Rape pyruvic dehydrogenase kinase gene and its application in rape

A technology of pyruvate dehydrogenase and rape, applied in the field of genetic engineering, to achieve the effect of increasing seed yield and oil content and reducing the growth period

Inactive Publication Date: 2006-05-17
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Rape pyruvic dehydrogenase kinase gene and its application in rape
  • Rape pyruvic dehydrogenase kinase gene and its application in rape
  • Rape pyruvic dehydrogenase kinase gene and its application in rape

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1--cloning and sequencing of genes

[0034] A sequence was isolated from the cDNA library by using primers, and BnPDK1 was obtained by sequencing.

[0035] 1. Extract rapeseed mRNA.

[0036] Extraction of RNA (TRIZOL TM Kit for RNA extraction)

[0037] Liquid nitrogen was used to triturate 100 mg of material.

[0038] (1) Add 1ml TRIZOL and place at room temperature for 5min.

[0039] (2) Add 200ul of chloroform, shake vigorously for 30s, and place at room temperature for 2min.

[0040] (3) 12000g, 15min, 4 degrees Celsius. Take the supernatant to a new tube, add 500ul of isopropanol, mix well and place at room temperature for 15min.

[0041] (4) 12000g, 15min, 4 degrees Celsius. Remove the supernatant and add 1ml of 70% ethanol.

[0042] (5) 7500g, 7min, 4 degrees Celsius. Remove supernatant and air dry.

[0043] (6) DEPC-H 2 O dissolved.

[0044] 2. Use the ZAPII-cDNA Synthesis kit of Strategene Company to construct a rapeseed cDNA library accord...

Embodiment 2

[0046] Embodiment 2--vector construction

[0047] After the synthetic full-length BnPDK1 DNA fragment was digested with restriction endonuclease sal1, it was reverse cloned into the plasmid PMD (such as figure 1 shown), and its insert was identified by DNA sequencing. pBnPDK1 contains a sequence encoding the full length of 367 amino acids of BnPDK1.

Embodiment 3

[0048] Embodiment 3--transformation and screening of plants

[0049] Agrobacterium Transformation of Rapeseed

[0050] 1. Preparation of sterile seedlings: the seeds were treated with 70% ethanol for 30 sec, ddH 2 O washing, soaking in 5% NaClO for 30min, ddH 2 After O washing 5 times, spread in MS medium (PH 5.8), agar concentration 0.8%. Rapeseed aseptic seedlings for use.

[0051] 2. Rapeseed petiole transformation: Inoculate LBA4404 (containing pMD-BnPDK1) on the solid medium YM, pick a single colony and culture it in 50 ml YM liquid medium two days later. Take the petiole of 5-day-old sterile seedlings and soak them in the bacterial solution for 10 minutes. Placed on co-culture medium for 2-3 days.

[0052] 3. After co-cultivation, the cotyledon petioles were washed twice with liquid MS medium, and then washed once with liquid MS medium added with Carb. The cotyledon petioles are transferred to selection medium. Subculture once every 15 days.

[0053] 4. When the ...

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Abstract

The present invention discloses one kind of rape pyruvic dehydrogenase kinase gene and its application in rape, and relates to gene engineering technology. The gene has nucleotide sequence coding the protein kinase catalysis area and mitochondrion locating sequence SEQ ID No. 1. The application of the gene in rape includes: 1. providing one kind of vector including proper promoter and translation controlling part; 2. providing one kind of host bacteria capable of being expressed in plant; 3. providing one method of introducing the vector to the plant cell; 4. providing one kind of plant transformed with the vector containing pyruvic dehydrogenase kinase gene; and 5. providing one transgenic rape capable of inhibiting the expression of rape pyruvic dehydrogenase kinase gene. The transgenic rape exhibits early blooming character, shortened growth period, and increased seed yield and oil bearing rate.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a rapeseed pyruvate dehydrogenase kinase gene sequence and its application in rapeseed. Background technique [0002] Pyruvate dehydrogenase (PDC) is widely distributed in microorganisms, plants and mammals. In animals this complex consists of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoate transacetylase (E2) and dihydrolipoate dehydrogenase (E3). The oxidative decarboxylation of pyruvate to form acetyl CoA is an irreversible step, which is at the branch point of the metabolic pathway and the central link connecting glycolysis and the tricarboxylic acid cycle, so it is under strict regulatory control. The regulation of the pyruvate dehydrogenase complex is mainly accomplished by phosphorylation or dephosphorylation of the kinase propionate dehydrogenase kinase (PDK) or the phosphatase propionate dehydrogenase lipase (PDP). PDK also participates in variou...

Claims

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Application Information

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IPC IPC(8): C12N15/53A01H5/00C12N9/04C12N15/82
Inventor 华玮王汉中李荣俊
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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