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Application of wheat tazcct2 gene in regulation of plant flowering time

A flowering time, plant technology, applied in the field of plant genetic engineering, can solve the problem of lack of direct molecular evidence

Active Publication Date: 2021-06-22
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The VRN2 gene of winter wheat can prevent flowering in autumn and winter, so that wheat can survive the winter in a state of vegetative growth, and it will not be able to head and flower until the spring temperature warms and the long sunshine comes. However, VRN2 is not a gene directly down-regulated by low temperature. During the vernalization process The expression of VRN1 increases with the duration of vernalization, and the expression of VRN1 inhibits the expression of VRN2, resulting in the expression of VRN3 / FT gene (Trevaskis et al., 2006; Casao et al., 2011b; Chen al., 2012 ; Muterko et al., 2015; Tan et al., 2016)
[0010] The functional research of wheat ZCCT1 is relatively sufficient, and it is considered to be a gene that forms the requirement of vernalization; ZCCT2 has only genetic evidence in tetraploid wheat and is related to flowering time, lacking molecular direct evidence

Method used

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  • Application of wheat tazcct2 gene in regulation of plant flowering time
  • Application of wheat tazcct2 gene in regulation of plant flowering time
  • Application of wheat tazcct2 gene in regulation of plant flowering time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Sequence analysis, cloning and vector construction of wheat TaVRN2 gene

[0069] Through bioinformatics analysis, the present invention finds the TaVRN2 genome of wheat, constructs Ubi overexpression vector and transforms Brachypodium distachyon to obtain transgenic plants.

[0070] The amino acid sequence encoded by the TaVRN2 gene was retrieved in Gremene, and through phylogenetic tree analysis, it was found that this gene had high homology with barley HvZCCT-Ha and HvZCCT-Hb, and belonged to the same subclade (such as figure 1 shown). Amino acid multiple sequence alignment showed that TaVRN2 is highly homologous to the CCT-domain protein of barley, that is, it has a zinc finger structure and a conserved CCT-domain region composed of 43 AAs (such as figure 2 shown).

[0071] According to the TaVRN2 gene sequence of wheat found, primers were designed and cloned. The cloning method was as follows:

[0072] (1) Extraction of RNA: Total RNA of Yannong 15 wh...

Embodiment 2

[0093] Example 2: Acquisition and Vernalization of Wheat TaVRN2 Gene Overexpression Transgenic Plants

[0094] Utilize the method mediated by Agrobacterium to infect the Brachypodium distachyon callus to obtain transgenic seedlings, the specific implementation method is as follows:

[0095] (1) Three days before infection, pick Agrobacterium (a single colony of Agrobacterium carrying a recombinant plasmid) and inoculate it in YEP medium containing 50mg / L spectinomycin, shake the bacteria overnight at 28°C and 200rpm, and take 1ml the next day The activated bacteria were plated onto the MGL medium containing acetosyringone, cultured in the dark for two days, and prepared for infection.

[0096] (2) Collect the bacterial cells on the culture medium into 20ml of infection solution, shake them at 28°C, and measure the concentration, so that the OD600 value is between 0.6-1.0, and the A600 is 0.06-0.1. Collect about 300 pieces of calluses induced by young embryos of Brachypodium d...

Embodiment 3

[0104] Example 3: Detection of the expression level of wheat TaVRN2 gene overexpression transgenic plants

[0105] According to the design requirements of qRT-PCR primers, gene-specific primers were designed using software such as Beacon Designer 7 and Primer Premier 5.0. Select the SYBR Green Design option, create a file, input the sequence, run the BLASTsearch sequence and Template structure search tools, and run the Primer search tool to select the optimal primer sequence (first ensure the specificity of the primers and then consider avoiding the influence of the template structure). Primers were synthesized at Shanghai Sangon Bioengineering Service Co., Ltd. and purified by PAGE.

[0106] Application qRT-PCR special 96-well plate (Axygen, the United States) and high light transmittance sealing film (Axygen, the United States), fluorescent quantitative PCR instrument Icycler real-time PCR system (Bio-Rad, the United States) for qRT-PCR analysis, each Each sample was repeat...

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Abstract

The invention discloses an application of a wheat TaZCCT2 gene in regulating plant flowering time; the wheat TaZCCT2 gene includes: TaZCCT2‑A2, TaZCCT2‑B2 and TaZCCT2‑D2; the nucleotide sequence of the TaZCCT2‑A2 is shown in the sequence listing As shown in SEQ ID NO.4, the nucleotide sequence of TaZCCT2‑B2 is shown in SEQ ID NO.5, and the nucleotide sequence of TaZCCT2‑D2 is shown in SEQ ID NO.6. The present invention found for the first time that under the condition of TaZCCT2 gene overexpression, the plants showed no flowering phenotype under long-day conditions, and could not flower under saturated vernalization conditions, and could not flower after prolonged vernalization time, which belonged to the function of wheat TaZCCT2 gene new discoveries.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of a wheat TaZCCT2 gene in regulating plant flowering time. Background technique [0002] Flowering time is extremely important to the formation of crop yield: crops need an appropriate flowering time to meet the requirements of temperature, light and rainfall during flowering and fruit setting (Distelfeld et al., 2009; Jung et al., 2009). The current study shows that there are six flowering pathways in plants: vernalization pathway, autonomous pathway, photoperiod pathway, gibberellin (GA) pathway, age pathway and ambient temperature pathway (Fornara et al., 2010). The vernalization pathway means that plants need a period of low temperature before flowering to be able to flower and set fruit. After years of research, multiple vernalization-related functional genes have been cloned and identified in different plants and their regulatory networks ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H5/02A01H6/46
CPCC07K14/415C12N15/827
Inventor 安海龙孙晋浩孙德慧王敬丹赵翔宇张岳民
Owner SHANDONG AGRICULTURAL UNIVERSITY
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