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Method for rapidly identifying bidirectional promoter of rice

A bidirectional promoter and rice technology, applied in the biological field, achieves the effect of short time-consuming, simple operation, and shortened experiment time

Inactive Publication Date: 2020-07-10
YANGZHOU UNIV
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Problems solved by technology

However, in the transient transformation system of protoplasts, whether the bidirectional promoter can simultaneously promote the expression of genes on both sides has not been reported yet.

Method used

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  • Method for rapidly identifying bidirectional promoter of rice
  • Method for rapidly identifying bidirectional promoter of rice
  • Method for rapidly identifying bidirectional promoter of rice

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Experimental program
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Embodiment Construction

[0024] The identification method of this embodiment is as follows:

[0025] 1. Positive control vector: Use the pCambia1301 vector as a template to amplify the Ubiquitin promoter; use the vector pGWB454 as a template to amplify the RFP gene; use restriction enzymes NcoI and EcoRI to linearize the p1300LV vector, and use a seamless ligation kit The above three fragments were quickly connected, transformed into Escherichia coli DH5α, and positive transformants were screened. Obtain the positive control vector GFP-RFP reporter (such as figure 2 shown).

[0026] 2. Negative control vector: Use restriction enzymes KpnI and BamHI to linearize the GFP-RFP reporter vector, insert two mini35s or a non-functional sequence into the linearized vector, transform it into E. coli DH5α after ligation, and screen for positive transformation son. Obtain 2 negative control vectors mini35s reporter and promoterless reporter (such as figure 1 shown).

[0027] 3. Selection of candidate bidir...

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Abstract

The invention discloses a method for rapidly identifying a bidirectional promoter of rice. The method comprises the following steps: connecting two ends of a bidirectional promoter OsBDP1 with two different fluorescent proteins GFP and RFP respectively, connecting two ends of a bidirectional promoter OsBDP2 with two different fluorescent proteins GFP and RFP respectively, and observing fluorescentsignals at two sides after protoplast transformation to detect activity of the bidirectional promoters. According to the identification method, on one hand, the experimental time is shortened by utilizing transient transformation, and on the other hand, the workload is reduced by simultaneously detecting activity of fluorescent proteins at two sides, so that an efficient way is provided for rapidly identifying the bidirectional promoter in rice.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for quickly identifying rice bidirectional promoters. Background technique [0002] A bidirectional promoter refers to a DNA sequence that is located between the two TSSs of a pair of "head-to-head" genes, is rich in high GC and generally does not exceed 1.0Kb in length, and can drive the expression of downstream genes of the sense strand and the antisense strand, such as figure 1 shown. In the field of genetic engineering, the bidirectional promoter is used to control gene transcription in two directions, and two different target genes are connected at its two ends. This not only reduces the difficulty of constructing the vector, but also reduces the introduction of transcriptional regulatory elements, avoiding the transgene "co-suppression" phenomenon that may be caused by the existence of homologous sequences between the regulatory elements. Therefore, bidirectional promoters can be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66
CPCC12N15/66C12N15/82
Inventor 刘鹏李鑫琪张韬
Owner YANGZHOU UNIV
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