A gene for regulating poplar leaf morphogenesis and its application
A technology of poplar, gene, applied in the field of plant genetic engineering and biology
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Embodiment 1
[0048] Example 1 Clone PagKNAT2 / 6b Gene
[0049] Using 84K (P.alba X P.glandulosa) Populus albadensis as material, use RNeasy Plant Mini kit and RNase-free DNase I kit (Qiagen, Hilden, Germany) to extract the total RNA of one-month soil culture seedlings, each sample About 1.0 μg of RNA was taken, and the first strand of cDNA was synthesized by using the SuperScript III first-strand synthesis system (LifeTechnologies, Carlsbad, CA, USA). Referring to the published Populus trichocarpa genome sequence, primers were designed using Primer 5 software (the amplicon included start codon and stop codon) for full-length gene amplification (GATEWAY linker is introduced into the primer);
[0050] Wherein, the PagKNAT2 / 6b ORF forward primer is shown as sequence 3 in the sequence listing, see Table 1 below;
[0051] Table 1
[0052]
[0053] The sequence of the PagKNAT2 / 6b ORF reverse primer is shown as sequence 4 in the sequence listing, see Table 2 below;
[0054] Table 2
[0055]...
Embodiment 2
[0063] Embodiment 2 Construction of PagKNAT2 / 6b gene plant expression vector
[0064] Using cloning technology to construct an overexpression vector of the PagKNAT2 / 6b gene, using specific PCR primers (the PagKNAT2 / 6b ORF primer in Example 1), and using 84K cDNA as a template, PCR amplification was carried out, and the PagKNAT2 / 6b gene ORF was constructed into the entry vector , the entry vector is PDNOR207, and the sequence is shown as sequence 5 in the sequence listing, see Table 5 below;
[0065] table 5
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[0067]
[0068]
[0069] The reaction system is Fresh PCR product (fresh PCR product) 150ng; PDNOR207 vector (vector) 75ng; BP Clonase (cloning enzyme) II enzyme mix (mixed enzyme) 0.8μl; sterile ddH 2 Make up to 4 μl with O; the reaction procedure is: react at 25°C for more than 5h.
[0070] Pick positive clones from the screening culture plate for PCR detection and sequencing verification. After the entry vector with PagKNAT2 / 6b gene is linearized by...
Embodiment 3
[0080] The genetic transformation of embodiment 3PagKNAT2 / 6b gene
[0081] By electric shock method, the PMDC32-PagKNAT2 / 6b overexpression vector constructed is transferred in the Agrobacterium GV3101, and by Agrobacterium-mediated, the PagKNAT2 / 6b gene is transferred into poplar (Yinden poplar, 84K poplar, the same below), The transformation steps are as follows: the hybrid poplar clone 84K tissue culture seedlings used for genetic transformation were cultivated at a temperature of 23-25°C, a light of 16 / 8h (day / night), and a light intensity of 50 μM m -2 the s -1 Cultured under the condition of , the Agrobacterium containing the PMDC32-PagKNAT2 / 6b expression vector infects 84K leaf discs at OD600=0.6~0.8, and the infected leaf discs are grown in adventitious bud induction medium (SIM, Murashige-Skoog (MS) Add 0.5mg / l 6-benzyl aminopurine (6-BA, 6-benzyl aminopurine) and 0.05mg / l naphthalene acetic acid (NAA, naphthaleneacetic acid)) to the minimal medium, in dark conditions...
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