Construction of CRISPR-Cas9 system of tomato PSY 1 gene and application thereof

Abstract

The invention belongs to the field of gene engineering and genetic modification and relates to construction of a CRISPR-Cas9 system of a tomato PSY 1 gene and an application thereof. The construction includes the following steps: (1) according to a cDNA conserved sequence of the tomato PSY 1 gene, designing a DNA sequence of sgRNA, which is represented as the SEQ ID No.1; (2) designing a primer combination used for amplifying a tomato PSY 1 gene target zone in the sgRNA; and (3) performing sgRNA amplification with the primer and purifying an amplified product to obtain a CRISPR-Cas9 Level-1 carrier and a CRISPR-Cas9 Level-2 carrier, which contain the sgRNA of the specific targeted tomato PSY-1 gene. The construction is beneficial to researching on effect that the gene participates in fruit maturity of tomatoes, so that the molecular mechanism of the fruit maturity of tomatoes can be known furthermore. The system can be used for producing PSY 1 gene deleted transgenic tomatoes, and has important effect of culturing a new tomato variety which is tolerant to storage and transportation in future.

Description

technical field

[0001] The invention belongs to the field of genetic engineering and genetic modification, in particular, relates to a tomato PSY 1 Gene CRISPR-Cas9 system construction and its application. Background technique

[0002] tomato( Solanum lycopersicum Mill), also known as tomato, foreign persimmon, tomato, June persimmon, maola fruit, etc., belongs to the genus Tomato of the family Solanaceae and is native to South America. Tomato is currently the most widely cultivated and consumed vegetable crop in the world, and it is also one of the main types of vegetables grown in my country. Lycopene is a natural carotenoid synthesized by plants, which is widely found in vegetables such as tomatoes, peppers, carrots, and pumpkins. It has the highest content in tomato, with a fresh weight content of 0.2-20mg per 100g. Lycopene is an isomer of carotene, synthesized through the isoprene metabolic pathway, and can be converted into delta-carotene, gamma-carotene, alpha-...

Examples

Embodiment 1

[0028] Example 1: tomato PSY 1 sgRNA design for gene CRISPR-Cas9

[0029] 1. Find the PAM (proto adjacent motif) motif

[0030] exist PSY 1 The PAM motif, namely NGG, was found in the first exon region of the gene sequence.

[0031] 2. Determine the sgRNA sequence

[0032] The 20 bp sequence at the 5' end of the PAM position is the sgRNA sequence. Generally, the U6 promoter is used in tomato CRISPR-Cas9 to transcribe the sgRNA, so the 20 bases at the 5' end of NGG are preferentially G, that is, G(N) 19 NGG. tomato PSY 1 The sgRNA sequence of the first exon region of the gene is shown in SEQ ID NO.1.

[0033] 3. sgRNA primer synthesis

[0034] The sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0035] 4. sgRNA amplification reaction system

[0036] Add deionized water to the synthesized primers to make the concentration 10 μM, and then prepare the sgRNA amplification reaction system as follows:

[0037]

[0038] 5. sgRNA amplification reaction c...

Embodiment 2

[0042] Example 2: Tomato PSY 1 gene sgRNA connected to CRISPR-Cas9 Level 1 carrier

[0043] 1. Purification of sgRNA amplification products

[0044] Using GenElut TM PCR Clean-Up Kit (SIGMA) was used to purify PCR products.

[0045] 2. CRISPR-Cas9 Level 1 ligation reaction system

[0046]

[0047] 3. CRISPR-Cas9 Level 1 ligation reaction conditions

[0048]

[0049] 4. Transform the ligation product into E. coli

[0050] Take 40 μL of Escherichia coli DH5α (DH8142) competent cells and mix with 5 μL of Level 1 ligation product, and put them in ice bath for 15 min, then in 42°C water bath for 45 s, and then in ice bath for 5 min. Then add 600 μL of LB medium, shake and culture at 37°C for 1.5 h. Pipette 100 μL of culture solution and spread evenly on LB plate (containing 100 mg·L -1 Xgal and 100 mg·L -1 Carbenicilin) ​​and incubated overnight at 37°C.

[0051] 5. Select monoclonal shaking bacteria

[0052] Select the white monoclonal colony on the LB plate and pu...

Embodiment 3

[0058] Example 3 Tomato PSY 1 gene sgRNA connected to CRISPR-Cas9 Level 2 vector

[0059] 1. CRISPR-Cas9 Level 2 ligation reaction system

[0060]

[0061] 2. CRISPR-Cas9 Level 2 ligation reaction conditions

[0062]

[0063] 3. Transform the ligation product into E. coli

[0064] Take 40 μL of Escherichia coli DH5α (DH8142) competent cells and mix them with 5 μL of Level 2 ligation product, and put them in an ice bath for 15 min, then in a 42°C water bath for 45 s, and then in an ice bath for 5 min. Then add 600 μL of LB medium, shake and culture at 37°C for 2 h. Pipette 100 μL of culture solution and spread evenly on LB plate (containing 100 mg·L -1 Kanamycin) at 37°C overnight.

