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Alfalfa melatonin synthesis gene msasmt and its application in regulating the synthesis of melatonin and flavonoids in plants

A technology of alfalfa and melatonin, applied in the field of genetic engineering, can solve the problems of cloning, expression and application of the key enzyme gene for melatonin synthesis, and the growth and development regulation function of endogenous melatonin.

Active Publication Date: 2021-01-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing research, there is no research on the cloning, expression and application of the key enzyme gene of melatonin synthesis in alfalfa, and there is no research on the regulation function of endogenous melatonin on the growth and development of plants in alfalfa

Method used

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  • Alfalfa melatonin synthesis gene msasmt and its application in regulating the synthesis of melatonin and flavonoids in plants
  • Alfalfa melatonin synthesis gene msasmt and its application in regulating the synthesis of melatonin and flavonoids in plants
  • Alfalfa melatonin synthesis gene msasmt and its application in regulating the synthesis of melatonin and flavonoids in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Cloning of alfalfa melatonin synthesis key enzyme gene MsASMT

[0045] Cloning of alfalfa melatonin synthesis key enzyme gene MsASMT comprises the following steps:

[0046] 1. Extraction of total RNA from alfalfa leaves and synthesis of cDNA

[0047] (1) Take about 100 mg of the leaves of alfalfa seedlings Zhongmi No. 1, grind them into powder in liquid nitrogen, and extract the total plant RNA according to the Trizol method. After verification by agarose gel electrophoresis, high-quality RNA with complete 28S and 18S was obtained. And measure RNA concentration and purity with Nanodrop2000, OD 260 / OD 280 All between 1.8-2.0, OD 260 / OD 230 All >2.0, it proves that the obtained RNA is of good quality and can be used for the synthesis of the first strand of cDNA and the cloning of downstream genes.

[0048] (2) Using 1 μg of the above RNA sample as a template, use the PrimeScript RT reagent Kit (TaKaRa) kit to carry out reverse transcription according to t...

Embodiment 2

[0068] Construction of embodiment 2 MsASMT overexpression vector and acquisition of transgenic alfalfa

[0069] This example provides the construction of an overexpression vector for the key enzyme gene MsASMT in the synthesis of alfalfa melatonin and the method for obtaining transgenic alfalfa, which specifically includes the following steps:

[0070] 1. Construction of MsASMT plant overexpression vector

[0071] (1) Extract the plasmid from the Escherichia coli liquid containing the pMD18T-MsASMT plasmid with correct sequencing using the TaKaRa plasmid mini-extraction kit, and measure the concentration of the plasmid with NanoDrop2000.

[0072] (2) Since the upstream and downstream primers of gene cloning have restriction enzyme recognition sites of BamH I and Kpn I respectively, these two enzymes were selected to perform double enzyme digestion on the correctly sequenced plasmid samples and the plant overexpression vector pZh01, respectively. The system is as follows: 1 μg o...

Embodiment 3

[0094] Example 3 Phenotype and biological function analysis of transgenic alfalfa

[0095] 1. Analysis of melatonin content in transgenic alfalfa

[0096] Take about 1.5-2 g of the above-ground parts of the wild type and each transgenic plant, and repeat 3 times for each plant. After grinding with liquid nitrogen, leaching with methanol overnight, centrifuging at 8000 g for 10 min, passing the supernatant through a 0.25 μm filter membrane into a brown vial, using high-efficiency The content of melatonin in each transgenic and wild-type alfalfa was determined by liquid chromatography (HPLC). The result is as Figure 8 As shown, the melatonin content in the transgenic alfalfa overexpressing MsASMT was significantly increased. Using SPSS to analyze the correlation between the level of melatonin and the expression of MsASMT gene, the correlation coefficient is 0.720, and the corresponding significance is 0.029, indicating that there is a significant positive correlation between ...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a medicago sativa melatonin synthesis gene MsASMT and an application thereof in regulation and control of synthesis of plant melatonin and flavonoids. According to the invention, melatonin synthesis key enzyme MsASMT of medicago sativa is obtained through cloning, the amino sequence of the MsASMT is as shown in SEQ ID NO.1, and the CDS sequence of the coding gene is as shown in SEQ ID NO.2. The MsASMT gene has the functions of regulating and controlling the accumulation of melatonin, the synthetic metabolismof flavonoids, terpenoids, alkaloids, polyphenols or phenylpropanoids and the growth and development of plants. The discovery of the MsASMT gene and functions thereof provides a theoretical basis anda new method for the breeding of high-quality new varieties of medicago and the quality breeding of other forage grass.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to MsASMT, a key enzyme for alfalfa melatonin synthesis, its coding gene and its role in regulating plant melatonin, flavonoids, terpenoids, alkaloids, polyphenols or Synthesis of phenylpropanoids and their application in plant growth and development. Background technique [0002] Alfalfa (Medicago sativa L.) is an important perennial leguminous forage in the world. It has been called the "king of forage" because of its excellent grass quality, rich nutrition and high protein content. Alfalfa is widely cultivated in the world and is one of the important perennial forage crops in the world. The planting area in the world reaches 3.2×10 7 hectare. The alfalfa planting area in China is about 3.8×10 6 hectares, mainly distributed in Northwest, North China and Northeast China. Alfalfa is not only rich in protein, but also contains high vitamins and flavonoids and other ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/04A01H5/12A01H6/54
CPCC12N9/1007C12N15/8243C12Y201/01004
Inventor 张蕴薇岑慧芳刘斯佳黄艳华田丹阳崔鑫管聪刘华玥
Owner CHINA AGRI UNIV
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