Gene participating in alpha-Farnesene biosynthesis of prunus persica and application of gene
A biosynthesis and gene technology, applied in the field of plant molecular biotechnology and genetic engineering, can solve the problem of identifying synthetic genes
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Embodiment 1
[0018] The present invention will be further described below with reference to specific embodiments and drawings, but the embodiments do not limit the protection scope of the present invention. Example 1: PpTPS2 gene cloning
[0019] (1) Experimental method
[0020] Taking the amino acid sequence of MdAFS-RG1 with α-Farnesene synthetic function in apple as the reference sequence, the Blastp algorithm was used to search for homologous sequences in the peach genome database Peach Genome V2.0. Design primer pairs SEQ: NO. 2 and SEQ: NO. 3, using peach cDNA as a template, PCR amplification to obtain the PpTPS2 sequence SEQ: NO. 1, and sequencing verification. The PCR reaction system is 50μl, and the components are: 0.5μl Taq enzyme (Roche), 5μl buffer (10×), 4μl dNTP (2.5mM), upstream and downstream primers (10μM, Invitrogen) each 2μl, 4μl cDNA, 32.5μl H 2 O. The RT-qPCR reaction program is 95°C for 5min; 95°C for 30s, 58°C for 30s, 72°C extension for 1.5min, 35 cycles; the last 72°C...
Embodiment 2
[0023] Example 2: PpTPS2 expression and α-Farnesene content
[0024] (1) Experimental method
[0025] 1. Peach fruit material
[0026] After the commercial maturity of peach fruit is picked, the leaves are selected from mature leaves with neat and normal leaf shapes, and the leaves and pulp tissue are frozen in liquid nitrogen and stored in a refrigerator at -80°C for later use. In order to further clarify the correlation between gene expression and substance content, fruits and leaf tissues were used as materials to carry out ultraviolet B (UV-B) and methyl jasmonate (MeJA) treatments. Set up three biological replicates, each with 5 fruits, or 20 leaves.
[0027] 2. RNA extraction and cDNA synthesis
[0028] The total RNA of peach fruits and peach leaves was extracted by CTAB method. After removing genomic DNA contamination with TURBO DNase Kit (Ambion), 1.0 μg RNA was taken to synthesize cDNA according to the instructions of iScript cDNA Synthesis Kit (Bio-Rad). After the RNA test ...
Embodiment 3
[0032] Example 3: Subcellular localization
[0033] (1) Experimental method
[0034] 1. Vector construction
[0035] Combining the primer pair SEQ:NO.6 and SEQ:NO.7, using PCR technology to amplify to obtain PpTPS2, the PCR system and reaction procedure are the same as in Example 1. The PCR product and the target vector pCAMBIA super 1300-RGFP were digested with restriction enzymes BamHI and SalI and then respectively connected. Transform Escherichia coli, select the bacteria and send for testing and verification, then transform the plasmid confirmed by sequencing into the Agrobacterium strain GV3101::pSoup by electroporation.
[0036] 2. Tobacco leaf infection
[0037] The transformed Agrobacterium was coated on a plate, cultured at 28°C for 2 days, and then a single clone was selected and transferred to 5ml LB culture medium, kanamycin (50mg / L) and gentamicin (25mg / L) were added, and cultured to OD 600 After 0.8-1.0 centrifugation (4000g, 5min). Equal volume of penetrant (10mM MES...
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