Genetic Control of Axillary Bud Growth in Tobacco Plants
A technology of tobacco plants and expression vectors, which is applied in the field of tobacco plants and can solve the problems of unrealized inhibition and expensive chemicals
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0116] Example 1: Sampling, RNA preparation and sequencing
[0117] Tobacco seeds from TN90 (Burley variety) were germinated. After 4 weeks, transfer the seedlings to 4-inch pots. At the layby stage (8-10 fully expanded leaves), a total of 10 different samples were collected, including axillary buds before truncation (Aa), axillary buds after truncation (Ab, Ac, Ad and Ae ( 2h, 6h, 24h and 72h), roots before truncation (Ra), roots after truncation (Rb, Rc (24h and 72h)), young leaves at truncation (YL), and branch apical meristems (ST) for next-generation sequencing analysis. Each time point is represented by three independently collected samples. These three samples serve as biological replicates.
[0118]RNA from the above samples was isolated using the RNeasy Plant Mini Kit (Qiagen, MA) and tested for quality using the Agilent Plant RNA Nano Kit and 2100 Bioanalyzer (Agilent Technologies, CA). Thirty cDNA libraries were constructed and indexed using the TrueSeq RNA Libra...
Embodiment 2
[0119] Example 2: RNA sequence analysis
[0120] Gene expression data from 5 axillary buds, 3 roots and 1 each of young leaf and stem apical meristems were analyzed to identify genes associated with axillary bud development compared to other tissues. Gene reads were mapped to our internal tobacco encyclopedia (tobaccopedia) genome database (Table 1). EdgeR in the CLC Genome Workbench was used to implement differential gene expression. Gene expression data were filtered for axillary bud-specific expression from other tissues. All p-values were FDR adjusted and a cutoff of FDR-corrected p-values <0.05 was used. Then, filter the results for tall axillary bud expression. A list of differentially expressed candidate genes used for sucker controls is listed in Table 2.
[0121] Table 1: Mapping of next-generation sequencing reads using the internal Tobacco Encyclopedia database
[0122] sample Mapped reads %position sample Mapped reads %position Aa1...
Embodiment 3
[0125] Example 3: Confirmation of Expression of Selected Candidate Genes
[0126] To confirm the expression patterns of the selected candidate genes, measurements were made from 10-16 different tissue samples (6 axillary bud samples (2h, 6h, 12h, 24h and 72h before and after truncation), truncated Transcripts of young leaves, mature leaves, senescent leaves, midribs, stems before truncation, stems 24 hours after truncation, shoot apical meristem, roots before truncation and 24 hours after truncation relative change. Briefly, total RNA was isolated using TRI reagent (Sigma-Aldritch, St. Louis, Mo). To remove DNA impurities, purified RNA was treated with RNase-free DNase (Turbo DNA Free, Ambion, Austin, TX). For first-strand cDNA synthesis, approximately 10 μg of total RNA was transcribed using the High Capacity cDNA Kit (Applied Biosystems, Foster City, CA). To measure the levels of selected gene transcripts in samples, RT-PCR was performed using SYBR GreenPCR Master Mix (Ap...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More