A kind of plant pollen-specific promoter psp1 and its application
A pollen-specific, plant pollen technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of affecting plant growth and development, poor control of the temporal and spatial specificity of target gene expression, and insignificant improvement effects, so as to avoid biosafety problems, Broad application prospects and the effect of agricultural economic value
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Embodiment 1
[0038] Example 1. Cloning of rice pollen-specific promoter PSP1
[0039] According to the whole genome sequence of rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided in NCBI, the specific amplification primers of PSP1 sequence were designed, and according to the characteristics of the selected pCambia1305 expression vector and PSP1 sequence, the specific primers were added at both ends of the specific primers. specific enzyme cleavage site ( figure 1 ). The specifically designed primers are: forward primer (P1F) with a KpnI restriction site (GGTACC) at the 5' end, and a reverse primer (P1R) with a NcoI restriction site (CCATGG) at the 5' end. The primer sequences are as follows:
[0040] P1F forward primer: 5'-CGGggtaccAACCAAACCTGTGGCTGC-3'KpnI
[0041] P1R reverse primer: 5'-CATGccatggCGCTCTGTGACGCCAT-3'NcoI
[0042] Then, the genomic DNA of the rice variety Nipponbare was extracted, and the primers (P1F and P1R) designed above were used to amplify the seque...
Embodiment 2
[0043] Example 2. Construction of plant expression vector driven by promoter PSP1
[0044] The positive clones obtained in Example 1 and identified by sequencing were subjected to plasmid extraction, the extracted plasmid was double digested with KpnI and NcoI, and the PSP1 promoter fragment was recovered. At the same time, KpnI and NcoI were used to double-enzyme digestion and linearization of pCambia1305, and the pCambia1305 backbone was recovered. The PSP1 promoter fragment recovered after enzyme digestion and the recovered pCambia1305 backbone were connected with T4 ligase (purchased from NEB Company), The plant expression vector PSP1-GUS-pCambia1305 ( image 3 ), the PSP1-GUS-pCambia1305 expression vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation method.
Embodiment 3
[0045] Example 3. Transforming rice with expression vector driven by promoter PSP1 GUS gene
[0046] The recombinant expression vector PSP1-GUS-pCambia1305 was transferred into mature rice embryos by Agrobacterium-mediated callus transformation method of rice mature embryos. The transformation method was as follows: (1) Induction of callus from rice mature embryos: The seeds of Nipponbare were peeled, then surface-sterilized with 75% alcohol for 2 minutes, then soaked in 30% NaClO solution for 30 minutes, and repeated once, and then washed with sterilized water for 4-5 times. Then the seeds were placed on the induction medium for cultivation, and the induced callus was cultivated in the dark at 26 degrees for transformation ( Figure 4 A). (2) Co-cultivation of rice callus and Agrobacterium: The EHA105 strain identified in Example 2 containing the PSP1-GUS-pCambia1305 expression vector was activated, enriched and resuspended to adjust OD600=0.3-0.5. The callus was collected ...
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