Method for quickly screening paddy transgenes by novel fusion tag
A technology of fusion tags and transgenics, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of high-cost screening difficulties, larger T-DNA transformation vectors, and reduced transformation efficiency, and achieve fast and effective. Screening and identification, improving the screening accuracy, and the stable effect of screening itself
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Embodiment 1
[0041] Embodiment 1 Transformation of pCAMBIA1300-UR vector is used for T-DNA transformation vector construction
[0042]Based on the existing vector pCAMBIA1300, we designed a new fusion tag, combined with the newly designed promoter and terminator sequences, and transformed the new pCAMBIA1300-UR vector (abbreviated as 1300-UR, its nucleotide sequence As shown in SEQ ID NO: 1, the nucleotide is a circular sequence). The transformation process was entrusted to Ningbo Academy of Agricultural Sciences, referring to the PCR and point mutation techniques in the "Molecular Cloning Experiment Guide", and then transforming Escherichia coli DH5α. In order to select positive transformants and improve the efficiency of sequencing hits, especially to exclude the case of vector self-ligation, before the transformants are sequenced, bacterial liquid PCR must be performed first, and the following two specific detection primers need to be synthesized:
[0043]
[0044] The PCR detection...
Embodiment 2
[0048] Embodiment 2 Agrobacterium-mediated transgene and identification technology
[0049] The 1300-UR plasmid obtained in Example 1 was transformed into Agrobacterium tumefaciens EHA105 strain (purchased from ATCC) by electroporation. The specific transformation conditions were: electric shock at 2500V, adding 800 μl of YEP medium (without antibiotics), at 28°C Recover at 250rpm for 2 hours, then under sterile conditions, take 100μl and spread it on the YEP culture plate containing 50mg / l kanamycin and 50mg / l streptomycin, culture at 28°C for 36 hours, select the transformants, and inoculate Into the YEP liquid medium containing 50mg / l kanamycin and 50mg / l streptomycin, and shake culture at 28°C and 250rpm for 20-36h until the OD600 of the bacterial solution is 0.9. Take 500 μl of the cultured bacterial solution in a 1.5ml centrifuge tube, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant. Then, resuspend with 30 ml of AAM bacterial infection solution (conta...
Embodiment 3
[0053] Embodiment 3 comparative experiment
[0054] Taking the vector constructed in Example 1 and the pCAMBIA1300 vector (which can be purchased from CAMBIA, Australia), a transcription factor gene (its sequence is shown in GenbankNM_001185683, with a size of 3129bp) was cloned and introduced into rice according to conventional molecular cloning methods, and transformed into With reference to the plant transgenic methods such as Example 2 and the screening process, it was found that the carrier constructed in Example 1 had a positive rate of 30%, and through the PCR identification of the target gene, no phenomenon that the rearward recombination only retained the label and lost the target gene was found; In the pCAMBIA1300 experiment, 2 cases of retaining the tag and losing the target gene were found, which were estimated to be driven by the 35S promoter).
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