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Phalaenopsis aphrodite R3-MYBx1 gene and application thereof in flower color adjustment

A Phalaenopsis, r3-mybx1 technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem that the color of tomato fruit cannot be changed, etc.

Active Publication Date: 2018-02-23
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Transgenic studies in tomato have shown that the gene can inhibit the accumulation of anthocyanins in leaves, stems and other tissues, but cannot change the color change of tomato fruit
In addition, the study of members of the same type as this gene in changing petal coloration has not been reported

Method used

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  • Phalaenopsis aphrodite R3-MYBx1 gene and application thereof in flower color adjustment
  • Phalaenopsis aphrodite R3-MYBx1 gene and application thereof in flower color adjustment
  • Phalaenopsis aphrodite R3-MYBx1 gene and application thereof in flower color adjustment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] This example mainly introduces the cloning process of the Phalaenopsis R3-MYB gene (including the two gene products of Phalaenopsis MYBx1 and MYBx2), and conducts a preliminary analysis of its expression pattern in Phalaenopsis plants. The specific process is as follows.

[0066] (1) Extract total RNA from Phalaenopsis petals and reverse transcribe it into cDNA. The specific process is described as follows.

[0067] Phalaenopsis RNA was extracted by the Trizol method, and the specific process was as follows:

[0068] Add 1.5mL Trizol to a 2mL centrifuge tube, take 1g~2g of the Phalaenopsis 'Big Pepper' petal sample after quick freezing in liquid nitrogen, grind it and add it to the centrifuge tube, mix quickly, vortex for 2min~3min; 25℃ water bath for 10min ;

[0069] Add 300 μL of chloroform, vortex and shake for 2 min to 3 min, and centrifuge at 12000 rpm for 15 min at 4 °C;

[0070] Slowly pipette 850 μL supernatant into a new 1.5 mL centrifuge tube, add 850 μL iso...

Embodiment 2

[0134] In order to further study and determine the role of Phalaenopsis MYBx1 gene in the regulation of Phalaenopsis flower color, the inventors further constructed related transgenic vectors and transformed petunia by using gateway technology. The relevant experiments are briefly introduced as follows.

[0135] (1) Vector construction

[0136] First, the PCR product of the MYBx1 gene was integrated into the entry clone pDONR207 by using Gateway™ BP Clonase™ II Enzyme mix. The operation steps are as follows:

[0137] Reaction system: PCR product 4 μL (Example 1) + pDONR207 vector 1 μL + BP enzyme 1 μL; overnight at room temperature (or 1 hour at 25°C);

[0138] Add 1 μL of protein kinase to the reaction solution, and react at 37°C for 10 min.

[0139] To transform the reaction product into Escherichia coli JM109, refer to the JM109 Competent Transformation Manual of Takara Company for this process, or refer to the following steps for details:

[0140] Dissolve Escherichia co...

Embodiment 3

[0206] In this example, the gateway technology was used to construct the yeast two-hybrid vector, and then a preliminary analysis was carried out on whether MYBx1 acts on the components of the MBW complex regulating anthocyanin biosynthesis. The related experiments are briefly introduced as follows.

[0207] First, use the fusion vector (entry vector) of MYBx1 gene and pDONR207 to exchange MYBx1 into pGBKT7 / GW and pGADT7 / GW through LR reaction to create the destination vector for yeast two-hybrid (refer to Example 2 for specific operations), other yeast The two-hybrid vector is a vector previously preserved in our laboratory.

[0208] However, it should be noted that the GAL4 gene fused in the pGBKT7 / GW and pGADT7 / GW vectors is located downstream of the target gene. Therefore, the PCR product used to construct the pDONR207 fusion vector is the full-length gene sequence of MYBx1 without a stop codon. PCR The primers used for cloning are as follows:

[0209] F: 5'-GGGGACAAGTTT...

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Abstract

The invention belongs to the technical field of plant variety cultivation, and specifically relates to a phalaenopsis aphrodite R3-MYBx1 gene and application thereof in flower color adjustment. An open reading frame of the gene contains a 162bp base, and a base sequence of the gene is shown as SEQ ID NO.1. The MYBx1 gene participates in a petal coloration process, but the MYBx1 gene can express both white petals and red petals; phalaenopsis aphrodite petal coloration can be affected by a plurality of transcription factors, and the MYBx1 is one of regulation factors and participates in the petal coloration process. Application of the gene in overexpression in petunia shows that the gene is related to an anthocyanin content, and the stronger expression of the gene is, the stronger anthocyanin synthesis inhibition capacity is. The MYBx1 is the gene which is researched and shown for the first time to have an obvious and specific anthocyanin degrading function. Through overexpression in plants, the gene can create novel varieties with novel petal coloration characteristics.

Description

technical field [0001] The application belongs to the technical field of plant variety breeding, and specifically relates to a Phalaenopsis R3-MYBx1 gene and its application in flower color regulation. Background technique [0002] Plant petal color is one of the important ornamental traits, and Phalaenopsis ( Phalaenopsis aphrodite ) Petal color is also one of the main ways to reflect its value. Studies have shown that the coloring of petals is mainly caused by the accumulation of anthocyanins in petal cells. [0003] Anthocyanins are a class of flavonoids. The genes controlling anthocyanin synthesis in plants include anthocyanin synthesis structural genes and anthocyanin synthesis regulatory genes. Among the regulatory genes that control the synthesis of anthocyanins, it has been found that R2R3-MYB (Arabidopsis TT2, maize ZmC1, petunia AN2, AN4, DPL, PHZ, etc.), bHLH (Arabidopsis GL3, maize ZmR, ZmB , petunia AN1, etc.) and WD40 (Arabidopsis TTG1, petunia AN11) and oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/02A01H6/82
CPCC07K14/415C12N15/8205C12N15/827
Inventor 张和臣王利民符真珠董晓宇张晶郑谊李艳敏王慧娟蒋卉高杰
Owner HENAN ACAD OF AGRI SCI
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