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

Pending Publication Date: 2021-11-30
CHONGQING UNIV OF ARTS & SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are also few reports on the members of the longan GRAS family. It is of great significance to explo

Method used

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  • Longan gene DlGRAS34, protein and application of longan gene DlGRAS34 and protein in regulating and controlling flowering of plants
  • Longan gene DlGRAS34, protein and application of longan gene DlGRAS34 and protein in regulating and controlling flowering of plants
  • Longan gene DlGRAS34, protein and application of longan gene DlGRAS34 and protein in regulating and controlling flowering of plants

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Experimental program
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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 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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Abstract

The invention discloses a longan gene DlGRAS34, a protein and an application of the longan gene DlGRAS34 and the protein in regulating and controlling flowering of plants. A cDNA nucleotide sequence of the longan gene DlGRAS34 is shown as SEQ ID No.1. The full length of an open reading frame of the longan gene DlGRAS34 is 1578bp, 525 amino acids are encoded, and the longan gene DlGRAS34 has a typical GRAS structural domain and a DELLA structure and belongs to GRAS protein. A qRT-PCR result shows that the gene has tissue expression specificity, has the highest relative expression quantity in pericarp, and has the second relative expression quantity in stem, leaf and flower organs; expression is down-regulated in the early flowering induction stage of 'Sijimi' longan, and expression is up-regulated in the later flowering stage of 'Shixia' longan. A transient expression result of the arabidopsis protoplast shows that a fluorescence signal is mainly concentrated on a cell nucleus. A transgenic arabidopsis thaliana result shows that a DlGRAS34-overexpressed transgenic plant shows a late flowering phenomenon that a wild type plant blooms in about 27 days, and a transgenic line blooms in 32-35 days; and meanwhile, rosette leaves become large, and plants become short. The results show that as a typical transcription factor, the protein coded by the DlGRAS34 is located in a cell nucleus, plant flowering is negatively regulated, and the overexpression of the DlGRAS34 gene can significantly inhibit plant flowering.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to the application of longan gene DlGRAS34 and protein in regulating plant flowering. 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): C12N15/29C12N15/82C07K14/415A01H5/02A01H6/00A01H6/20
CPCC07K14/415C12N15/827
Inventor 桑雪莲决登伟石胜友唐建民廖钦洪李哲馨
Owner CHONGQING UNIV OF ARTS & SCI
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