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Astragalus sinicus LHY gene and application thereof

A milk vetch and gene technology, applied in the field of genetic engineering and plant genetic breeding, can solve the problems of unclear functions of flowering-related genes and achieve good application prospects

Active Publication Date: 2020-05-19
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the flowering mechanism of milk vetch and the role of flowering-related genes in this process are still unclear

Method used

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  • Astragalus sinicus LHY gene and application thereof
  • Astragalus sinicus LHY gene and application thereof
  • Astragalus sinicus LHY gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Example 1 Cloning and sequence analysis of milk vetch LHY gene

[0035] The present invention uses RACE technology to obtain the full-length cDNA sequence of the LHY gene from milk vetch, which is 2865bp (SEQ ID NO: 1), contains an open reading frame of 2259bp, and the lengths of the 5' and 3' non-coding regions are 407bp and 3' respectively. 199bp. The PolyA tail signal peptide region is located at 2854-2859bp. The amino acid sequence of the encoded protein is shown in SEQ ID NO:2. The specific method is as follows: ...

Embodiment 2

[0101] Example 2 Detection of relative expression levels of LHY gene in various tissues of milk vetch

[0102] 1. Total RNA extraction

[0103] Milk vetch is a plant material grown in a greenhouse, and it is a fresh milk vetch tissue sample cultivated by collecting seeds from common milk vetch plant materials.

[0104] 2. Design and synthesis of fluorescent quantitative PCR primers

[0105] Primer Premier 6.0 and Beacon designer 7.8 software were used for quantitative PCR primer design, and then synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The internal reference gene used was 18S rDNA (GenBank: AF359603.1).

[0106] 3. Real-Time PCR (Q-PCR) statistical analysis of gene expression differences

[0107] Each sample was repeated three times, and the relative expression levels of each gene were compared with 2 (Ct内参基因－Ct目的基因) conduct statistical analysis. The results of LHY gene Q-PCR expression analysis are shown in Table 17.

[0108] Table 17 LHY gene Q-PCR exp...

Embodiment 3

[0112]Embodiment 3 The cultivation of transgenic Arabidopsis thaliana and its phenotype analysis

[0113] 1. Construction of a recombinant plasmid containing the milk vetch LHY gene (SEQ ID NO: 1) of the present invention.

[0114] 2. Transform Agrobacterium Competent

[0115] Transform the recombinant plasmids with correct sequencing into competent Agrobacterium. Colony PCR identification showed that the vector plasmid had been successfully transferred into Agrobacterium.

[0116] 3. Arabidopsis transformation process (inflorescence dipping method)

[0117] (1) Planting: Choose the rock with good water absorption and soft soil with nutrient soil (1:1 / 2) as the planting soil for Arabidopsis. For flower pots with a diameter of 9 cm, 100-150 seeds are sown in each pot. After sowing, cover the flowerpot with a film to provide a moist environment for the growth of the plant.

[0118] (2) Transplanting: 10-15 days after sowing, the Arabidopsis thaliana seedlings grow to the fo...

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Abstract

The invention provides an astragalus sinicus LHY gene and application thereof. The LHY gene of astragalus sinicus and a sequence of a protein encoded by the LHY gene are shown in SEQ ID NO: 1 and 2. The LHY gene is cloned from the astragalus sinicus for the first time, and the LHY gene is verified to have a function of regulating and controlling plant flowering, in particular, delaying plant flowering, in arabidopsis thaliana through an agrobacterium-mediated genetic transformation method. The LHY transgenic arabidopsis thaliana shows early bolting and early flowering, bolting days are 18.36 days later than wild type, and flowering days are 20.72 days later than wild type. This indicates that LHY gene of astragalus sinicus is closely related to flowering, and has a function of regulating flowering time. The gene is applied to plant property improvement, and has good application prospect. The LHY gene and the encoded protein thereof provide valuable resources for cultivating new plant varieties.

Description

technical field [0001] The invention relates to the fields of genetic engineering and plant genetic breeding, in particular to the milk vetch LHY gene and its application. Background technique [0002] Milk vetch (Astragalus sinicus L.) is a biennial herbaceous plant belonging to the Leguminosae family. It is mainly distributed in the Yangtze River Basin of China and is an important nectar source plant. Milk vetch is of great significance to the sustainable utilization of soil resources. [0003] Flowering is the key transformation of plants from vegetative growth to reproductive growth, and it is also the central link to realize the alternation of generations, which largely determines the success of plant reproduction. According to the morning and evening of flowering, milk vetch can be divided into extra early flowering type, early flowering type, medium flowering type and late flowering type. The flowering time directly determines the length of the effective growing seas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20A01H6/54
CPCC07K14/415C12N15/827
Inventor 张贤王建红曹凯徐静斯林林
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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