Wheat salt-tolerant gene TaOPR and application thereof
A wheat salt tolerance gene and gene technology, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problem that the role of OPR genes has not yet been reported, and achieve the effect of improving salt tolerance.
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Embodiment 1
[0030] Example 1, Taopr Clone
[0031] 1.1 Extraction of wheat total rna
[0032] 1. Put the tissue material in the mantle of the liquid nitrogen pre -cold, and grind it into powder in the liquid nitrogen;
[0033] 2. Wait for liquid nitrogen to dry, and immediately transfer to a 2ml centrifugal tube. Add a Trizol extract of 1ml of Invitrogen for each 100mg of materials.Full division, room temperature for 5 minutes;
[0034] 3. Add 0.2ml chloroform (chloroform), mix it for 15 seconds, and place it for 10 minutes;
[0035] 4.4 ℃, 12000rpm centrifugal 15 minutes;
[0036] 5. Carefully suck the upper water phase with a pipette, add a new 1.5ml centrifugal tube, add 500 μL of isopropyl (1: 1 volume), fully mix, -20 ° C, precipitate 30min or overnight;
[0037] 6.4 ℃, 12000rpm centrifugal 10min, carefully discard the liquid liquid;
[0038] 7. RNA precipitation was washed with 75%ethanol with 1ml.4 ° C for 8000rpm centrifugal 10min collection;
[0039] 8. Repeat 75%ethanol to wash RNA ...
Embodiment 2
[0093] Examples 2. Common nuclear expression analysis
[0094] 2.1 Construction of the Eukary Election Carrier
[0095] The expression carrier used is PET32A, and the receptor bacteria is de3.Choose Hin D III and ECO R the dual enzyme cutting on PET32A and PMD18 -T carrier containing destination genes, respectively, recover large segments and destination genes of the carrier, and use T 4 After the DNA connection is connected, the E. coli DH10B feels state cells. After identifying the reorganization, the primary expression carrier with a targeted gene is obtained, and then the receptor bacteria BL21 (DE3) feels for protein expression.
[0096] 1. Enzyme cutting of PET32A and PMD18 -T
[0097] The alkali cracking method extracts the plasmids of PET32A and PMD18 -T, and each takes 10ul for each Hin diii and ECO R Ⅰ dual enzyme cut.The plasmid extraction method is as follows, and the plasmid dual enzyme cutting system is as follows:
[0098] ECO Rs 1 μL
[0099] Hin DIII 1 μL
[010...
Embodiment 3
[0160] Example 3. Construction of plant expression carrier (35S promoter)
[0161] 3.1 35S boosted sub -plant expression carrier construction
[0162] Use plant expression vector PSTART, select SAC I and Bam H the dual enzyme cutting on PSTART and PMD18-T carrier containing destination genes, respectively, recover large segments and destination genes of the carrier, and use T 4 After the DNA connection is connected, the E. coli DH10B feels state cells. After identifying the reorganization, a plant expression carrier with a target gene is obtained.
[0163] (1) The plasmid PStart empty carrier and PMD18-T SAC I and Bam Hi dual enzyme cut
[0164] The alkaline crack extracts PStart empty carrier and PMD18-T plasmid, each takes 10 μg enzyme cutting, the enzyme cutting system is as follows:
[0165] Bam H -1 μL
[0166] SAC I 1 μL
[0167] PSTART carrier / PMD18-T plasmid 1 ~ 2 μL
[0168] 10 × Buffer K 1μL
[0169] DDH 2 O to add to 20 μL
[0170] Cut at 30 ° C constant temperature w...
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