Strawberry auxin synthesis rate-limiting enzyme fayuc11 gene and its application
An auxin and rate-limiting enzyme technology, applied in the field of plant biology, can solve the problems of low average yield and low ratio
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Embodiment 1
[0044] Example 1 Isolation of FaYUC11 gene and construction of virus-induced gene silencing vector
[0045] Based on the sequence information of the whole family of YUC genes in diploid forest strawberry (Liu et al. 2014), the dynamic expression patterns of the whole family genes were resolved in the octoploid cultivated strawberry fruit (receptacle and achene). It was found that FaYUC11 was the only highly expressed member of this family in achenes, and its dynamic expression pattern was consistent with that of auxin accumulation in achenes.
[0046] Design PCR amplification primers according to the sequence of the forest strawberry homologous gene, forward primer OF:
[0047] 5'-AAAATGGAGAACAATGTGTTTGGGA-3'; reverse primer OR:
[0048] 5'-GGACTAGACCTCTCTTGCAGCAT-3', as shown in SEQ ID No.3 and 4, respectively.
[0049] Using the wild-type Jiuxiang Strawberry fruit cDNA as a template, the full-type gold high-fidelity enzyme (from Beijing TransGene Company) was used for PCR ...
Embodiment 2
[0054] Embodiment 2 The acquisition of virus-induced gene silencing strawberry
[0055] The pBSK-in-dYUC11 recombinant plasmid was further digested with KpnI enzyme and SacI enzyme, and the bidirectional hairpin structure of the FaYUC11 gene fragment was cloned into the CP protein behind the 2×35S promoter of tobacco rattle virus pTRV2 (Liu et al. 2002). Downstream, the pTRV2-dYUC11 recombinant vector was obtained. Agrobacteria containing RNA1 and RNA2 of tobacco rattle virus were mixed.
[0056]Inoculate GV3101 Agrobacterium containing target gene virus-induced silencing expression vector and empty pTRV1 and pTRV2 vectors in 5mL YEP medium containing 50 μg / mL Kan, 10 μg / mL Rif and 50 μg / mL Gen and culture overnight at 28°C, and the next day follow Inoculate in induction medium (YEP, containing 10mM / L MES, 20μM / L AS, 50μg / mL Kan, 10μg / mLRif and 50μg / mL Gen) at a ratio of 1:50, shake the bacteria at 28°C until logarithmic phase (OD Value 0.6-0.8), 5000r / min centrifuge for 10m...
Embodiment 3
[0057] Example 3 Identification of virus-induced gene silencing strawberry
[0058] In order to detect whether tobacco rattle virus successfully infects strawberry, the total RNA of strawberry fruit was extracted from wild-type and injected virus original vector and recombinant FaYUC11 virus-induced silencing vector, and cDNA was synthesized by reverse transcription using random primers.
[0059] According to Jia et al. 2011, specific primers for tobacco rattle virus RNA2 were provided for RT-PCR reaction to identify virus-infected strawberry fruits. PCR product is carried out electrophoresis detection, the result finds, the strawberry fruit that the recombinant virus vector of virus original vector and FaYUC11 infects can amplify virus-specific band (such as figure 2 ), but there was no band for this purpose in wild-type strawberry. It was preliminarily determined that tobacco rattle virus had infected strawberry fruit by microinjection method.
[0060] Further, a new pair...
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