Glehnia littoralis phospholipase gene promoter and application thereof
A technology of promoter and vegetable phospholipid, applied in application, genetic engineering, hydrolase and other directions, can solve the problems of amplifying gene function, functional redundancy, affecting plant growth and development, etc.
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Embodiment 1
[0024] Embodiment 1: the acquisition of the promotor of the coral dish phospholipase gene
[0025] 1. Use the CTAB method to extract the genomic DNA of Anthophyllum (the leaves of Anchophylla were collected from the Germplasm Resource Garden of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province in May 2020).
[0026] 2. Design and synthesize the following specific primers according to the sequence of Corona phospholipase:
[0027] SP1: 5'-TCTGTTGCACCATGAGACGTAGCC-3' (SEQ ID NO. 3):
[0028] SP2: 5'-GGAAAACCACCTGTCACAAACCGC-3' (SEQ ID NO.4);
[0029] SP3: 5'-TCAGCGAATCCTTCCATTGTCGGG-3' (SEQ ID NO. 5).
[0030] Genome DNA of Corona genus was used as a template, and thermal asymmetric PCR reaction was carried out with the degenerated primer AP3 in the chromosome walking kit (Genome Walking, Takara, Dalian). The flanking sequences were obtained by nested PCR three times, and the PCR products were recovered and DNA sequencing. The specific operation steps an...
Embodiment 2
[0032] Example 2: Construction of the pCAMBIA1301 plant recombinant expression vector containing the antisense sequence of the above-mentioned promoter
[0033] According to the antisense sequence and pCAMBIA1301 carrier map of the Anchophylla phospholipase gene promoter obtained in Example 1, select the HindIII and BglII double restriction sites, and use the Anchophylla genomic DNA as a template, and design primers to carry out PCR amplification, double restriction digestion, Carrier connection. The ligation product was transferred into Escherichia coli competent cells, cultured overnight at 37°C on a plate of 50 mg / L kana antibiotics, and a single positive colony was picked for plasmid sequencing verification. The obtained recombinant vector is to replace the 35S promoter on the pCAMBIA1301 vector with the antisense sequence (SEQ ID NO.2). After the replacement, the β-glucuronidase gene (GUS) is expressed by the above antisense sequence. figure 2 shown.
Embodiment 3
[0034] Example 3: Using the above-mentioned plant recombinant expression vector to express foreign protein in Arabidopsis
[0035] The recombinant vector constructed in Example 2 was transformed into Agrobacterium GV3101, and the flowers were dipped in method [Clough, S.J.andBent, A.F. (1998) Floral dip: A simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana.Plant Journal, 16,735 -743] transformed Arabidopsis, the seeds of which were grown on the MS plate containing 50mg / L of hygromycin for one week, and the plants that could grow normally were transgenic positive plants, and the genomic DNA of wild-type and transgenic plants was extracted for further PCR identification, the seeds of transgenic plants identified as positive by PCR continued to be screened, selfed and propagated to obtain T3 homozygous lines, and the homozygous lines were used for β-glucuronidase (β-glucuronidase, GUS) staining observation . The results are attached image 3 Show...
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