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Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof

A technology of pyruvate carboxylase and nucleic acid molecules, applied in the field of C4 cycle, can solve problems such as unreported plant research

Inactive Publication Date: 2005-12-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

About sugarcane pepc gene genetic transformation C 3 Plant research has not been reported

Method used

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  • Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof
  • Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof
  • Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning and sequencing of the sugarcane pepc gene of embodiment 1

[0034] 1.1 Construction of sugarcane genome library and sequencing of sugarcane pepc gene

[0035] Sugarcane genomic DNA was extracted from young leaves of sugarcane plants by CTAB method. Sugarcane total RNA was extracted according to the instructions of the Trizol kit. The sugarcane genome library was constructed as follows:

[0036] 1) Sugarcane genomic DNA was completely digested with XbaI endonuclease at 37°C overnight;

[0037] 2) 10-40% sucrose gradient centrifugation to collect 9kb DNA fragment components containing pepc gene;

[0038] 3) Ligate the fragment with the XbaI cut point to the λFix II vector (experimental operation follows the instructions for constructing the FixII library from Stratagene, USA).

[0039] 4) The ligated DNA was packaged with the packaging protein of GigapackIII Gold-4 (Product No. #200201) of Stratagene Company (the experimental operation was in accordance with t...

Embodiment 2

[0074] Example 2 Construction of plant expression vectors pc30SNP and pc30SNPr

[0075] 1.1 Strain transformation and plasmid extraction

[0076] Transform Escherichia coli XL1 strain with 1 μL of plasmid pBI121 (commercially purchased, activated and preserved by Key Laboratory of Sugarcane Physiology, Ecology and Genetic Improvement, Ministry of Agriculture) and pUCP (cloning vector containing sugarcane PEPC gene cloned in Example 1 above) to pick a single colony Expand the culture and extract and purify a large number of plasmids.

[0077] 1.2 Construction of intermediate expression vector pC30SN

[0078] Since the restriction sites at both ends of the pepc gene did not match those of the plant expression vector, the intermediate expression vector pC30SN with CaMV35S promoter and Nos terminator was constructed to facilitate the cloning of the pepc gene. The construction process is as follows: insert the CaMV35S promoter on pBI121 (commercially purchased by CLONTECH Company...

Embodiment 3

[0126] Example 3 pC30SNP, pC30SNPr into Agrobacterium

[0127] Using the direct transformation method, pC30SNP and pC30SNPr were respectively introduced into Agrobacterium LBA4404 and A281 (Invitron, commercially purchased from USA, activated and preserved by the Key Laboratory of Sugarcane Physiology, Ecology and Genetic Improvement of the Ministry of Agriculture), and the pepc gene was used as a probe, labeled with DIG, to detect pepc Whether the gene is introduced into Agrobacterium.

[0128] 1.1 Direct conversion method

[0129] 1.1.1 Preparation of Agrobacterium Competent Cells

[0130] 1) Agrobacterium LBA4404 (Str + , Rif + ), EHA105 (Str + , Rif + ), A281 (Str + , Rif + , Nal + ) single colony was inoculated in 5 mL of YEP liquid medium supplemented with corresponding antibiotics, cultured overnight at 28°C and 250 r / m.

[0131] 2) Inoculate 1 mL of each overnight culture into 20 mL of antibiotic YEP (Str 25 mg / L, Rif 25 mg / L, A281 plus Nal 40 mg / L), and conti...

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Abstract

The invention relates to the nucleic acid molecule and method for improving C3 plant photosynthesis by utilizing transgenic technology, wherein the nucleic acid molecule is the nucleic acid molecule which encodes sugar cane phosphoenolpyruvate pyruvate carboxylase (PEPC), and the method consists of leading the enzyme-sugar cane phosphoenolpyruvate pyruvate carboxylase (PEPC) participating C4 photosynthesis channels into plants having C3 type photosynthesis channels such as rice, so as to provided them with C4 photosynthesis channels, and make them have higher efficiency of photosynthetic activity. The invention also relates to the expression vectors for converting the C3 plants, and the use of the method.

Description

technical field [0001] The present invention relates to a kind of coding sugarcane pepc gene cloning, high-efficiency expression carrier and the method for transforming plant thereof, especially relate to the method that will participate in C 4 Enzyme of photosynthetic pathway—sugarcane phosphoenolpyruvate carboxylase (PEPC) gene introduced into C 3 Type photosynthetic pathway plants thus providing C 4 loop method. Background technique [0002] Three types of photosynthetic pathways are known to exist in higher plants, namely C 3 , C 4 and CAM type. have C 4 Type photosynthetic pathway plants (hereinafter sometimes referred to as C 4 The leaf tissues of plants) contain mesophyll cells and bundle sheath cells present around the vascular bundles, forming a special leaf tissue structure called Kranz-type anatomy. C 4 Plants use the action of phosphoenolpyruvate carboxylase (hereinafter sometimes referred to as PEPC) located in the cytoplasm of mesophyll cells to fix car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52
Inventor 张木清陈如凯周会
Owner FUJIAN AGRI & FORESTRY UNIV
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