Ovule-specific promoter pmei_d and its application
A specific promoter and promoter technology, applied in the field of plant genetic engineering, can solve problems such as limited promoters
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Embodiment 1
[0042] Example 1 Extraction and quantitative PCR analysis of each tissue RNA of upland cotton
[0043] The EASYspin plant RNA rapid extraction kit (Aidlab) was used to extract RNA from each tissue of cotton. According to the RevertAid First Strand cDNA Synthesis Kit (MBI) manual, reverse transcription of the first strand of cDNA was used as a template for quantitative RT-PCR analysis. The relative expression of the target gene was analyzed using iQ SYBR Green Supermix (BIO-RAD) reagent. The Ghhis3 gene (AF024716) of upland cotton was selected as the internal standard gene, and the primers were Ghhis1 and Ghhis2, as shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; GhPMEI gene quantitative PCR primers were, PMEI-1 and PMEI-2, They are respectively shown in SEQ ID NO.4 and SEQ ID NO.5. The amplification conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 30 s, and 40 cycles.
[0044] Calculate ...
Embodiment 2
[0045] Cloning of embodiment 2 upland cotton PMEI_D promoter and plant expression vector construction
[0046] Using the new plant genomic DNA rapid extraction kit of Adelaide Biological Company, the genomic DNA of upland cotton Jimian 14 was extracted according to the instructions, and the specific primers PMEI_D-up and PMEI_D-dn (SEQ ID NO .6 and 7), using the above-mentioned DNA as a template to amplify the promoter sequence. The amplification system is as follows: 10×PCRbuffer for KOD Plus 5μL, 25mmolMgSO 4 5μL, 2mmol / L dNTPs 2μL, primer PMEI_D-up (5μmol / L) 2μL, primer PMEI_D-dn (5μmol / L) 2μL, KOD Plus polymerase 1U / μL, upland cotton DNA about 60ng, double distilled water to make up to 50μL . The amplification program is: 94°C, 2min; 94°C, 15sec, 53°C, 30sec, 68°C, 2min, 35 cycles. After the amplification was completed, agarose gel electrophoresis was performed to recover the corresponding DNA bands, and the recovered products were stored in a -20°C refrigerator for la...
Embodiment 3
[0049] Embodiment 3 Genetic transformation of Arabidopsis thaliana and screening of transformants
[0050] Arabidopsis Genetic Transformation
[0051] (1) The genetic transformation of Arabidopsis thaliana was carried out by flower soaking method (Clough and Bent, 1998). The specific steps are as follows: Agrobacterium strains stored at -80°C were activated and cultured on a YEB solid medium plate containing corresponding antibiotics at 28°C for 36-48 hours; Cultivate in liquid YEB medium at 28°C and 200rpm on a shaker; inoculate an appropriate amount of activated bacterial liquid into 250mL of YEB liquid medium containing corresponding resistance, shake at 28°C and 200rpm until OD600=1.2-1.5; Centrifuge at 5000rpm for 10min to collect the cells; resuspend the cells in an equal volume of osmotic medium; soak the Arabidopsis flowers in full flowering stage with the resuspended bacteria solution for 1min; place the soaked Arabidopsis under dark conditions for 1d, and then Grow...
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