Longan gene DlGRAS34, protein and application of longan gene DlGRAS34 and protein in regulating and controlling flowering of plants
A longan and gene technology, applied in the application field of longan gene DlGRAS34 and protein in regulating plant flowering
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Embodiment 1
[0026] The cloning of embodiment 1 target gene
[0027] Materials and methods
[0028] 1.1 Plant material
[0029] Three groups of 'Sijimi' longan and the main cultivar 'Shixia' longan with the same growth vigor and tree age (9 years) were selected as sampling trees, and 'Shixia' longan showed seasonal flowering as other cultivars. , SF). Its flowering needs a period of low temperature induction (ie "vernalization") to start, and generally only blooms and bears fruit once a year. The 'Sijimi' longan has the characteristic of perpetual flowering (PF), which can continuously induce flowering and bear fruit in four seasons a year. Dormant buds, flowers and fruits can exist on the same branch, and its flowering induction is not affected. External environmental influence. It is an excellent material for studying the analysis mechanism of longan flowering induction. In this study, three terminal buds at critical stages of flower induction were selected for sampling. The three ...
Embodiment 2
[0038] Example 2 Subcellular Localization Analysis
[0039] Primers were designed according to the cloned DlGRAS34 gene sequence (terminator removed) (Table 1), and the full-length ORF of DlGRAS34 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 pBWA(V)HS-osgfp and DlGRAS34 plasmids were digested with EcoRI 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-DlGRAS34-osgfp plasmid. Then it was transferred into Arabidopsis protoplasts by PEG-mediated method (Yoo S D, Cho Y H, Sheen J.Arabidopsis mesophyllproto-plasts: a versatile cell system for trans...
Embodiment 3
[0040] Example 3 Overexpression vector construction and functional verification of transgenic Arabidopsis
[0041] Using specific PCR primers OEGRAS34-S / OEGRAS34-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 DlGRAS34 target gene was constructed by T4 DNA ligase, and named pBI121-DlGRAS34. The constructed overexpression vector pBI121-DlGRAS34 was transformed into the Agrobacterium strain GV3101 by the liquid nitrogen freeze-thaw method, referring to the literature (Clough, Steven J, Bent, et al. Floral dip: simplified method for Agrobacter...
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