Rice ossdy1 gene, biological material, method, expression vector for transformation host and its application in stress resistance and stable yield

A rice and expression technology, applied in the field of plant genetic engineering, can solve problems such as limiting plant growth and development, changing water efficiency of stomatal plants, stress resistance, and yield reduction.

Active Publication Date: 2022-03-15
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traits affecting stomata often lead to changes in plant water-use efficiency and stress resistance
Drought and water shortage are the biggest limiting factors in food production. Reducing the opening and density of stomata can achieve the purpose of water saving and drought resistance. Reducing water loss by closing stomata depends on adversity signals such as plant hormone ABA and ethylene, ABA and its metabolites tends to limit plant growth and development resulting in reduced yield

Method used

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  • Rice ossdy1 gene, biological material, method, expression vector for transformation host and its application in stress resistance and stable yield
  • Rice ossdy1 gene, biological material, method, expression vector for transformation host and its application in stress resistance and stable yield
  • Rice ossdy1 gene, biological material, method, expression vector for transformation host and its application in stress resistance and stable yield

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Experimental program
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Effect test

Embodiment 1

[0030] The cloning of embodiment 1 rice OsSDY1 gene

[0031] 1. Obtaining the genome sequence of the rice OsSDY1 gene

[0032] Extract the leaf DNA of rice variety Zhonghua 11 (ZH11), use this DNA as a template, use forward primer OsSDY1-F (sequence shown in SEQ ID NO.5) and reverse primer OsSDY1-R (sequence shown in SEQ ID NO.5) 6), PCR amplification was carried out with KOD PlusNeo (Code: KOD-401, TOYOBO) to obtain the amplified product, namely SEQ ID NO.1 in the sequence listing, OsSDY1 gene sequence.

[0033] 2. Obtaining the sequence of the coding region of the rice OsSDY1 gene

[0034] Total RNA was extracted from leaves of rice variety ZH11, using PrimeScript TM RT reagent Kit with gDNAEraser (Perfect Real Time) (Code: RR047A, Takara) to synthesize cDNA, using this cDNA as a template, using forward primer OsSDY1-CDS-F (sequence shown in SEQ ID NO.7) and reverse primer OsSDY1-CDS-R (sequence shown in SEQ ID NO.8), was amplified by PCR with KOD-Plus-Neo (Code: KOD-401,...

Embodiment 2

[0035] Example 2 Identification of Rice CRISPR-Cas9 Gene Editing Strains

[0036]Plants to be tested: the recipient variety ZH11 and the OsSDY1 gene mutant strain obtained through gene editing.

[0037] Extract the leaf genome DNA of plant to be tested, take this DNA as template, utilize primer: OsSDY1-CPR-F (sequence as shown in SEQ ID NO.9) and OsSDY1-CPR-R (sequence as shown in SEQ ID NO.10 ) for PCR amplification, and the genomic DNA of the recipient variety ZH11 was used as a negative control.

[0038] The PCR amplification product was subjected to 1% agarose gel electrophoresis, the positive control showed a band of about 459bp, and the negative control had no band. Sequence the strains with positive bands to analyze whether there are insertions, deletions or substitutions at the editing site. Some sequencing results are as follows: figure 2 . Select the positive strain and record it as Ossdy1-1.

Embodiment 3

[0039] The identification of the stomatal development phenotype of embodiment 3 transgenic lines

[0040] Plants to be tested: ZH11, positive homozygous gene edited plant Ossdy1-1.

[0041] 1. After culturing in the incubator for about 25 days, the plants to be tested were moved to the greenhouse for soil cultivation, with 4 plants per pot and 3 pots per plant line.

[0042] 2. After the rice plants are fully heading in step 1, cut the blade fragments about 4 cm long in the middle of the main fringe sword leaf, and quickly put them into the standard fixative solution FAA (70% alcohol solution: formalin: glacial acetic acid). =18:1:1) in a plastic bottle, the amount of fixative is about 30 times that of the leaves, to ensure a certain gap between the leaves, and to store in airtight storage for more than 18 hours.

[0043] 3. Take out the leaves fixed in step 2, rinse with 70% alcohol for about 5 minutes, remove the fixative in the tissue, put them into a plastic bottle contai...

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Abstract

The invention discloses a rice OsSDY1 gene, a biological material, a method, an expression vector for transforming a host and an application thereof in stress resistance and stable production. It specifically relates to the application of the OsSDY1 gene in stress resistance and stable yield of rice. Through candidate gene screening and other methods, a gene OsSDY1 related to controlling stress resistance and stable yield of rice was cloned. The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the protein sequence encoded by the gene is shown in SEQ ID NO.3 . Through the phenotypic identification of the transgenic rice materials at the booting stage and mature stage, the results showed that the knockout or mutation of the OsSDY1 gene caused the phenotypes of the transgenic rice, such as decreased stomatal conductance, decreased photosynthetic rate, shorter plant height, and decreased yield. The invention confirms the biological function of the OsSDY1 gene and the way and method of its application.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a rice OsSDY1 gene, a biological material, a method, an expression vector for transforming a host, and its application in stress resistance and stable production. Background technique [0002] With the increase of population and the development of social economy, the demand for food in my country shows a rigid growth. It is particularly necessary to actively develop and utilize the drought-resistant and water-saving mechanism encoded in the plant genome to improve the drought-resistant and stress-resistant and stable yield of crops. Plant height and yield are important agronomic traits of crops, especially rice. Stomata play a vital role in photosynthesis as the formation of crop yield and in the basic physiological process of crop resistance and stability. Stomata are the main portals for gas exchange between plants and the external environment, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8261C12N15/8271
Inventor 梁允宽弓路平
Owner WUHAN UNIV
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