and application. The protein is a protein of the following 1) or 2): 1) a protein composed of the amino acid sequence shown in sequence 2 in the sequence listing; Substitution and / or deletion and / or addition of proteins derived from 1) associated with stachyose synthase. The present invention constructs the antisense expression vector of STS, a gene related to stachyose synthesis, and transfers it into cucumber plants. Experiments show that the activity of stachyose synthase in cucumber plants transformed into the STS antisense expression vector is significantly reduced. , indicating that the protein STS and its coding gene related to stachyose synthesis can be used to regulate sugar metabolism in cucumber and improve cucumber quality, and can be widely used in the research of sugar metabolism in plants.

Description

technical field [0001] The invention relates to a protein related to stachyose synthesis, its coding gene and application. Background technique [0002] In recent years, my country's vegetable industry has developed rapidly. According to statistics from the Ministry of Agriculture, in 2005, the country's vegetable sown area was 17.7207 million hectares, with a total output of 564.5149 million tons and a per capita share of 432kg. According to the statistics of FAO, in 2005, my country's vegetable sowing area and output both ranked first in the world, among which the vegetable sowing area accounted for 43% of the world, and the total output accounted for 49% of the world. At present, my country's vegetable sown area accounts for more than one-third of the world's total vegetable sown area, and its output accounts for about 50% of the world's total output. It is the country with the largest vegetable sown area and the largest output in the world today. The development of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/29A01H5/00C12N1/19C12N15/11A01H1/00C12N1/21C12N5/10C07K14/415C12N1/15
Inventor 张振贤孟凡珍胡丽萍眭晓蕾王绍辉黄金丽孙建磊
Owner CHINA AGRI UNIV
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