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Plant anthocyanin metabolism related gene Rd3GTs as well as encoding protein and application thereof

An anthocyanin and gene technology, applied in the field of bioengineering, can solve the problems of restricting the utilization regulation and improvement of Rhododendron 3GT, restricting the research of 3GT evolution, etc.

Active Publication Date: 2021-10-01
GUIZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous improvement of cDNA cloning technology, domestic researchers have successively cloned the cDNA or genomic DNA sequence of the 3GT gene from many plants, but there is no report on the cloning and functional research of the 3GT gene of Rhododendron lantana. It limits people's research on the evolution of 3GT, and also limits people's utilization of Rhododendron 3GT and the regulation and improvement of anthocyanin biosynthesis

Method used

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  • Plant anthocyanin metabolism related gene Rd3GTs as well as encoding protein and application thereof
  • Plant anthocyanin metabolism related gene Rd3GTs as well as encoding protein and application thereof
  • Plant anthocyanin metabolism related gene Rd3GTs as well as encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] In this example, the Rhododendron lantana Rd3GTs gene was cloned.

[0047] The total RNA of Rhododendron lantana was extracted, and then sent to transcriptome sequencing. According to the results of transcriptome sequencing, two sets of primers were designed. The primer sequences are shown in Table 1.

[0048] Table 1 Sequences of primers for cloning Rhododendron lantana Rd3GTs gene

[0049]

[0050] The nucleotide sequence of primer 3GT-1F1 in table 1 is shown in SEQ ID NO:5; The nucleotide sequence of primer 3GT-1F2 is shown in SEQ ID NO:6; The nucleotide sequence of primer 3GT-6F1 is shown in SEQ ID NO:6 Shown in ID NO:7; The nucleotide sequence of primer 3GT-6F2 is shown in SEQ ID NO:8.

[0051] Using the total RNA of Rhododendron lantana as a template for PCR amplification, the primer pair 3GT-1F1 and 3GT-1F2 can be used to clone the amplified fragment of 1425bp, and the coding gene Rd3GT1 of Rd3GT1 of Rhododendron lantana Rd3GT1 was obtained by ORFFinder softw...

Embodiment 2

[0055] In this example, the enzyme activity of Rhododendron lantana Rd3GTs was detected.

[0056] 1. Construction of prokaryotic expression vector

[0057] (1) Using the cloned Rd3GT1 and Rd3GT6 genes as templates, using primer pairs DJ-3GT1-32 / 28F and DJ-3GT1-32 / 28R with EcoRI restriction sites, and primers with BamH I and HindIII enzymes respectively The primers for the cleavage site were used for PCR amplification of DJ-3GT6-32 / 28F and DJ-3GT6-32 / 28R. The primer sequences are shown in Table 2.

[0058] Table 2 Sequences of primers for amplification of Rhododendron lanana Rd3GTs gene

[0059]

[0060]

[0061] The nucleotide sequence of primer DJ-3GT1-32 / 28F in table 2 is shown in SEQ ID NO:9; The nucleotide sequence of primer DJ-3GT1-32 / 28R is shown in SEQ ID NO:10; Primer DJ- The nucleotide sequence of 3GT6-32 / 28F is shown in SEQ ID NO:11; the nucleotide sequence of primer DJ-3GT6-32 / 28R is shown in SEQ ID NO:12.

[0062] The PCR program is 94°C for 8min; 94°C for...

Embodiment 3

[0091] This example studies the effect of Rd3GTs on the synthesis of Arabidopsis and tobacco anthocyanins.

[0092] 1. Construction of binary expression vector

[0093] (1) Using the cloned Rd3GT1 and Rd3GT6 genes as templates, PCR amplification was performed using primers with BamHI and XbaI restriction sites, respectively. The sequences of the primers are shown in Table 4.

[0094] Table 4 Sequences of primers for amplification of Rhododendron lantana Rd3GTs gene

[0095]

[0096] The nucleotide sequence of primer DJ-3GT1-121F in table 4 is shown in SEQ ID NO:13; The nucleotide sequence of primer DJ-3GT1-121R is shown in SEQ ID NO:14; The nucleotide sequence of primer DJ-3GT6-121F The nucleotide sequence is shown in SEQ ID NO:15; the nucleotide sequence of primer DJ-3GT6-121R is shown in SEQ ID NO:16.

[0097] The PCR program is 94°C for 8min; 94°C for 30s, 54°C for 1.5min, 72°C for 8min, 30cycles.

[0098] (2) A large number of gene fragments were obtained by PCR ampl...

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Abstract

The invention discloses a rhododendron delavayi anthocyanin metabolism related gene Rd3GTs, and belongs to the technical field of bioengineering. The nucleotide sequence of the Rd3GTs gene in the invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The invention also discloses a recombinant Rd3GTs protein expressed by the Rd3GTs gene in escherichia coli cells. The protein can catalyze cyanidin in vitro to react to generate cyanidin 3-O-glucoside, and the protein has the function of flavonoid 3-O-glycosyltransferase. The invention further discloses application of the Rd3GTs gene in transgenic plants, the Rd3GTs gene is transferred into an arabidopsis mutant and tobacco, the anthocyanin phenotype of the arabidopsis mutant is successfully recovered, and the color of tobacco petals is deepened. The Rd3GTs gene provided by the invention has potential application value in the aspects of flower color modification of transgenic plants and flowers and improvement of medicinal components of medicinal plants.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a plant anthocyanin metabolism-related gene Rd3GTs and its encoded protein and application. Background technique [0002] Anthocyanins are one of the most important pigment substances affecting the color presentation of plants, which can give plants a series of different colors from red to purple. Studies have shown that in addition to imparting color to flowers, fruits and other tissues, anthocyanins also have many important functions. It can protect plant cells from ultraviolet rays, resist pathogens and herbivores, act as a signal molecule to promote the interaction between plants and microorganisms, affect the growth and development of pollen, and the transport of hormones in plants. In addition, a large number of experiments have proved that there is also a close relationship between anthocyanins and human health. It has biological activities such as anti-oxidation, ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/02A01H6/20A01H6/82
CPCC12N9/1048C12N15/825
Inventor 孙威张艳孙世宇鞠志刚乙引
Owner GUIZHOU NORMAL UNIVERSITY
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