Gene engineering application for elsholtziasplendens metallothionin gene EhMT1
A metallothionein and genetic engineering technology, applied in the field of metal binding proteins in response to heavy metal stress in plants, to achieve the effect of improving tolerance
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Embodiment 1
[0028] Seeds of Cystis haizhou were collected from the copper mining area. When the seedlings of Pansia haizhou grew to the 7-8 leaf stage, they were treated with 50 μM CuSO 4 After 48 hours of treatment, the leaves were taken immediately and placed in liquid nitrogen for cryopreservation. Part of the leaves were taken, crushed with a mortar, added to a 1.5ml centrifuge tube filled with lysate (purchased from Beijing Tiangen Company), shaken fully, and then transferred into a glass homogenizer. After homogenization, transfer to a 1.5ml centrifuge tube to extract total RNA. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer. The three fragments amplified by RT-PCR and RACE were spliced to obtain the full-length sequence, and primers at both ends were designed according to the sequence
[0029] Primer P1: 5′-ACGTCGATCACTAAGAAAAG-3′
[0030] Primer P2: 5′-CTTAGACGCATAAACACGAA-...
Embodiment 2
[0035] The primers at both ends of EhMT1 designed in Example 1 were used to conduct semi-quantitative RT-PCR to analyze the expression of the aboveground and underground parts of the A. haizhou seedlings, and the expression of the 18SrRNA gene (Sancenon et al., 2003) was used as the internal reference. The results showed that the expression of EhMT1 in the seedlings of A. haizhouensis in 50μM CuSO 4 It reaches a maximum at 48 hours under treatment.
Embodiment 3
[0037] According to the full-length sequence of EhMT1 obtained in Example 1, primers P3 and P4 for amplifying the complete coding frame were designed, and the sequences were as follows:
[0038] Primer P3: 5'-G TCTAGA AAGAAAATGTCGAGTGGA-3′
[0039] Primer P4: 5′- CCCGGG AGGCAGTATATCTGACAG-3′
[0040] Restriction endonuclease sites were introduced in order to construct expression vectors. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the cDNA of EhMT1 was cloned into the intermediate vector pGEM-T, and further cloned into the binary expression vector pBI121, and the expression was identified under the premise of ensuring the correct reading frame. vector, which is then transformed into Agrobacterium, and then into tobacco. After the obtained transgenic plants are verified by PCR and RT-PCR, the heavy metal resistance evaluation of the plants is carried out. T of the transgenic plant 0 Generation and control plants were treated wi...
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