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Application of arabidopsis thaliana gene At-UGE2, overexpression mutant strain and deletion mutant strain in plant character adjustment

A deletion mutation and transgenic plant technology, applied in the application field of transgenic plants, can solve the problems of changing the resistance of raffinose content and increasing expression, and achieve the effects of delaying senescence and enhancing plant yield-related traits

Inactive Publication Date: 2017-05-10
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, in the reported gene chip data of Arabidopsis in response to adversity stress, low temperature, drought, high salt, etc. can induce the expression of UGE gene to increase; the expression of rice UGE1 gene is induced by drought, high salt and ultraviolet stress. , and is responsible for the allocation of sugars under nitrogen deficiency conditions; the UGE1 gene of rice was transformed into Arabidopsis thaliana, although the morphological characteristics of Arabidopsis were not changed, but the raffinose content and the response to abiotic stress resistance

Method used

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  • Application of arabidopsis thaliana gene At-UGE2, overexpression mutant strain and deletion mutant strain in plant character adjustment
  • Application of arabidopsis thaliana gene At-UGE2, overexpression mutant strain and deletion mutant strain in plant character adjustment
  • Application of arabidopsis thaliana gene At-UGE2, overexpression mutant strain and deletion mutant strain in plant character adjustment

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Experimental program
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Effect test

Embodiment 1

[0041] The acquisition and expression detection of At‐UGE2 gene deletion mutant strains uge2‐1 and uge2‐2.

[0042] (1) PCR identification of At‐UGE2 gene deletion mutants uge2‐1 and uge2‐2 homozygotes:

[0043] The numbering of the Arabidopsis thaliana At‐UGE2 gene described in the present invention in GeneBank is AT4G23920, the full-length coding region sequence is 1053bp, encodes 350 amino acids, and its nucleotide sequence and amino acid sequence are as shown in Sequence Table 1 and 2 (SEQ ID No 1: At-UGE2 gene coding region nucleotide sequence and SEQ ID No 2: At-UGE2 gene coding amino acid sequence), mainly expressed in the cytoplasm, catalyzing the conversion of UDP-D-galactose and UDP-D-glucose, Involved in the biosynthesis of cell wall carbohydrates.

[0044] The present invention purchased two T-DNA insertion mutant strains of the At-UGE2 gene (seed numbers: CS25040 and CS859980) from the Arabidopsis Biological Resource Center (ABRC) ( figure 1), its flanking seque...

Embodiment 2

[0060] Construction and identification of At‐UGE2 gene overexpression mutant 35S::UGE2:

[0061] The specific method of constructing At‐UGE2 overexpression mutant 35S::UGE2 is as follows: extract total RNA from Arabidopsis leaves grown for four weeks under normal conditions at 22°C, and clone At ‐The coding region of the UGE2 gene, and then the gene fragment confirmed to be completely correct after sequencing was constructed on the overexpression vector pBI121 connected with the CaMv 35S constitutive expression promoter ( Figure 3A ), using the liquid nitrogen freeze-thaw method to transfer the expression vector into Agrobacterium ( Figure 3B ), and then genetically transform Arabidopsis thaliana by infecting inflorescences with Agrobacterium. The first-generation seeds harvested were screened for resistance using kanamycin at a final concentration of 50 mg / L and verified by PCR amplification of the target fragment. and three generations of seeds were also screened for ant...

Embodiment 3

[0063] The phenotypes of At‐UGE2 gene deletion mutants and overexpression mutants were verified by galactose toxicity test.

[0064] Excessive galactose in cells can cause serious galactose toxicity to plants, mainly manifested in inhibiting plant growth, reducing chlorophyll content, and yellowing seedlings. At‐UGE2 gene mainly catalyzes the conversion of UDP‐grape to UDP‐galactose in Arabidopsis, and the functional defect of At‐UGE2 gene may lead to galactose toxicity in plants after a certain concentration of galactose is applied exogenously. Therefore, the present invention sterilizes the seeds of Arabidopsis wild type (Col-0), overexpression mutant strains 35S::UGE2-1, 35S::UGE2-2 and deletion mutant strains uge2-1, uge2-2 The divisional points were planted on the MS medium with galactose concentration increasing in order of 0mM, 50mM, 100mM, 150mM, and 200mM. After vernalization at 4°C for 1 day, they were cultured under normal conditions (temperature 22°C, humidity 50%-...

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Abstract

The invention provides application of arabidopsis thaliana UDP-glucose 4-isomerase gene At-UGE2, an overexpression mutant strain 35S:UGE2 and a deletion mutant strain uge2 in adjustment of plant yield correlated characters, adversity resistance characters and the like. The function of biological synthesis and conversion of the UDP-glucose and UDP-galactose, which are catalyzed by UDP-glucose 4-isomerase, in adjustment of plant growth and development as well as adversity stress response is disclosed, a high-quality gene is provided for plant variety improvement, a new method and a new mechanism for improving plant yield characters and adversity resistance abilities are explained, and important economic significance and application value are achieved.

Description

[0001] Technical field: [0002] The invention belongs to the field of plant genetic engineering, and relates to an Arabidopsis thaliana UDP‐glucose 4‐isomerase gene At‐UGE2 and its overexpression mutant 35S::UGE2 and deletion mutant uge2 in regulating plant yield-related traits and stress resistance Application of transgenic plants for traits. [0003] Background technique: [0004] In the process of plant growth and development, it is often affected by adverse environmental factors such as low temperature, high temperature, drought, salinity, etc. These factors often constitute serious abiotic stress to the growth process of plants. Since the movement of plants is limited, they can only survive by adapting to environmental changes. Therefore, during the long process of adaptive evolution, plants have continuously formed a set of physiological mechanisms to resist adversity. The study of the physiological changes and molecular mechanisms of plants under adverse conditions is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/61A01H5/00
CPCC12N15/8205C12N9/90C12N15/8222C12N15/8261C12N15/8273C12N15/8291C12N15/8293C12Y501/03002
Inventor 李唯奇毕丽莎张洁
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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