Application of gene OsPIL16 in simultaneously improving tolerance of rice to drought stress and salt stress

A drought stress, transgenic rice technology, applied in the fields of drought stress tolerance and salt stress tolerance of rice, can solve the problem of unknown gene function, and achieve the effects of improving drought resistance, drought and salt stress tolerance, and improving expression level

Active Publication Date: 2015-11-04
SHANDONG RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the function of th

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  • Application of gene OsPIL16 in simultaneously improving tolerance of rice to drought stress and salt stress
  • Application of gene OsPIL16 in simultaneously improving tolerance of rice to drought stress and salt stress
  • Application of gene OsPIL16 in simultaneously improving tolerance of rice to drought stress and salt stress

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Embodiment 1

[0022] Embodiment 1: Isolation and cloning are used to construct the cDNA fragment of OsPIL16 gene plant expression vector

[0023] In order to analyze the function of OsPIL16 gene, we constructed the overexpression vector of OsPIL16 gene cDNA, the specific steps are as follows.

[0024] Total RNA was extracted from leaves of rice variety Nipponbare (a publicly reported variety) using TRIZOL reagent (Invitrogen). The specific steps are as follows: put 20 mg leaves into a liquid nitrogen pre-cooled mortar, add liquid nitrogen to quickly grind into powder, put the powder into a 1.5ml centrifuge tube, quickly add 1ml Trizol (Invitrogen) and invert to mix well, stand at room temperature Leave for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. ...

Embodiment 2

[0027] Embodiment 2: Construction and genetic transformation of OsPIL16 gene plant expression vector

[0028] In order to better analyze the function of the OsPIL16 gene, the applicant increased the expression level of the cDNA sequence of the OsPIL16 gene in the rice variety Nipponbare through overexpression technology. The function of the gene was studied according to the phenotype and physiological characteristics of the transgenic plants.

[0029] In order to construct the plant expression vector of OsPIL16 gene, the present application first constructed the plant expression vector of pCAMBIA1390-Ubi. Firstly, the genomic DNA of maize was extracted by the following method, and the total DNA was extracted from the leaves of maize variety Ludan 981 (a variety reported publicly). The specific steps are as follows: take a small amount of leaves (2-3cm long), put them into a 1.5ml centrifuge tube, freeze them with liquid nitrogen, and grind them quickly. Add 200ul DNA Extract...

Embodiment 3

[0091] Example 3: Detection of the insertion site of the OsPIL16 gene in transgenic rice plants

[0092] The rice variety Nipponbare and three OsPIL16 transgenic rice lines were used as materials for Southern hybridization. The specific steps are as follows: Genomic DNA was extracted by the CTAB method. For specific steps, refer to Murray and Thompson, Nucl. Acids Res, 1980, 8:4321-4325. 100 μg of genomic DNA was digested with HindШ, and then separated by 0.8% agarose gel electrophoresis. DNA was transferred to a nylon membrane Hybond-N+; Amersham Co. Ltd., NJ, USA. Because the exogenous gene OsPIL16 and the resistance selectable marker gene HPTII were in the same T-DNA region, we used the HPTII gene as a probe to identify the insertion site of the OsPIL16 gene. With the pCAMBIA1390-Ubi plasmid as a template, the upstream primer HPTIIF1 (5'-TTC AGC TTC GAT GTA GGA GG-3', SEQ ID NO: 7) and the downstream primer HPTIIR1 (5'-TAC ACA GCC ATC GGT CCA GA-3', SEQ ID NO: 8). Primer...

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Abstract

The invention discloses an application of a gene OsPIL16 in simultaneously improving tolerance of rice to drought stress and salt stress. In the application, a cDNA segment of the gene OsPIL16 of Nipponbare rice is adopted as an applied gene, the applied gene is transformed into rice in a forward direction, the expression level of the gene OsPIL16 is improved, and the genetically-modified positive rice plant has the salt tolerance and drought tolerance both improved. Under the salt stress (200mM NaCl) condition, the survival rate of a reference material is 1.3%, while the survival rate of genetically-modified rice seedlings is 68-75%, which is obviously higher than that of the reference material; under the drought (20% PEG4000) condition, the survival rate of the reference material is 18%, while the survival rate of the genetically-modified rice seedlings is 81-87%. With the adoption of the application provided by the invention, a new thought is provided for culturing rice varieties with multiple resistances and high resistances, and a theoretical basis is also provided for improving the stress resistance of other crops by utilizing the heterologous gene technology.

Description

technical field [0001] The invention relates to the field of agricultural science, in particular, the invention relates to improving rice drought stress tolerance and salt stress tolerance by increasing the expression of rice OsPIL16 gene. Background technique [0002] Rice is the most important food crop in the world, and 50% of the world's population uses rice as a staple food. Rice production plays an important role in ensuring global food security, reducing poverty and rural employment. For a long time, rice has been the grain variety with the largest sown area, the largest total output, and the highest per unit yield in my country. It is the staple food of about 65% of the population in China, and plays a leading role in grain production and consumption. Drought and salt damage are the most serious abiotic stress hazard factors in rice production. Rice production is directly affected by the distribution of water resources. Most of the cultivated land in my country is ...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 谢先芝李亚萍解丽霞周晋军程惠敏
Owner SHANDONG RICE RES INST
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