A longan single fruit weight trait regulation gene dlcnr8 and its protein and application
A technology for regulating genes and proteins, which is applied in the direction of recombinant DNA technology, application, genetic engineering, etc., and can solve the problem of low map accuracy
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Embodiment 1
[0027] The cloning of embodiment 1 target gene
[0028] Materials and methods
[0029] 1.1 Plant material
[0030] Three groups of 'Sijimi' longan with the same growth vigor and tree age (9 years old) were selected as sampling trees, and the flowers, flower buds, leaves, pericarp, pulp, roots, seeds, stems and young fruits (after flowering) of 'Sijimi' longan were collected. 60d whole fruit) and other organs were used as materials for tissue expression analysis. Three groups of F1 generation large-fruited strain FD105 and small-fruited strain FD21 longan with the same growth vigor and tree age (10 years old) were selected as sampling trees, and the longan pulp of 60, 70, 80, 90 and 100 days after flowering was taken as materials Analysis of fruit development was performed. All experiments were repeated three times, and the samples were immediately frozen in liquid nitrogen and transferred to a -80°C refrigerator for storage and use.
[0031] 1.2 Cloning and bioinformatic a...
Embodiment 2
[0040] Example 2 Subcellular Localization Analysis
[0041] Primers were designed according to the cloned DlCNR8 gene sequence (terminator removed) (Table 1), and the full-length ORF of DlCNR8 was amplified, and the PCR reaction procedure was as above. The PCR product was detected by 1% agarose gel electrophoresis, purified, connected to the pMD18-T vector, and transformed into DH5α. Single colonies were picked, and plasmids were sequenced after PCR detection. Then the pBWA(V)HS-osgfp and DlCNR8 plasmids were digested with EcoR I respectively, and enzyme ligation was carried out after recovery. The enzyme-linked plasmid was transformed into Escherichia coli DH5α, and after positive detection, the correct strain was selected for sequencing, and then extracted to obtain the pBWA(V)HS-DlCNR8-osgfp plasmid. Then it was transferred into Arabidopsis protoplasts by PEG-mediated method (YooS D, Cho Y H, Sheen J.Arabidopsis mesophyll proto-plasts: a versatile cell system for transien...
Embodiment 3
[0042] Example 3 Construction of overexpression vector and functional verification of transgenic tomato
[0043] Using specific PCR primers OECNR8-S / OECNR8-A (Table 1), longan cDNA was used as a template for PCR amplification. A BamH I restriction site is added to the 5' end of the primer, and a Sac I restriction site is added to the 5' end of the anti-primer. The obtained PCR product was ligated with pMD19-T vector and sequenced. Finally, the plasmids with correct sequencing were extracted, pBI121 and the plasmids with correct sequencing were double-digested with BamH I and Sac I, respectively, and a plant expression vector containing the DlCNR8 target gene was constructed by T4 DNA ligase, and named pBI121-DlCNR8. The constructed overexpression vector pBI121-DlCNR8 was transformed into Agrobacterium strain GV3101 by liquid nitrogen freeze-thaw method, referring to literature (Arshad W, Waheed M T, Mysore K S, et al. Agrobacterium-mediated transformation of tomato with rolB ...
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