A rice stamen-specific expression promoter osanth2 and its application
A promoter and stamen technology, which is applied in the field of biotechnology and crop genetic engineering, can solve the problems of limiting the ability to improve stamen traits, and the genetic resources of stamen-specific promoters are not very rich, so as to achieve the effect of improving rice fertility
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Embodiment 1
[0034] Acquisition of OsAnth2 Promoter Containing Restriction Sites
[0035] Step 1. Design of primers
[0036] According to the whole genome sequence of rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided by NCBI, the amplification primers were designed according to the sequence of rice OsAnth2 gene, and the enzyme cutting sites of the primers were designed according to the characteristics of the selected vector and target gene.
[0037] In this embodiment, rice binary expression vector pCAMBIA1391( figure 1 In part A, from CAMBIA, openly used vectors, Anhui Academy of Agricultural Sciences, Ministry of Agriculture, Rice Group of Supervision, Inspection and Testing Center for GMO Product Components) as an example, the target gene is the Gus gene, and the specifically designed primers are: forward primer (SEQ ID No: 2) with HindIII at the 5' end, restriction site (AAGCTT), reverse primer (SEQ ID No: 3) with BamHI at the 5' end, restriction site (GGATCC), the prim...
Embodiment 2
[0054] In order to verify that the sequence in the sequence listing SEQ ID No.1 of the present invention still has a promoter function after removing the 22 bp at the front end and the 22 bp at the back end, the present invention also carried out experiments similar to the above Example 1 on this sequence using other primers , that is: use the rice variety Nipponbare DNA as a template, use new forward primers and reverse primers to amplify the promoter; recover the target fragment amplified by PCR, connect it to the PGEM-T-Easy vector, and transfer it to the activated sensory State cells; then, connect it with the pCAMBIA1391 fragment with T4 ligase to obtain the corresponding crop expression vector, and use the freeze-thaw method to transfer the crop expression vector into Agrobacterium tumefaciens; perform Agrobacterium-mediated rice genetic transformation on mature rice seeds , cultivate the regenerated plant culture; finally carry out the staining verification.
[0055] It...
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