[0065] 4. Select monoclonal shaking bacteria

[0066] On the LB plate, pick the white monoclonal into 10 mL LB medium (containing 100 mg·L -1 Kanamycin), cultured overnight at 37°C. 3shown. PCR program: 98°C, 30 s; 94°C, 10 s; 60°C, 30 s; 72°C, 30 s; 35 cycles; 72°C, 2min; 4°C, Hold. ...

technical field

[0001] The invention belongs to the field of genetic engineering and genetic modification, in particular, relates to a tomato PSY 1 Gene CRISPR-Cas9 system construction and its application. Background technique

[0002] tomato( Solanum lycopersicum Mill), also known as tomato, foreign persimmon, tomato, June persimmon, maola fruit, etc., belongs to the genus Tomato of the family Solanaceae and is native to South America. Tomato is currently the most widely cultivated and consumed vegetable crop in the world, and it is also one of the main types of vegetables grown in my country. Lycopene is a natural carotenoid synthesized by plants, which is widely found in vegetables such as tomatoes, peppers, carrots, and pumpkins. It has the highest content in tomato, with a fresh weight content of 0.2-20mg per 100g. Lycopene is an isomer of carotene, synthesized through the isoprene metabolic pathway, and can be converted into delta-carotene, gamma-carotene, alpha-...

Examples

Embodiment 1

[0028] Example 1: tomato PSY 1 sgRNA design for gene CRISPR-Cas9

[0029] 1. Find the PAM (proto adjacent motif) motif

[0030] exist PSY 1 The PAM motif, namely NGG, was found in the first exon region of the gene sequence.

[0031] 2. Determine the sgRNA sequence

[0032] The 20 bp sequence at the 5' end of the PAM position is the sgRNA sequence. Generally, the U6 promoter is used in tomato CRISPR-Cas9 to transcribe the sgRNA, so the 20 bases at the 5' end of NGG are preferentially G, that is, G(N) 19 NGG. tomato PSY 1 The sgRNA sequence of the first exon region of the gene is shown in SEQ ID NO.1.

[0033] 3. sgRNA primer synthesis

[0034] The sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0035] 4. sgRNA amplification reaction system

[0036] Add deionized water to the synthesized primers to make the concentration 10 μM, and then prepare the sgRNA amplification reaction system as follows:

[0037]

[0038] 5. sgRNA amplification reaction c...

Embodiment 2

[0042] Example 2: Tomato PSY 1 gene sgRNA connected to CRISPR-Cas9 Level 1 carrier

[0043] 1. Purification of sgRNA amplification products

[0044] Using GenElut TM PCR Clean-Up Kit (SIGMA) was used to purify PCR products.

[0045] 2. CRISPR-Cas9 Level 1 ligation reaction system

[0046]

[0047] 3. CRISPR-Cas9 Level 1 ligation reaction conditions

[0048]

[0049] 4. Transform the ligation product into E. coli

[0050] Take 40 μL of Escherichia coli DH5α (DH8142) competent cells and mix with 5 μL of Level 1 ligation product, and put them in ice bath for 15 min, then in 42°C water bath for 45 s, and then in ice bath for 5 min. Then add 600 μL of LB medium, shake and culture at 37°C for 1.5 h. Pipette 100 μL of culture solution and spread evenly on LB plate (containing 100 mg·L -1 Xgal and 100 mg·L -1 Carbenicilin) ​​and incubated overnight at 37°C.

[0051] 5. Select monoclonal shaking bacteria

[0052] Select the white monoclonal colony on the LB plate and pu...

Embodiment 3

[0058] Example 3 Tomato PSY 1 gene sgRNA connected to CRISPR-Cas9 Level 2 vector

[0059] 1. CRISPR-Cas9 Level 2 ligation reaction system

[0060]

[0061] 2. CRISPR-Cas9 Level 2 ligation reaction conditions

[0062]

[0063] 3. Transform the ligation product into E. coli

[0064] Take 40 μL of Escherichia coli DH5α (DH8142) competent cells and mix them with 5 μL of Level 2 ligation product, and put them in an ice bath for 15 min, then in a 42°C water bath for 45 s, and then in an ice bath for 5 min. Then add 600 μL of LB medium, shake and culture at 37°C for 2 h. Pipette 100 μL of culture solution and spread evenly on LB plate (containing 100 mg·L -1 Kanamycin) at 37°C overnight.

[0065] 4. Select monoclonal shaking bacteria

[0066] On the LB plate, pick the white monoclonal into 10 mL LB medium (containing 100 mg·L -1 Kanamycin), cultured overnight at 37°C. 3shown. PCR program: 98°C, 30 s; 94°C, 10 s; 60°C, 30 s; 72°C, 30 s; 35 cycles; 72°C, 2min; 4°C, Hold. ...

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