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Cloning and application of an important gene shmdh1 for tolerance to manganese toxicity in plants
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A plant, manganese-tolerant technology, applied in the field of plant biology
Inactive Publication Date: 2015-11-18
SOUTH CHINA AGRI UNIV
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However, the key genes controlling Mn toxicity tolerance in Stylophyllum have not been reported yet
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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, according to 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'(SEQIDNO:3) and the downstream homologous primer 5'-GATTCCCTTCAAGCAAGCATCG-3'(SEQIDNO:4), using the TRIzol method to extract total RNA from the root system of the Manganese-tolerant genotype Fine-stem of Stylosmanthus styloides, and then reverse transcription After forming cDNA, it was 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 mMdNTP, 0.12 μL of Ex-Taq (TAK...
[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] Referring to the SCeasycomp transformation kit (Invitrogen Company) method, 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 screene...
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Abstract
The invention discloses a key gene that promotes plant tolerance to manganese poisoning ShMDH1 cloning and its application. Should ShMDH1 The nucleotide sequence such as SEQ? ID? As shown in NO:1, the amino acid sequence of the encoded protein is shown as SEQ? ID? NO:2 shown. ShMDH1 The gene regulates the synthesis and secretion of malic acid in stylophyllum, which plays an important role in enhancing the manganese tolerance of plants.
Description
technical field [0001] The invention relates to the field of plant biotechnology, in particular to a manganese toxicity-resistant gene ShMDH1 cloning and its application. Background technique [0002] More than 50% of the potential arable soils in the world are acidic soils, mainly distributed in tropical, subtropical and temperate regions (Kochian et al., 2004). In acidic soils, in addition to phosphorus deficiency and aluminum toxicity, manganese toxicity is also an important obstacle to crop growth and yield (deCarvalhoe et al., 1980; Horst et al., 1988; Raghothama, 1999; Vance et al., 2003). Manganese is one of the essential trace elements for plant growth (Mukhopadhyay and Sharma, 1991). However, excess manganese can also cause toxic effects on plants. Symptoms of manganese toxicity generally manifest as brown manganese oxidation spots on leaves, reduction of chlorophyll content, weakening of photosynthesis and other symptoms, which ultimately inhibit plant growth an...
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