Plant light energy conversion efficiency associated protein and encoding gene and application thereof
A transformation efficiency, plant technology, applied in plant genetic improvement, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as slow growth, block assembly process, unstable assembly intermediates, etc.
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Embodiment 1
[0049] Embodiment 1, the discovery of LPA3 gene
[0050] 1. Mutant screening, phenotypic and genetic analysis
[0051] By measuring the leaf area of Arabidopsis photosynthetic function-deficient mutant lpa3 at different growth stages, it was found that the growth rate of lpa3 was only about 12% of the wild type, the leaf color was yellow, and the pigment content was significantly reduced. Chlorophyll fluorescence dynamics analysis using PAM2000 showed that the Fv / Fm value decreased to 0.42, and the maximum photochemical efficiency of PSII was about half of that of the wild type. Through the PCR amplification verification of the Bar gene carried in the T-DNA element, it was confirmed that there was indeed a T-DNA insertion in lpa3 and a homozygote was screened.
[0052] 2. Cloning of LPA3 gene
[0053] Map-based cloning was used to map the mutation site to a new gene. A new gene was obtained by RT-PCR and sequencing analysis. The new gene encodes the protein shown in Sequ...
Embodiment 2
[0058] Embodiment 2, tissue localization of LPA2
[0059] 1. Detection of GFP fusion protein
[0060] 1. Construction of the carrier
[0061] The GFP gene (source NCBI GeneID: 7011691, plasmid: pCmGFP) was amplified by PCR (primers: 5'-GCCTCTAGAATGAGTAAAGGAGAAGAAC-3' and 5'-AAGCTTCTCGAGTTGTATAGTTCATCC-3'). The PCR product and vector pPUC18 (Taraka Company; Cat. No. D3218) were digested with XbaI and HindIII. The pPUC18-GFP intermediate vector was identified after ligation, transformation and identification.
[0062] 2. Amplify the sequence CLPA3 comprising the LPA3 leader peptide (the DNA sequence encoding the N-terminal region of the LPA3 protein; encoded from the 1-150th amino acid residue at the amino terminal), using CLPA3 as a template (primer: 5'-ATAGGATCCATGGCTATGGCGATTGC-3 ' and 5'-TCCTCTAGAAATCATTATGCCATC-3') for PCR amplification, BamHI and XbaI double enzyme digestion of the PCR product and pPUC18-GFP vector, after ligation, transformation, and identification of ...
Embodiment 3
[0078] Example 3, the acquisition and identification of overexpression plants
[0079] 1. Obtaining of LPA3 overexpression plants
[0080] 1. Cloning of LPA3 gene
[0081] Using the cDNA of wild-type Arabidopsis as a template, PCR amplification was performed with specific primer pairs, and the recovered PCR amplification product was connected to the pGEM-T vector (promega product pGEM-T easy Vector system), and transformed into Escherichia coli DH5α ( Beijing Tianwei Times Company) was competent, and positive clones were selected for sequencing. The results showed that the PCR amplified product had the full-length cDNA of the LPA3 gene shown in Sequence 2 of the sequence listing.
[0082] Specific primer pairs:
[0083] Upstream primer::5'-AGGATCCATGGCTATGGCGATTGCGATG-3';
[0084] Downstream primer: 5'-CCCGGGTCAGTTACGCCAATTAGATGATG-3'.
[0085] 2. Construction of recombinant expression vector
[0086] Digest pBI121 (Takara) with BamHI and SmaI to recover the backbone vect...
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