Nucleic acid molecule, vector, cell and primer, application of nucleic acid molecule, vector, cell and primer, and plant high-purity cloned seed sorting method based on dual regulation and control

A nucleic acid molecule and recombinant vector technology, which is used in nucleic acid vectors, botanical equipment and methods, and the introduction of foreign genetic material using vectors. It can solve problems such as inability to obtain cloned seeds, achieve broad application and market prospects, and improve screening efficiency Effect

Pending Publication Date: 2022-03-15
HUNAN HYBRID RICE RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method provided by the invention can improve the high-purity sorting of cloned seeds based on dual regulation, and solve the problem that the existing heterosis fixed sorting technology cannot obtain cloned seeds with required purity, so as to realize the method of hybrid non-fusion system application

Method used

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  • Nucleic acid molecule, vector, cell and primer, application of nucleic acid molecule, vector, cell and primer, and plant high-purity cloned seed sorting method based on dual regulation and control

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Embodiment approach

[0060] According to a preferred embodiment of the present invention, the nucleic acid molecule further comprises an expression cassette E2, and the expression cassette E2 carries a regulatory gene capable of recognizing the first regulatory site and the second regulatory site and excising the first regulatory site and the second regulatory site. An element between a first regulatory site and a second regulatory site.

[0061] According to a preferred embodiment of the present invention, the expression cassette E2 includes the following elements from upstream to downstream: a third promoter, a regulatory gene and a fourth terminator, more preferably, the expression cassette E2 includes from upstream to downstream The following elements: pollen-specific expression promoter, regulatory gene and fourth terminator.

[0062] According to the present invention, the pollen specific expression promoter may include but not limited to PG47; preferably PG47, the sequence of the PG47 promo...

Embodiment 1

[0128] This example is used to illustrate the vector construction of the apomictic vector p68C

[0129] 1. Synthetic expression cassettes E1, E2 and E3

[0130] Synthetic expression cassette E1, the sequence of which is shown in SEQ ID NO.2: including embryo-specific expression promoter OsESP1 (SEQ ID NO.2), Loxp-FRT fusion recognition site (SEQ ID NO.3), double-strand break inducing enzyme I -SceI site (SEQ ID NO.4), NOS terminator (SEQ ID NO.5), pollen-specific expression promoter pG47 (SEQ ID NO.6), lethal gene ZmAA1 (SEQ ID NO.7), IN2-1 Terminator (SEQ ID NO.8), double-strand break inducing enzyme I-SceI site (SEQ ID NO.4), Loxp-FRT fusion recognition site (SEQ ID NO.3), red fluorescent protein gene DsRed2 (SEQ ID NO. NO.9), NOS terminator (SEQ ID NO.5).

[0131] Synthetic expression cassette E2, the sequence of which is shown in SEQ ID NO.12: pollen-specific expression promoter pG47 (SEQ ID NO.23), recombinase coding gene Cre (SEQ ID NO.11) and NOS terminator (SEQ ID NO...

Embodiment 2

[0149] This example is used to illustrate the acquisition of transgenic plants

[0150] Select the rice expression vector p69C Agrobacterium single colony and inoculate it on the LB medium containing 50mg / L kanamycin. After dark culture at 26°C for 2 days, wash the Agrobacterium cells with NB-AS liquid medium, and culture at 28°C with 180rpm liquid shaking for 90- 120min. Adjust the colony concentration to OD600 of 0.8-1.0, transform the hybrid rice, sterilize the hybrid rice seeds, select the seeds with plump grains, soak them in 75% alcohol for 30s, pour off the alcohol, rinse with sterile water, and rinse with HgCl 2 Disinfect for 8 minutes, wash 2 times with sterile water, soak for 1 minute each time, and soak for 1 hour with sterile water. The sterilized seeds were inoculated on the induction medium, and grown under light for 7 days to obtain sterile callus.

[0151]Gather the sterile callus together, put it into the Agrobacterium suspension, soak for 5-10min, take it o...

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Abstract

The invention belongs to the technical field of plant molecular biology and agricultural biology, and particularly relates to a nucleic acid molecule, a vector, a cell and a primer, application of the nucleic acid molecule, the vector, the cell and the primer and a plant high-purity cloned seed sorting method based on dual regulation. A pollen specificity regulation expression cassette E2; carrying out parthenogenesis on an embryo autonomously-generated gene expression cassette E3 to obtain an asexual embryo without a selection marker; and creating a gene editing expression cassette E4 of the MiMe mutant, and introducing the gene editing expression cassette E4 into a plant. E2 regulates gene expression, deletes elements between recognition sites in E1 and regulates expression of selection marker genes to obtain active pollen with the selection marker, and when recognition is invalid, the pollen death gene is expressed, so that the pollen without the selection marker is inactivated. According to the method, the seeds with fixed heterosis can be efficiently sorted from the hybrid progeny, and the industrialization requirement of a heterosis utilization one-line method is met.

Description

technical field [0001] The invention belongs to the fields of plant molecular biology and agricultural biotechnology, and specifically relates to a nucleic acid molecule, a recombinant vector, a transgenic cell, the application of the nucleic acid molecule, the recombinant vector or the transgenic cell, and a primer , a method for cultivating transgenic plants, a method for identifying said transgenic plants, a method for sorting seeds of high-purity clones of plants based on dual regulation, and a method for screening apomictic lines. Background technique [0002] A complete plant includes sporophytic generations (2n), which have specific reproductive structures (floral organs), in which pistils and stamens undergo meiosis and genetic recombination to produce megaspores (1n) and microspores (1n), respectively; The large and small spores undergo several mitosis to form female and male gametophytes, and the female gametophyte (embryo sac) contains 7 cells (1 egg cell, 2 syner...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N5/10C12N15/11C12Q1/6895A01H5/00A01H6/46A01H1/02
CPCC12N15/8212C12N15/8217C12N15/8234C12N15/65C12Q1/6895A01H1/02C12N2800/80C12N2800/30
Inventor 曹孟良夏玉梅余木兰唐宁詹祎捷淡俊豪卜小兰王耀石跃斌
Owner HUNAN HYBRID RICE RES CENT
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