Visual plasmid for replacing fluorescent proteins and visualization method for evaluating plant protoplasts transformation and transformation efficiency by replacing fluorescent proteins

A visual plasmid for replacing a fluorescent protein, wherein the visual plasmid is a pUC-tdDODA plasmid or a pUC-DODA plasmid;

• wherein genes for replacing the fluorescent protein in the pUC-tdDODA plasmid;
• comprise, the tdTomato gene shown in SEQ ID NO.1 and the DODA gene shown in SEQ ID NO.2;
• wherein genes for replacing the fluorescent protein in the pUC-DODA plasmid;
• comprise, the DODA gene shown in SEQ ID NO.2;
• wherein a vector skeleton of the visual plasmid comprises a 35S promoter and a NOS terminator;
• wherein the tdTomato gene and the DODA gene in the pUC-tdDODA plasmid are connected by the DNA linkage unit shown in SEQ ID NO. 3;
• wherein, during construction of the plasmid pUC-tdDODA, primers used for amplifying the tdTomato gene are shown in SEQ ID NO.6 and SEQ ID NO.7;
• wherein, during construction of the plasmid pUC-tdDODA, primers used for amplifying the DODA gene with the DNA linkage unit as described above are shown in SEQ ID NO.8 and SEQ ID NO.9;
• wherein, primers used for amplifying the DODA gene are shown in SEQ ID NO.10 and SEQ ID NO. 11;
• wherein, a process for making visual plasmid for replacing a fluorescent protein, wherein the visual plasmid is the pUC-tdDODA plasmid comprises:
• using a PLV23 (TDTOMATO) plasmid as a DNA template to amplify TDTOMATO and use primer pairs as shown in SEQ. ID. NO 6 and SEQ. ID. NO. 7 in the PCR reaction:
• PUC-TD F:
• SEQ ID NO. 6
• 5′-TTTACGAACGATAGACTAGTATGGTGAGCAAGGGCGAGG-3′,;
• MID-TD R:
• SEQ ID NO. 7
• 5′-CTTATACAGCTCGTCCATGCCGT-3′,.
• using a RUBY plasmid as a DNA template to amplify F2A and DODA, to use primer pairs as shown in SEQ. ID NO. 8 and SEQ. ID NO. 9 in the PCR reaction:
• MID-DODA F:
• SEQ ID NO. 8
• 5′-GCATGGACGAGCTGTATAAGGGTTTCAGGAGCTACAAATTTTTCTC
• TT-3′,;
• PUC-DODA R:
• SEQ ID NO. 9
• 5′-CGATCGGGGAAATTCTCTAGATTAAGCAGATGTGAACTTGTATGA
• TC-3′,;
• recovering amplified PCR products by using an agarose gel DNA recovery kit;
• wherein the vector has a skeleton plasmid of pUC-2×35S-EGFP, and using Restriction enzymes SpeI and XbaI to linearize the vector and cut out the EGFP gene;
• wherein a process for making visual plasmid for replacing a fluorescent protein, wherein the visual plasmid is the pUC-DODA plasmid comprises:
• using a Ruby plasmid as a DNA template to amplify DODA and using primer pairs as shown in SEQ. ID. NO. 10 and SEQ. ID. NO. 11 in the PCR reaction:
• PUC-DODA F:
• SEQ ID NO. 10
• 5′-TTTACGAACGATAGACTAGTATGAAGATGATGAATGGTGAAGAT-
• 3′,;
• PUC-DODA R:
• SEQ ID NO. 11
• 5′-CGATCGGGGAAATTCTCTAGATTAAGCAGATGTGAACTTGTATGAT
• C-3′,;
• recovering the two amplified PCR products by using the agarose gel DNA recovery kit;
• wherein the skeleton plasmid of the vector is pUC-2×35S-EGFP, and using restriction enzymes SpeI and XbaI to linearize the vector and cut out the EGFP gene;
• wherein construction of the pUC-tdDODA plasmid and the pUC-DODA plasmid further comprises:
• a PCR reaction system comprises:
• Max Super-Fidelity DNA Polymerase 1 μL;
• 2×Max buffer 10 μL;
• dNTP Mix 0.5 μL;
• upstream primers PUC-TD F, MID-DODA F and PUC-DODA F RUBY F 0.8 μL;
• template DNA 1 μL;
• double distilled water 20 μL;
• wherein the PCR amplification steps comprise:
• (1) predenaturation at 95° C. for 5 min with a cycle number of 1;
• (2) denaturation 95° C. for 15 sec with a cycle number of 33;
• (3) annealing at 58° C. for 15 sec with a cycle number of 33;
• (4) extension at 72° C. for 45 sec with a cycle number of 33;
• and (5) extension at 72° C. for 5 min with a cycle number of 1;
• and
• wherein the enzyme digestion system comprises:
• 10× enzyme digestion buffer 10 μL;
• SpeI 1 μL;
• XbaI 1 μL;
• vector 2000 ng;
• double distilled water 100 μL;
• and
• placing a reaction system in a 37° C. incubator for enzyme digestion overnight.