A gene that regulates the occurrence of adventitious roots in Populus chinensis, detection primers, expression vectors and applications
An expression vector, the technology of Populus chinensis, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of little research on woody plants, little research on adventitious root development, etc.
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Embodiment 1
[0050] The acquisition of PuMYC2 gene of Populus chinensis:
[0051] 1.1 Obtaining the target gene sequence
[0052] According to the sequence of PuMYC2 in Populus trichocarpa, primers were designed, and the sequence is shown in Table 1.
[0053] Table 1 Primer sequences for target gene cloning
[0054]
[0055] Utilize the CTAB method to extract the total RNA of Populus ussurensis kom. tissue-cultured seedlings cut for 3 weeks, and reverse-transcribe it into cDNA, use the cDNA as a template, and use the KOD enzyme system to clone the target gene. The reaction conditions are: Pre-denaturation at 95°C for 3min; denaturation at 98°C for 10s, annealing at 60°C for 30s, extension at 68°C for 90s, the number of cycles was 35; final extension at 68°C for 7min, detected by 1.2% agarose gel electrophoresis. The PCR system is shown in Table 2:
[0056] Table 2 The present invention is used for target gene cloning PCR reaction system
[0057]
[0058]
[0059] 1.2 Target ge...
Embodiment 2
[0063] Obtaining PuMYC2 Transgenic Populus chinensis
[0064] 2.1 Construction of overexpression vector
[0065] First, primers for restriction sites were designed according to the multiple cloning sites of the pBI121-GFP vector and the sequence characteristics of PuMYC2 itself. The restriction endonuclease selection is SalI and SpeI, and the primers are shown in Table 3:
[0066] Table 3 Restriction Primer Sequence
[0067]
[0068] PCR was carried out using the recombinant plasmid extracted from the positive colonies obtained in Example 1 as a template.
[0069] (1) PCR reaction system, as shown in Table 4:
[0070] Table 4 PCR reaction system used for target gene cloning in the present invention
[0071] template 100ng PuMYC2-Sal-F (10μM) 0.6μL PuMYC2-Spe-R (10μM) 0.6μL 2×PCR buffer for KOD 10μL dNTPs (2mM) 4μL KOD Polymerase 0.4μL wxya 2 o
to20μL
[0072] Reaction procedure:
[0073] Pre-denaturation at...
Embodiment 3
[0091] Phenotypic Observation of Adventitious Root Occurrence Regulated by Transgenic Populus chinensis
[0092] The wild-type and PuMYC2 overexpression poplar transgenic lines MYC2-OE1, MYC2-OE3 and MYC2-OE8 with the same growth state were microcutted into normal 1 / 2MS medium, and the number of adventitious roots was observed after 3 weeks. Time, rooting rate and adventitious root phenotype. Three independent experiments were performed per line, and each experiment was biologically replicated 60 times.
[0093] Counting the number of indefinite roots, the results are as follows Image 6 As shown, the average number of adventitious roots in PuMYC2 overexpressed plants is 10, and the average number of adventitious roots in WT is 7, indicating that the number of adventitious roots in PuMYC2 overexpressed plants is more than that of the wild type; in terms of the formation time of adventitious roots, PuMYC2 overexpressed plants have the earliest adventitious roots , Adventitiou...
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