Longan flowering period regulation gene DlCO14 as well as protein and application thereof

A technology for regulating genes and regulating proteins, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2021-11-26
CHONGQING UNIV OF ARTS & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the members of the longan COL family, especially the mechanism of action and expression pathways of specific genes in the process of flower formation. What role they play in the flower formation of longan is worthy of further study

Method used

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  • Longan flowering period regulation gene DlCO14 as well as protein and application thereof
  • Longan flowering period regulation gene DlCO14 as well as protein and application thereof
  • Longan flowering period regulation gene DlCO14 as well as protein and application thereof

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Experimental program
Comparison scheme
Effect test

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 DlCO14 gene sequence (terminator removed) (Table 1) to amplify the full length of the ORF of DlCO14, 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 DlCO14 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-DlCO14-osgfp plasmid. Then it was transferred into Arabidopsis protoplasts by PEG-mediated method (Yoo S D, Cho Y H, Sheen J.Arabidopsis mesophyll proto-plasts: a versatile cell system for transient gen...

Embodiment 3

[0040] Example 3 Overexpression vector construction and functional verification of transgenic Arabidopsis

[0041] Using specific PCR primers OECO14-S / OECO14-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 DlCO14 target gene was constructed by T4 DNA ligase, and named pBI121-DlCO14. The constructed overexpression vector pBI121-DlCO14 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: a simplified method for Agrobacterium-medi...

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Abstract

The invention provides a longan flowering period regulation gene DlCO14. A cDNA nucleotide sequence of the longan flowering period regulation gene DlCO14 is shown as SEQ ID No. 1. The full length of an open reading frame of the longan DlCO14 gene is 1038bp, 345 amino acids are encoded, and the longan DlCO14 gene has a typical CCT structural domain and belongs to COSTANS-like protein. A qRT-PCR result shows that the gene has tissue expression specificity, the relative expression quantity in flower buds is relatively high, and the relative expression quantity in pericarp, pulp and seed organs is lower than that in the flower buds; and the expression is down-regulated in the early flowering induction stage of 'Sijimi' and 'Shixia' longan. A transient expression result of the arabidopsis protoplast shows that a fluorescence signal is mainly concentrated on a cell nucleus. Results of transgenic arabidopsis show that a DlCO14 overexpressed transgenic plant shows a late flowering phenomenon: a wild type plant blooms in about 25 days, and a transgenic line blooms in 33-36 days; and meanwhile, the number of rosette leaves is increased, and the leaves become large. As a typical transcription factor, the protein coded by the DlCO14 is located in the cell nucleus and negatively regulates plant flowering, and overexpression of the DlCO14 gene can significantly inhibit plant flowering.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to the longan flowering stage regulating gene DlCO14 and its protein and application. Background technique [0002] Longan (Dimocarpus longana Lour.) is an important subtropical fruit tree of Sapinaceae, widely planted in many tropical and subtropical countries such as South Asia and Australia. Seasonal flowering and fruiting is an important issue affecting longan yield. Longan flower induction requires a certain period of low temperature (vernalization) and suitable drought conditions (Jue D, Sang X, Liu L, et al.Comprehensive analysis of the longan transcriptome reveals distinctregulatory programs during the floral transition[J].BMC genomics, 2019,20(1):1-18.), the annual production and income reductions caused by climate anomalies are very serious. In my country's main production areas, such as the western part of Guangdong and Hainan, because they do not have the lo...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/02A01H6/20
CPCC07K14/415C12N15/827
Inventor桑雪莲决登伟石胜友唐建民兰建彬张文林
OwnerCHONGQING UNIV OF ARTS & SCI