Method for transforming plant, the resultant plant and gene thereof
a technology of transforming plant and gene, applied in the field of transforming plant, the resultant plant and gene thereof, can solve the problems of insufficient other problems, and achieve the effect of reducing the expression of lime chlorosis
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
example 1
Extraction of Total RNA from FRE1 Introduced Transgenic Tobacco
[0179] Extraction of total RNA from FRE1 introduced transgenic tobacco was performed according to a method described in the reference (Naito et al., 1988).
[0180] Leaves 2 g of FRE1 introduced transgenic tobacco were put in the mortar, and liquid nitrogen was added thereto, then leaves were completely mashed. Three fold amounts of buffer for extraction and equal amount of phenol / chloroform (1:1) were added to the debris and suspended, then centrifuged at 8000 rpm for 15 minutes, and extracted with chloroform once. Ethanol precipitation was conducted at -80.degree. C. for 30 minutes, and centrifuged at 4.degree. C. for 30 minutes. Precipitate was washed with 70% ethanol and dried in vacuum. The precipitate was dissolved in DEPC treated water 1 ml, centrifuged at 13500 rpm for 3 minutes, and the supernatant was transferred to a new tube, further 10 M LiCl, 1 / 4 volume, was added and allowed to stand on ice for 2 hours. The m...
example 2
Purification of Poly(A)+RNA and Synthesis of cDNA
[0185] Poly(A)+RNA was purified from total RNA 100 .mu.g obtained in example 1 by applying with Dynabeads Oligo (dT) 25 (DYNAL Inc.). This poly(A)+RNA was treated with reverse transcription reaction by M-MLV reverse transcriptase (TOYOBO Co. Ltd.) at 37.degree. C. for 1 hour using the following hybrid primer to obtain cDNA.
[0186] Hybrid primer (dT.sup.17 adapter primer):
1 5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'
example 3
RT-PCR and Confirmation of Base Sequence
[0187] PCR was conducted with the primer specific to hybrid primer and the 5' primer of FRE1 using cDNA obtained in example 2 as a template.
[0188] Reaction product of PCR was electrophoresed with 0.8% agarose gel, and the obtained band was cloned to pT7Blue(R) vector (Takara Corp.). Colony was shake cultured in LB medium for overnight, extracted the plasmid by alkaline-SDS method, and the base sequence of 7 clones, to which the insertion was confirmed by restriction enzyme treatment, was determined by using Bca BEST DNA polymerase ("Biotechnology Experiments Illustrated, Fundamentals of gene analysis") (Shujun-Sha).
2 Primer specific to hybrid primer: 5'-GACTCGAGTCGACATCG-3' 5' primer of FRE1: 5'-ACACTTATTAGCACTTCATGTATT-3'
[0189] Reaction condition for PCR:
[0190] (1) 95.degree. C. for 5 minutes;
[0191] (2) 95.degree. C. for 40 seconds;
[0192] (3) 55.degree. C. for 30 seconds;
[0193] (4) 72.degree. C. for 1 minute;
[0194] (5) 72.degree. C. for 10 mi...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Volume | aaaaa | aaaaa |
| Molar density | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More