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A kind of multi-gene assembly vector system and its multi-gene assembly method

A vector system and multi-gene technology, applied in the field of multi-gene assembly vector system and its multi-gene assembly, can solve the problems of difficulty in use, cumbersome complexity, difficulty and the like

Active Publication Date: 2021-02-19
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods are still difficult, time-consuming and labor-intensive to use in multi-gene assembly, and the efficiency is not high
For example, the MISSA multi-gene system requires the use of two recombination systems, Cre / loxP and Gateway, and the complex method of bacterial mating-assisted transfer to achieve gene transfer and fusion between two strains with a donor carrier and a recipient carrier, which is cumbersome and complicated; The multigene vector system established by Cre / loxP and homing endonuclease (Lin et al., 2003; ZL02134869.3) is much simpler. This system uses Cre / loxP to recombine and integrate the donor vector plasmid into the acceptor vector, and then Use a homing enzyme with low cutting efficiency to cut off the backbone of the supply vector, and connect it into the vector with a double-stranded DNA adapter, but this also makes the entire operation more difficult and less efficient in connecting multiple genes

Method used

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  • A kind of multi-gene assembly vector system and its multi-gene assembly method
  • A kind of multi-gene assembly vector system and its multi-gene assembly method
  • A kind of multi-gene assembly vector system and its multi-gene assembly method

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Experimental program
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Effect test

Embodiment 1

[0097] Construction of acceptance vector

[0098] S1. Construction of acceptance vectors pYL0380 and pYL1300H

[0099] Such as Figure 5 As shown in A, the sequence (EcoR I / Sac I / Kpn I / I-Ceu I / I-Ceu I / BamH I / PI-Psp I / Sal I / Pst I / loxP / I-Sce I / loxP1R / I-Ppo I / Hind III, image 3 B, this sequence is as shown in SEQ ID NO.4), after EcoR I and Hind III double enzyme digestion, connect into pCAMBIA0380 and pCAMBIA1300 plasmid (Australian CAMBIA company) through EcoR I and Hind III double enzyme digestion, obtain accepting vector pYL0380 and acceptor vector pYL1300H.

[0100] S2. Construction of acceptance vector pYLTAC380

[0101] Such as Figure 5 As shown in B, the TAC vector pYLTAC747 plasmid (disclosed in the literature "Lin et al., 2003. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci., 100:5962-5967") is Template, carry out PCR amplification with primer P1 / P2 (primer 5' end contains XbaI site), ...

Embodiment 2

[0107] Construction of supply vector I and supply vector II

[0108] S1. Construction of supply vector I plasmid pYL322d1

[0109] Such as Figure 6 As shown in A, use primers P7 / P8 (the 5' ends of the primers contain BssH II / EcoR V sites and Mlu I sites respectively) and P9 / P10 (the 5' ends of the primers contain BssHII and Mlu I sites respectively), and use Figure 5 The pYL1300H and pYLSV plasmids constructed in A (disclosed in the literature "Lin et al., 2003. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci., 100:5962-5967") were used as templates Perform PCR amplification to obtain BssHII / EcoR V / pBR322 replicon / Mlu I fragment and BssHII / Cm r / Mlu I fragment (insert fragment I). The intermediate vector d1-I was obtained by double digestion with BssH II and Mlu I and ligation; the commercially synthesized fragment containing BssH II site BssH II / loxP / PI-Sce I / loxP2R / MCS / loxP1L / I-Sce I / BssH II...

Embodiment 3

[0113] Construction of marker-free supply plasmid

[0114] Such as Figure 7 As shown, use long primers P15 / P16 (the 5' end of the primers contain Xba I / Asc I / attB1 sites and HindIII / Xba I / attB2 sites respectively), and use the supply vector II plasmid pYL322d2 as a template for PCR amplification to obtain Xba I / Asc I / attB1 / pBR322 Replicon / Amp r The backbone fragment of / attB2 / HindIII / Xba I was cut and self-ligated with Xba I to obtain the intermediate vector pYLMF-0.

[0115] Using the DNA of rice variety Zhonghua 11 as a template, use primers P17 / P18 to amplify the promoter PV4 of the anther-specific expression gene Villin 4 (gene number Os04g0604000) as fragment 1, and use primers P19 / P20 to use Escherichia coli NS3529 genomic DNA as a template Amplify the Cre gene coding region (sequence is the same as GenBank No.DQ340306) to obtain fragment 2, use primers P21 / P22 to amplify the Nos terminator Tnos from plasmid pCAMBIA1305 (CAMBIA company) as fragment 3, and use the abov...

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Abstract

The invention discloses a multigene assembly carrier system and a multigene assembly method thereof. The system includes a type 1 acceptor vector, a type 2 donor vector, and an optional donor plasmid capable of self-deleting selection markers. The system utilizes the Cre / loxP recombination system and two sets of irreversible recombination sites of mutant loxP. Under the action of Cre enzyme, the recombination of wild-type loxP sites is used to realize the integration of the donor vector into the acceptor vector, and then a set of mutant loxP The irreversible recombination of loxP automatically deletes the backbone of the donor vector, leaving only the target gene on the acceptor carrier; repeating the assembly of the two types of donor vectors and acceptor vectors alternately can realize the rapid assembly of multiple genes. The present invention is the most simple and efficient multigene carrier system at present. Therefore, the invention provides an operable technical platform for the multi-gene genetic engineering operation of important and complex biosynthetic pathways and important agronomic traits, and has important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multi-gene assembly carrier system and a multi-gene assembly method thereof. Background technique [0002] Important traits in plants (crops), such as many agronomic traits, complex metabolic synthesis pathways, important signal transduction pathways, and protein complexes with important functions, all involve the interaction of multiple genes and are controlled by multiple genes. Need to use the multi-gene transformation stacking technology (TransGeneStacking, TGS). However, the conventional transgenic technology can only complete the operation of a small number of (usually 1-3) genes. "Golden Rice" and "Purple Tomato" are two successful cases of multi-gene transformation crops reported so far, but they only use 2 genes. Therefore, multi-gene assembly and transformation technology is a hot and difficult point in genetic engineering research, and has important application prospec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66
CPCC12N15/63C12N15/66C12N2800/30
Inventor 刘耀光祝钦泷
Owner SOUTH CHINA AGRI UNIV
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