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Wheat TaARF12 gene and application thereof

A wheat and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing plant height, improving and improving germplasm resources

Active Publication Date: 2020-02-28
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are few studies on ARF genes in wheat, especially the functional research and production application of TaARF12 in wheat

Method used

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  • Wheat TaARF12 gene and application thereof
  • Wheat TaARF12 gene and application thereof
  • Wheat TaARF12 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Cloning of embodiment 1 wheat TaARF12 gene

[0052] 1. Extraction of total RNA from young ears of wheat

[0053] Total RNA was extracted from Chinese spring (Triticum aestivum L.) young panicle using Plant mini Kit (QIAGEN).

[0054] 2. Synthesis of the first strand of cDNA

[0055] Follow the operating instructions of reverse transcriptase EasyScript One-Step gDNA Removal and cDNA SynthesisSuperMix (Beijing Quanshijin Biotechnology Co., Ltd.).

[0056] 3. Clone the coding region sequence of TaARF12 from the cDNA of young panicle of Chinese spring (Triticum aestivum L.). Forward primer sequence such as SEQ ID NO.4, reverse primer sequence such as SEQ ID NO.5.

[0057] The PCR system and procedures were carried out with reference to the operating instructions of TransStart FastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.).

[0058] After the PCR product was gel-cut, recovered and purified, it was cloned and connected to the Blunt Zero Cloning vector...

Embodiment 2

[0059] Example 2 Construction of TaARF12 RNAi vector and identification and statistics of transgenic plants

[0060] a. Construction of pWMB006-TaARF12 intermediate vector

[0061] (1) Using primers SEQ ID NO.6 and SEQ ID NO.7, using the sequenced TaARF12-A plasmid as a template, amplify a specific sequence of TaARF12-A with BamH I and Kpn I restriction sites, double Digest the purified PCR product, and use the gel recovery kit to recover the target fragment;

[0062] (2) double enzyme digestion pWMB006 vector (see figure 1 ), using the gel recovery kit to recover the linearized carrier;

[0063] (3) Ligation, the reaction system is: 2 μL of the target fragment obtained in step (1), 2 μL of the linearized vector pWMB006, 1 μL of T4 ligase, 1 μL of T4 ligation buffer, and ddH 2 Make up to 10 μL with O, connect overnight at 16°C;

[0064] (4) Transformation of ligation products and selection of positive clones for sequencing.

[0065] (5) Another reverse complementary fragm...

Embodiment 3

[0074] Example 3 Detection of gene editing in TaARF12 CRISPR / Cas9 transgenic plants

[0075] (1) According to the location information of the target site, design primers spanning the target site, the primers for target site 1 are SEQ ID NO.19-20, and the primers for target site 2 are SEQ ID NO.21-22.

[0076] (2) Use the nucleotide sequence SEQ ID NO.13-14 to carry out PCR identification in transgenic wheat leaves, and the 430bp band represents the positive plant (see Figure 9 ).

[0077] (3) Perform PCR using primers SEQ ID NO.19-20 and SEQ ID NO.21-22 respectively in the DNA in the transgenic plants. The PCR system and procedures were carried out with reference to the operating instructions of TransStart FastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.).

[0078] (4) PCR products were recovered and purified by gel cutting and ligated to Blunt Zero Cloning vectors according to the Blunt Zero Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd.) cloning...

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Abstract

The invention provides a wheat TaARF12 gene and application thereof, and belongs to the field of molecular biology of crops. A designed primer clones a coding region sequence of TaARF12 from cDNA of Chinese spring young ears, and the coding region sequence of the TaARF12 gene in homologous chromosomes A, B and D is represented as SEQ ID NO. 1-3 respectively. A TaARF12-specific RNAi fragment is selected, an RNAi vector is created, and Fierder is transformed. A CRISPR / Cas9 vector is used to create a gene editing vector containing two target sites and transform wild-type Fierder. Obtained RANi transgenic lines and gene editing lines have similar phenotypes, with plants becoming shorter and ears becoming longer. It is indicated that the wheat TaARF12 gene can regulate the plant height and theear length, and the plants become shorter and the ears become longer after the gene is interfered and expressed.

Description

technical field [0001] The present invention relates to the field of crop molecular biology, more specifically, relates to the cloning of wheat TaARF12 gene, the construction method of TaARF12 RNAi (RNA interfering) vector, the application of CRISPR / Cas9-TaARF12 vector, and the role of TaARF12 gene in regulating plant height and ear length in the application. Background technique [0002] Common wheat is an important food crop, and more than 1 / 3 of the world's population uses wheat as a staple food. Wheat plant height and ear length have important effects on the formation of yield, and the study of its developmental genetics is helpful to understand the mechanism of yield formation. In the 1970s, the Green Revolution led by the use of wheat's semi-dwarf gene brought a great increase in global wheat production. Reducing the plant height can improve the lodging resistance of wheat, and the dwarf gene can be fully utilized in breeding to breed varieties with lodging resistanc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C12N15/113C12N15/82A01H5/00A01H6/46C12Q1/6895
CPCC07K14/415C12N15/1096C12N15/8218C12N15/8261C12Q1/6895C12Q2600/13C12Q2531/113
Inventor 李爱丽王芳耿帅锋毛龙
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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