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Genes regulating plant seed germination and seedling growth, their encoded proteins and their applications

A plant seed and gene technology, applied in the biological field, can solve the problems of limited research on CIPK23 and no CIPK23 involved in the normal growth and development of plants, and achieve the effects of accelerating germination rate, improving genetic characteristics, and improving the uniformity of unearthed

Active Publication Date: 2022-06-28
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although CIPK23 has been intensively studied in Arabidopsis and other plants, the research on CIPK23 in Solanaceae crops is extremely limited
At the same time, there is no report that CIPK23 is involved in the normal growth and development of plants.

Method used

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  • Genes regulating plant seed germination and seedling growth, their encoded proteins and their applications
  • Genes regulating plant seed germination and seedling growth, their encoded proteins and their applications
  • Genes regulating plant seed germination and seedling growth, their encoded proteins and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction of expression vector containing NtCIPK23 protein kinase gene

[0044] 1. Primers used for construction of expression vector of NtCIPK23 protein kinase gene

[0045] Primers used to clone NtCIPK23CDS sequences:

[0046] NtCIPK23-1F: GGCATGGGTTCAAGATCAAATAATGG

[0047] NtCIPK23-1R: CGCTCGCCTAACCATCTTTTAC

[0048] Primers used to clone the NtCIPK23 promoter sequence:

[0049] NtCIPK23pro-1F:AAGCTTGAGGCTTCTGCTGGTTGGAG

[0050] NtCIPK23pro-1R: GGATCCCTACCTCCAAACTTTCTATTTCTTACAG

[0051] Primers used for construction of NtCIPK23-CRISPR / Cas9 vector:

[0052] NtCIPK23CR-1Target-1F: GATTGTGATGTAGGGAGGACCCTTG

[0053] NtCIPK23CR-1Target-1R: AAACCAAGGGTCCTCCCTACATCA

[0054] NtCIPK23pro - Primers used to construct pBI101-ProNtCIPK23::GUS vector:

[0055] NtCIPK23pro-2F-HindⅢ: CCCAAGCTTGAGGCTTCTGCTGGTTGGAG

[0056] 2R-BamHI: CGGGATCCCTACCTCCAAACTTTCTATTTCTTACAG

[0057] 2. This study adopts the method of homologous cloning. By using the known CDS ...

Embodiment 2

[0059] Example 2. Transformation and screening of transgenic plants containing NtCIPK23 gene

[0060] 1. Primers used for NtCIPK23 gene transformation and screening of transgenic plants

[0061] For positive identification of NtCIPK23 overexpressing material:

[0062] NtCIPK23-1F: GGCATGGGTTCAAGATCAAATAATGG

[0063] pCHF3-R: ATTCTGGTGTGTGCGCAATG

[0064] For positive identification of NtCIPK23 promoter transgenic material:

[0065] pBI101-F: CCGATTCATTAATGCAGCTG

[0066] NtCIPK23pro-2R: GGATCCCTACCTCCAAACTTTCTATTTCTTACAG

[0067] For positive identification of NtCIPK23 CRISPR / Cas9 transgenic material:

[0068] pORE-F: TTAGGTTTACCCGCCAATA

[0069] NtCIPK23CR-1Target-1R: AAACCAAGGGTCCTCCCTACATCA

[0070] For detection of NtCIPK23 editing sites:

[0071] NtCIPK23-1-UTR2F: ACAAGAGGATGGGATTTGT

[0072] NtCIPK23-1-145R: ATCACCAGTTTCAACATTCC

[0073] For NtCIPK23 amplification in qPCR:

[0074] NtCIPK23-qF: CCACTGACTATGAATGCTTT

[0075] NtCIPK23-qR: GCGCCCACTCTTCTCCCCA

...

Embodiment 3

[0083] Embodiment 3. Observation of GUS staining experiment

[0084] ProNtCIPK23::GUS transgenic seeds were sterilized and sown on 1 / 2 MS medium. Four days later, the radicle broke through the seed coat, germinated and began sampling. The seedlings at different germination stages were selected for GUS histochemical staining (the exposure process of radicle, the extension process of hypocotyl, the extension stage of cotyledons, the emergence stage of two true leaves and the growth stage of two true leaves). The samples were completely immersed in GUS staining solution (LEAGENE Lot. 1127A19) and incubated at 37°C for 24 hours. After that, the chlorophyll of the samples was completely removed with ethanol for microscopic observation. The result is as figure 2 b, GUS staining analysis showed that NtCIPK23 was expressed and regulated at the seed germination stage, and this gene may play an important role in the elongation of the hypocotyl and the expansion of the cotyledons.

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Abstract

The invention discloses a gene for regulating plant seed germination and seedling growth, its encoded protein and application. The inventors of the present invention identified and obtained the NtCIPK23 gene from the common tobacco (Nicotiana tabacum) cultivar Zhongyan 100, the nucleotide sequence of the NtCIPK23 gene is shown in SEQ ID NO.1, and proved by transgenic technology that overexpressing NtCIPK23 can Accelerate the germination rate of tobacco seeds and promote the growth of seedlings, while knocking out the gene will delay the germination rate of seedlings and inhibit the growth of seedlings. By using the present invention, the germination rate and uniformity of plant seeds can be controlled by adjusting the expression level of CIPK23, and the transgenic crops with moderate expression of CIPK23 gene and uniform emergence rate can be obtained by means of genetic engineering. The invention meets the requirements of sustainable agricultural development, and has important practical value and market prospect for cultivating economic crops that grow early, grow fast, and emerge neatly.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene for regulating plant seed germination and seedling growth, its encoded protein and its application. Background technique [0002] Calcium ions (Ca 2+ ) is a ubiquitous second messenger in plant cells. When plants are stimulated by the outside world, intracellular Ca 2+ Temporal and spatial specific changes occur in the concentration of , resulting in the formation of calcium signals. In this calcium signal will be sensed by calcium sensor, signal transduction to downstream effector proteins, causing physiological and biochemical reactions of plant cells, thus participating in the process of plant growth and development and stress response). Calcineurin B-like protein CBL (Calci-neurin B-like protein) is one of the calcium sensor proteins, which can efficiently interact with Ca. 2+ Binding and causing a conformational change to form a CBL-CIPK complex by combining with the t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/84A01H5/00A01H6/82
CPCC12N9/12C12Y207/11C12N15/8267
Inventor 王倩刘好宝安璐璐史素娟徐方正毛静静
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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