Litchi disease-resistant gene LcLTP as well as encoding protein and application thereof
A disease-resistant gene and protein-encoded technology, applied in the field of molecular plant pathology, can solve problems affecting the occurrence of diseases, and achieve the effect of enhanced expression and reduced disease spots
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Embodiment 1
[0034] Embodiment 1: Cloning and acquisition of LcLTP gene
[0035] (1) Through the analysis of the litchi genome, it was predicted that the litchi may encode the LcLTP gene. We designed primers (upstream primer LcLTP-F and downstream primer LcLTP-R), and successfully cloned the LcLTP gene in the litchi cDNA. Its nucleotide sequence is as follows: Shown in SEQ ID NO:1, the amino acid sequence of its encoded protein is shown in SEQ ID NO:2:
[0036] LcLTP gene (SEQ ID NO: 1; base composition: 80A, 87T, 109G, 66C):
[0037] ATGGGAAGCACCAAGGGAACATCACTAGGTTTTATGGTATTAGTAGTTGTAGCTGTTGTGGGGAAGTGGGAGGTGAAGATGGCTGGTGCAGAACTTAGTGCAGCCCAGTGCAAGGAAGAGAGGAGAATTGGGCTGAATGAGTGCAAGCCAGTGGTGTATGGGAAGCTTCCGTCGCCGTCGTGCTGTGAGCGTGTAAGGGTGAGTCATGTTGAATGTGTGTGCCCTGTCATTACACCTAAGTTGGCTGCTCTTATTGATCTCAACCGTGCCATCCGCCTCATCGAAGGCTGCGGTAGAAGAGTCCCTCGCCACTTCAAGTGTGGAAGTATCACAACTCCTTGA。
[0038] LcLTP protein (SEQ ID NO:2):
[0039] MGSTKGTSLGFMVLVVVAVGKWEVKMAGAELSAAQCKEERRIGLNECKPVVYGKLPSPSCCERVRVSHVE...
Embodiment 2
[0047] Example 2: Transient expression of LcLTP in tobacco
[0048] (1) Tobacco transient expression vector construction:
[0049] ① The target fragment is connected to the carrier: the amplified LcLTP target fragment is connected to the PVX vector with homologous recombinase (linkage site: SmaI) to obtain a recombinant plasmid containing the sequence of SEQ ID NO: 1; the GFP sequence (SEQ ID NO: 3) Use homologous recombinase to connect with PVX vector (connection site: SmaI), obtain recombinant plasmid PVX::GFP containing GFP green fluorescent protein, after breeding in Escherichia coli JM109 strain, extract the plasmid, and recombined plasmid PVX::GFP and the recombinant plasmid containing the sequence of SEQ ID NO:1 were transformed into Agrobacterium GV3101.
[0050] GFP gene (SEQ ID NO: 3):
[0051] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC...
Embodiment 3
[0055] Embodiment 3: Zoospores infect litchi leaves and branches
[0056] 1. Cultivation of Pythophthora lychee
[0057] The hyphae blocks of Pyrosophthora lychee strains preserved in the laboratory were inoculated on the carrot medium, and placed in the culture room at 27°C for one week.
[0058] 2. Preparation of zoospore suspension
[0059] Pour sterilized water on the Lychee downy mildew plate, use a brush to collect the litchi downy Phytophthora mycelium through the filter in a 50ml centrifuge tube, place it at 18°C for 2 hours until it releases zoospores, when the sporangia are observed under the microscope After releasing the zoospores, the zoospore suspension was collected by filtration.
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