Clone of plant manganese poison-resistant important gene ShMDH1 and application thereof
A plant, manganese-tolerant technology, applied in the field of plant biology
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Embodiment 1
[0047] ShMDH1 gene cloning
[0048] 1, ShMDH1 gene cloning
[0049] According to protein two-dimensional electrophoresis, a protein up-regulated by manganese poisoning was identified as malate dehydrogenase. Then, based on the reported nucleotide sequences of soybean (XM_003527163), alfalfa (AF020271), japonicus japonicus (BT146072) and castor (XM_002524216) genes for homology comparison, upstream homologous primers were designed in highly conserved regions5 '- ATTATTGCCGCTGAGGTTTTTCAAGA -3' (SEQ ID NO:3) and the downstream homologous primer 5'- GATTCCCTTCAAGCAAGCATCG -3' (SEQ ID NO:4), using the TRIzol method to extract total RNA from the root system of the fine-stem manganese-tolerant genotype of Stylophyllum styloides, Then it was reverse-transcribed into cDNA and used as a template for PCR amplification. The PCR reaction system was 20 μL, including 0.5 μL of 10 μM upstream and downstream primers, 2 μL of 10×PCR buffer, 1.6 μL of 2.5 mM dNTP, and Ex-Taq (TAKARA Company) ...
Embodiment 2
[0071] Research on genetically modified materials
[0072] 1. Obtaining genetically modified materials
[0073] 1) Obtaining transgenic Arabidopsis plants
[0074] will construct a good overexpression ShMDH1 The vector plasmid was transformed into Agrobacterium tumefaciens Gv3101, and transgenic Arabidopsis plants were obtained by Agrobacterium-mediated Arabidopsis inflorescence transfection method.
[0075] 2) Obtaining of transgenic yeast strains
[0076] According to the method of SC easy comp transformation kit (Invitrogen Company), the constructed yeast expression ShMDH1 Vector plasmid and pYES2 empty vector were transformed into yeast INVSC1 (Invitrogen).
[0077] 2. Detection of genetically modified materials
[0078] 1) Detection of transgenic Arabidopsis plants
[0079] After the T1 generation Arabidopsis seeds were screened for hygromycin resistance, the T1 generation transgenic plants were obtained, and the seeds were harvested; the T2 generation seeds were sc...
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