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Gateway inlet vector pEn-L4*-PrbcS-*T-GFP-L3*, construction method thereof and application thereof

A T-GFP-L3, T-GFP technology, applied in the field of molecular biology, can solve the problem of no chloroplast transfer peptide sequence and so on

Inactive Publication Date: 2010-09-15
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, in all entry cloning vectors containing attL4-gene2-attL3 fragments, there are very few restriction sites for suitable gene subcloning after attL4, and there is no chloroplast transfer peptide sequence behind the CaMV35S promoter, so the existing technology cannot be directly used at present. Use this entry cloning vector and the target vector pK7m34G2-8m21GW3 to construct a plant expression vector that is suitable for chloroplast genetic engineering and can express two target genes

Method used

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  • Gateway inlet vector pEn-L4*-PrbcS-*T-GFP-L3*, construction method thereof and application thereof
  • Gateway inlet vector pEn-L4*-PrbcS-*T-GFP-L3*, construction method thereof and application thereof
  • Gateway inlet vector pEn-L4*-PrbcS-*T-GFP-L3*, construction method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Introducing the HindIII site into the entry vector pEN-L4-2-L3 using point mutation technology

[0048] Using the purified plasmid pEN-L4-2-L3 (purchased from VIB / Gent, Belgium) as a template, a pair of complementary primers HindIII5 and HindIII3 ( figure 1 ), add 25ng of purified plasmid pEN-L4-2-L3 as a template in the point mutation reaction mixture, add 50ng of point mutation primers HindIII5 and HindIII3, 1μl dNTP (10mM), 5μl of 10×LongTaq reaction buffer and 1μl Long Taq polymerase (Tiangen Biochemical Technology), and double distilled water was added to make the final volume of the reaction 50 μl. Heat at 94°C for 3 minutes on the PCR instrument, then perform 18 cycles of reaction according to the program of 94°C for 30 seconds, 50°C for 30 seconds, 72°C for 3 minutes and 40 seconds, and finally extend the reaction at 72°C for 10 minutes. Synthesize the daughter chain containing the mutation site ( figure 2 A). After the reaction was completed, the...

Embodiment 2

[0049] Example 2: Using point mutation technology to change the PstI in the attL3 downstream multiple cloning site of the entry vector pEN-L4*-2-L3 to XhoI

[0050] Using the plasmid pEN-L4*-2-L3 as a template, a pair of (XhoI5 and XhoI3) complementary primers (XhoI5 and XhoI3) for point mutations were designed according to the sequence near the PstI site ( image 3 ), and entrusted Shanghai Sangong Synthetic Co., Ltd. Add 25ng of purified plasmid pEN-L4*-2-L3 to the point mutation reaction mixture as a template, and at the same time add 50ng of point mutation primers XhoI5 and XhoI3, 1μldNTP (10mM), 5μl of 10×Long Taq reaction buffer and 1μl of Long Taq polymerase (Tiangen Biochemical Technology), and double distilled water were added to make the final volume of the reaction 50 μl. Heated at 94°C for 3 minutes on a PCR instrument, followed by 18 cycles of reaction at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 4 minutes, and finally extended the reaction at 72°C f...

Embodiment 3

[0051] Example 3: Construction of entry vector pEN-L4*-PrbcS-*T-GFP-L3*

[0052] Use HindIII and XhoI to cut pEN-L4*-2-L3* and pENTR*-PrbcS-*T-GFP (pENTR*-PrbcS-*T-GFP construction method see another patent application of the applicant, application No. 200710066422.9), separated the cut vector and insert fragment by agarose gel electrophoresis, recovered the vector fragment pEN-L4*-L3* (2.6kb) and pENTR*-PrbcS-*T-GFP was cleaved to generate a PrbcS-*T-GFP DNA fragment (2.4kb), and then pEN-L4*-L3* and PrbcS-*T were combined with a ligase kit from TaKaRa -GFP was ligated to obtain the entry vector pEN-L4*-PrbcS-*T-GFP-L3*( Figure 5 ). Conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with kanamycin (Kan, 50 μg / ml), cultivate overnight at 37°C, and screen for Km resistance. Recombinant colonies of resistant recombinants, plasmids were extracted from colonies...

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Abstract

The invention relates to a gateway inlet vector for the gateway technology, and in particular to a gateway inlet plasmid vector (pEn-L4*-PrbcS-*T-GFP-L3*). The vector comprises sequences L4 and L3, rubisco, a small subunit gene photo-inductive promoter (PrbcS), a chloroplast stroma positioning sequence (T*) and a green fluorescent protein (GFP) reporter gene, which are required for the LR recombination reaction in the gateway technology. A construction method and application of the vector are that: one plant expression vector connected in series with two target gene expression boxes can be quickly constructed by using the vector and another gateway inlet vector containing L1 and L2 sequences through the LR recombination reaction so as to realize conversion operation of two target genes through one conversion event. The expression of the target gene can be controlled with the PrbcS promoter in the vector and high-level expression of the target gene in the leaves of a plant can be realized by substituting the GFP gene in the vector with the target gene.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and relates to an entry vector for pathway cloning (Gateway) technology, in particular containing L4 and L3 sequences, light-inducible promoter (PrbcS) and chloroplast matrix positioning required for LR recombination reaction of pathway cloning technology Sequence (T*) and the Gateway entry vector of the reporter gene GFP, the present invention also relates to the construction and application of the entry vector. Background technique: [0002] Although many of the classic binary vectors used in Agrobacterium-mediated genetic transformation experiments contain chloroplast transfer peptide sequences, due to the large molecular weight of such vectors (greater than 10kb), it is difficult to pass classical restriction enzyme digestion / The ligation method and the molecular manipulation of subcloning the target gene into these binary vectors are all quite difficult. In addition, some situations that ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N15/82
Inventor 陈丽梅肖素勤王莎莎梁峰李莉孙振严金平李昆志
Owner KUNMING UNIV OF SCI & TECH
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