Gene transient expression method with in-vivo jujube fruits as materials

A transient expression and gene technology, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of low conversion rate, poor repeatability, time-consuming, etc., and achieve the effect of cost saving and rapid cost

Active Publication Date: 2020-09-04
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the defect of prior art, provide a kind of gene transient expression method that takes body jujube fruit as material, utilize this method to directly carry out functional veri...

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  • Gene transient expression method with in-vivo jujube fruits as materials
  • Gene transient expression method with in-vivo jujube fruits as materials
  • Gene transient expression method with in-vivo jujube fruits as materials

Examples

Experimental program
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Effect test

Embodiment 1

[0047] This example takes the transient expression of a candidate adenylyl cyclase gene as an example, and introduces a gene transient expression method using in vivo jujube fruit as a material, including the following steps.

[0048] Step 1, cloning the target gene to be verified: extract the RNA of jujube tissues and organs, obtain cDNA through reverse transcription as a template for PCR amplification, design primer A based on the sequence information of the target gene to be verified, and perform PCR amplification to obtain the target gene DNA.

[0049] When the sequence information of the target gene to be verified in step 1 is an unknown sequence and comes from the jujube whole genome database, the homologous cloning method is used to clone the target gene to be verified, and the specific operation is as follows.

[0050] Step 1-A: Take 1g of the fruit of Dongzaobanhong period, add liquid nitrogen and grind it into powder, use the polysaccharide and polyphenol plant total...

Embodiment 2

[0089] This example takes the transient expression of a candidate adenylyl cyclase gene as an example, and introduces a gene transient expression method using in vivo jujube fruit as a material, including the following steps.

[0090] Step 1, cloning the target gene to be verified: extract the RNA of jujube tissues and organs, obtain cDNA through reverse transcription as a template for PCR amplification, design primer A based on the sequence information of the target gene to be verified, and perform PCR amplification to obtain the target gene DNA.

[0091] When the sequence information of the target gene to be verified in Step 1 is a known sequence, and the sequence information is derived from the corrected sequence information of the target gene in Step 1-E of Example 1, the specific operation is as follows.

[0092] Step 1-a: Same as Step 1-A in Example 1.

[0093] Step 1-b: designing primer A based on the sequence information of the target gene;

[0094] The gene sequence...

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Abstract

The invention relates to a gene transient expression method with in-vivo jujube fruits as materials. The method comprises the following steps of performing homologous cloning according to sequence information in an NCBI database to obtain a target gene, integrating the target gene into a plant overexpression plasmid vector through enzyme digestion-connection, and transferring a recombinant plant vector into agrobacterium GV3101; carrying out shake culture on the agrobacterium in an LB culture medium at 28 DEG C until OD600 reaches 1.0-1.2, centrifuging and collecting bacteria, pouring out theLB culture medium, and resuspending thalli by using a penetrating fluid containing MgCl2, MES and acetosyringone for later use; injecting the agrobacterium penetrating fluid into the in-vivo jujube fruits in the half-red period by using a medical injector; and verifying the function of the target gene by observing the phenotypic changes of the jujube fruits and detecting the expression quantity ofthe target gene and the content of related metabolites. The method avoids the problems of poor repeatability, low conversion rate, long time and the like of stable genetic transformation of ziziphusjujuba mill, can verify the gene function in a short time, is simple and quick, and saves the cost.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and in particular relates to a gene transient expression method using in vivo jujube fruit as a material. Background technique [0002] jujube( Ziziphus jujuba Mill.) is Rhamnaceae (Rhamnaceae) jujube ( Ziziphus Mill.) plant, native to the middle and lower reaches of the Yellow River in my country, has been cultivated for more than 3,000 years. Since the reform and opening up, the jujube industry has developed rapidly and has become the largest dried fruit tree species in my country, accounting for 99% of the world's output. Jujube fruit is extremely nutritious. According to the measurement, the content of important therapeutic components such as cyclic adenosine monophosphate (cAMP), vitamin C, and carbohydrates in jujube fruit ranks among the top in all kinds of fruits. my country is in an international leading position in jujube research. In 2014, our research group took the lea...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/08A01H6/00C12Q1/6895
CPCC12N15/8205C12Q1/6895C12Q2600/158
Inventor 刘孟军刘志国袁野
Owner HEBEI AGRICULTURAL UNIV.
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