Gene for improving content of beta-carotene, encoded protein and application thereof
A carotene and gene technology, applied in the field of genes and their encoded proteins and applications, can solve the problems that have not been reported, the increase of hypervitamin Aemia, etc.
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Embodiment 1D
[0038] The acquisition of embodiment 1DXS and DXR gene
[0039] 1. Acquisition of DXS gene
[0040] (1) Utilize the plant RNA extraction kit provided by OMEGA company to extract the total RNA of Cassia officinalis seeds, carry out 1.2% agarose gel electrophoresis to the extracted total RNA of Cassia officinalis to detect its extraction amount and concentration, the results are shown in figure 1 , figure 1 The middle band M is Marker, and band 1 and band 2 are the detection results of total RNA.
[0041] (2) Synthesis of cDNA: The total RNA extracted above was reverse-transcribed using PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) kit to obtain cDNA.
[0042] 2 forward primers (DXS-3GSPW and DXS-3GSPN) of the 3' end RACE were designed, and 2 reverse primers were 3RP and 3RNP, and the cDNA obtained above was used as the 3' end RACE template to clone the 3' end primer of CODXS The two-round reaction system is as follows:
[0043] The first round of PCR: Te...
Embodiment 2
[0057] Example 2 Construction of expression vector
[0058] 1. Construction of PBI212-CODXS vector
[0059] The CODXS and pBI121 plasmids recovered above were digested with BamH I at the same time. The digestion system was as follows: CODXS / pBI121 plasmid 25 μL; 10×K Buffer 5 μL; BamH I 3 μL; ddH 2 O 17 μL (Total 50 μL). Mix in a flash, react at 30°C for 4 hours, and directly recover and purify the reaction solution to obtain 30 μL of purified products, which are then digested with SnaB I. The enzyme digestion system is as follows:
[0060] CODXS / PBI121 (first digestion product) 30 μL; SnaB I 3 μL; 10×Basal 5 μL; 0.1% BSA 5 μL; ddH 2 O 7 μL (Total 50 μL).
[0061] After centrifugation and mixing, react at 37° C. for 4 h, then use agarose gel (1%) electrophoresis to cut the gel and recover, and purify CODXS and carrier pBI121.
[0062] Use T4 DNA ligase to ligate the digested and purified CODXS to the PBI121 vector, and after centrifugation and mixing, connect overnight at ...
Embodiment 3
[0069] The acquisition of embodiment 3 transgenic tobacco
[0070] 1. Transformation of Agrobacterium with recombinant plasmids PBI212-CODXS and PBI121-CODXR
[0071] (1) Take 50 μL of prepared competent GV3101, place at 4°C, add 20 μL of recombinant plasmids PBI121-CODXS and PBI121-CODXR respectively, and mix by pipetting;
[0072] (2) Place the mixed bacterial solution in step (1) at 4°C for 30 minutes, then transfer it to liquid nitrogen for 5 minutes, then place it at 37°C for 5 minutes, and then place it at 4°C for 5 minutes;
[0073] (3) Add 1 mL of LB liquid medium to the bacterial liquid in step (2), then place it at 28°C, 200 rpm, and shake for 2-3 hours;
[0074] (4) Centrifuge the bacteria solution in step (3), discard the supernatant, collect the bacteria, add 200 μL LB liquid medium to resuspend the bacteria, and spread it on the LB solid plate (containing Kan 50mg / L and Rfi 50mg / L L), cultivated at 28°C for 48h;
[0075] (5) Pick positive clones for expanded c...
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