Heat-resistance related protein of plant maintaining stability of thylakoid membrane and application thereof
A plant and thylakoid technology, applied in the fields of biotechnology and botany, can solve the problem of undiscovered thylakoid membrane thermostable protein and other problems
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Embodiment 1
[0085] Construction of p35S:HsfA2 vector
[0086] The cDNA coding sequence of HsfA2 was amplified by RT-PCR. Specific steps are as follows:
[0087] Using the RNeasy Plant Mini kit (purchased from Qiagen), the total RNA of col-O type Arabidopsis was isolated according to the instructions, and the isolated total RNA was reverse-transcribed using M-MLV reverse transcriptase (purchased from TOYOBO) , to obtain cDNA.
[0088] Using cDNA as a template, PCR reaction was performed using Primer star DNA polymerase (purchased from Takara Company) to amplify the cDNA coding region sequence of HsfA2. The primers used are as follows:
[0089] Upstream 121A2-F -UP 5′-AA TCTAGA CTCTGAGCTTATGGATTTGAG-3' (SEQ ID NO.: 2), wherein the underlined part ( TCTAGA ) part is restriction site XbaI;
[0090] Downstream 121A2-R-DO 5'-AAGAGCTCGACCGCAAACAAGTAGATGTG-3' (SEQ ID NO.: 3), wherein the underlined part (GAGCTC) Part of it is the enzyme cutting site SacI.
[0091] The resulting DNA amp...
Embodiment 2
[0116] get transgenic plants
[0117] The Agrobacterium obtained in Example 1 was transformed into col-O type of wild-type Arabidopsis thaliana by spraying method to produce transgenic plants. As a result, 35 transgenic lines were obtained, all of which could grow normally on the PNS medium containing kanamycin.
[0118] The overexpression strain of HsfA2 is screened by the method of RT-PCR, and the primers used are as follows:
[0119] HsfA2+5'-ATGGAAGAACTGAAAGTGGAAATG-3' (SEQ ID NO.: 4);
[0120] HsfA2-5'-GATCAAATCTTTCTGAATCCATAAG-3' (SEQ ID NO.: 5).
[0121] The results showed that the screened lines were all HsfA2 transgenic plants.
Embodiment 3
[0123] Construction of rps1 transgenic plants constitutively expressing HsfA2 gene (35S:HsfA2rps1)
[0124] The rps1 mutant (purchased from salk) obtained in Example 1 was transformed with Agrobacterium, and the rps1 transgenic plant (35S:HsfA2rps1) constitutively expressing HsfA2 was obtained. The seeds of homozygous transgenic plants of the T2 generation with a Kan resistance segregation ratio of 3:1 were used for subsequent experiments.
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