Application of wheat TaLCT1 gene silencing in regulation and control of cadmium stress tolerance of wheat
A gene and cadmium-resistant technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as anemia, affecting the hematopoietic system, mutation, etc.
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Embodiment 1
[0039] Example 1 Construction of TaLCT1 gene silencing recombinant vector
[0040] The method for constructing the TaLCT1 gene silencing recombinant vector, its steps are as follows:
[0041] 1.1 Extract total RNA from wheat leaves and reverse transcribe it into cDNA.
[0042] 1.2 Using the cDNA obtained in step 1.1 as a template, the nucleotide sequence of which is shown in SEQ ID No.2, select the RNAi fragment targeting the TaLCT1 gene, and perform PCR amplification with primers TaLCT1-F and TaLCT1-R, to obtain The PCR product of the site; the primers are
[0043] TaLCT1-F:5-GG GGTACCACTAGT CTCTCGTGAAAGACTCGC-3
[0044] TaLCT1-R:5-CG GGATCCGAGCTC TCGGGGAAGCAGGACCAA-3.
[0045] 1.3 The PCR product was constructed on the pTCK303 vector in two steps. For the first time, use Spe I (restriction enzyme) and Sac I (restriction enzyme) to digest the pTCK303 plasmid and the RNAi fragment (PCR product) of TaLCT1 with restriction sites. The digestion system is as follows:
[004...
Embodiment 2
[0075] Example 2 Carrying out Agrobacterium Infection and Transformation to Wheat
[0076] Adopt the GV3101 recombinant vector that embodiment 1 makes to carry out Agrobacterium infection transformation to wheat, and its steps are:
[0077] 2.1 Inoculation of Agrobacterium tumefaciens on LB solids
[0078] Activate Agrobacterium tumefaciens twice on the medium plate, then pick a single colony of the bacterial strain and inoculate it into 5ml containing 20mg·L -1 Rifampicin and 50mg·L -1 In the LB liquid culture medium of kanamycin, under the conditions of temperature 26°C and shaking speed 160-200rpm, culture for 5-8 hours, then transfer 1ml to 50ml containing 20mg L -1 Rifampicin, 50mg·L -1 Kanamycin and 110umol L -1 In the LB liquid medium of acetosyringone, under the conditions of temperature 26°C and shaking speed 160-200rpm, after culturing for 12-16 hours, the A600 value is 0.75-1.0 for later use;
[0079] 2.2 Select plump wheat seeds, wrap them in gauze, rinse them...
Embodiment 3
[0084] Example 3 Identification of transgenic plants
[0085] Cultivate transformed wheat plants with the seeds harvested in Example 2. When the transformed wheat plants grow to three leaves and one heart, cut the young leaves of a single plant and put them into 0.2mL sterilized centrifuge tubes respectively, and carry out corresponding identification, and then Add GUS staining solution to submerge the leaves of the plants, place in a 37°C incubator for 6-12 hours, rinse the samples with 50%, 75%, and 100% ethanol successively, soak for 5 minutes each time, then add 100% ethanol to soak until completely decolorized, The expression of GUS was observed under a stereomicroscope, and photographed and recorded. Such as figure 1 As shown, the wild control has no blue color, but the transformed strains 2, 3, and 4 can all be stained blue, indicating that the target gene has been integrated into the genomes of these plants.
[0086] In order to further identify positive plants, the...
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