ntferl Gene Affecting Tobacco Pigment Content and Its Application
A technology of pigment content and tobacco, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of tobacco leaf green miscellaneous gas, unfavorable chemical components, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0040] This embodiment mainly affects the tobacco pigment content NtFERL The process of gene acquisition is briefly introduced as follows:
[0041] (1) Tobacco RNA extraction and cDNA synthesis
[0042] For RNA extraction, please refer to the following steps:
[0043]Take the 4-week-old young leaves of cultivated tobacco K326 as samples, and after being fully ground into powder with liquid nitrogen, take about 100 mg of powdered material and place it in a 1.5 ml centrifuge tube filled with 1.0 ml of TRIZOL reagent, add 200 μL of chloroform; oscillate to mix, and after centrifugation, carefully remove the upper aqueous phase and transfer to another centrifuge tube; add 500 μL of isopropanol, precipitate, centrifuge to separate RNA, wash with 75% alcohol, and dry at room temperature. Add an appropriate volume of RNase free water to fully dissolve;
[0044] The extracted total RNA was treated with DNase I, and the 10 μL digestion reaction system was designed as follows:
[00...
Embodiment 2
[0078] Utilize the obtained in embodiment 1 to influence tobacco pigment content NtFERL gene, the inventor further constructed the gene silencing RNAi interference vector TRV2- NtFERL , the relevant construction process is briefly introduced as follows.
[0079] A relatively specific nucleic acid fragment in this gene (nucleotide sequence 2017-2405 in the sequence table SEQ ID NO.1) was selected as the guide sequence of VIGS, and primers were designed to obtain this sequence. This nucleic acid fragment is then inserted into the VIGS vector to construct TRV2- NtFERL Carrier, the specific construction process is:
[0080] (1) Obtain the target fragment, select a more specific nucleic acid fragment in this gene (nucleotide sequence 2017-2405 in the sequence table SEQ ID NO.1) as the guide sequence of VIGS, design primers and obtain them as in Implementation 1 The full length of the gene was used as the template for PCR amplification to obtain the target gene fragment. The prim...
Embodiment 3
[0105] Using the VIGS silencing vector TRV2- constructed in Example 2NtFERL Taking Nicotiana benthamiana as an example, the inventor further carried out a cultivation test to silence the factors that affect the tobacco pigment content in the plant. NtFERL Genes and related experimental procedures are briefly introduced as follows.
[0106] (1) Tobacco seeds were sown in seedling pots to grow seedlings, and seedlings were divided two weeks after germination, planted in plastic pots (10cm×10cm), and daily fertilizer and water management was carried out at 22°C under 16 h light / 8 h dark conditions etc.; grow for 4 weeks, select seedlings with consistent growth and group into groups for subsequent use;
[0107] (2) will contain TRV1, TRV2, TRV2- NtFERL , TRV2- PDS Agrobacterium single colony (other plasmid construction process can refer to the above construction process) into 5 mL of LB medium (containing 50 mg / L kanamycin, 50 mg / L rifampicin), 28 ℃, 180 r / min Overnight shak...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More