CN113881687A

CN113881687A - Application of rice cold tolerance gene OsICE2 in improving rice cold tolerance

Info

Publication number: CN113881687A
Application number: CN202111312999.XA
Authority: CN
Inventors: 张振宇; 党姝; 杨祥波; 于晓明; 王晓航
Original assignee: Jilin Agricultural Science and Technology College
Current assignee: Jilin Agricultural Science and Technology College
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-01-04

Abstract

The invention relates to the technical field of rice breeding, in particular to application of a rice cold-resistant gene OsICE2 in improving the cold resistance of rice, wherein a pCsV1300-OsICE2 overexpression vector is constructed by an enzyme cutting method based on a cloned rice cold-resistant gene OsICE2, and an agrobacterium-mediated method is utilized to create OsICE2 transgenic rice. After low-temperature stress treatment, determining the dry weight and the survival rate of the transgenic rice; experimental results show that the rice OsICE2 gene can improve the cold stress tolerance of rice, provide important basis and material for improving the cold stress tolerance of rice and realizing large-area planting of rice under low-temperature conditions, and also provide an example for research on development of cold stress tolerance genes.

Description

Application of rice cold tolerance gene OsICE2 in improving rice cold tolerance

Technical Field

The invention relates to the technical field of rice breeding, in particular to application of a rice cold tolerance gene OsICE2 in improving the cold tolerance of rice.

Background

Rice is widely distributed around the world as one of three major food crops. Up to now, the planting area reaches 1.8 hundred million hectares, which is one of important food crops. Rice is a cold-sensitive and temperature-loving crop, so that the requirement on heat supplied to the environment in the rice growth process is stricter. Therefore, when the temperature of the growing environment is too low, stress is applied to the growth of the rice, and the normal operation of metabolism and physiological functions of the rice is damaged in the growing and developing process of the rice and is in the environment with the temperature lower than the proper growing temperature for a long time or a short time, so that abnormal development is caused, the yield and the quality of the rice are influenced, and even the plant is dead in severe cases. The low-temperature cold damage is a common problem in the main production area of rice in the world, and if a meteorological disaster of the low-temperature cold damage is encountered in the growth and development process of the rice, the low-temperature cold damage not only has great harm to the rice production, but also has great influence on the formation of the yield and the quality of the rice. The low temperature cold damage problem is particularly prominent in northeast China, Japan and Korean east Asia rice district. As a main cultivated variety in the northeast region, japonica rice has good quality, high taste value and large demand, has high economic benefit when being sold as commercial grain, gradually becomes the characteristic of the development of the northern rice region in China, and has important significance in developing high-quality, high-yield and high-efficiency agriculture.

Genetic transformation of rice by agrobacterium-mediated transformation is the major rice transformation method at present. The T-DNA can be inserted into the plant genome by the agrobacterium chemotactic under natural conditions, and the agrobacterium-mediated method based on the T-DNA has high transformation rate, high transgenic single copy ratio and stable transformant. The research on rice transgenosis mostly improves the salt tolerance, disease resistance and the like of rice, and the research on the cold tolerance of rice is very little, so that the method is an important direction worthy of transgenic breeding exploration.

Disclosure of Invention

In order to solve the problems, the rice OsICE2 gene is cloned, an OsICE2 overexpression vector is constructed, and an agrobacterium-mediated method is utilized to create OsICE2 transgenic rice, so that important basis and material are provided for improving the cold resistance of rice, large-area planting of rice under a low-temperature condition is realized, and an example is provided for research on the aspect of developing cold resistance genes.

In order to achieve the purpose, the invention adopts the technical scheme that:

the application of the rice cold-resistant gene OsICE2 in improving the cold resistance of rice is characterized in that a pCsV1300-OsICE2 overexpression vector is constructed by an enzyme cutting method based on the cloned rice cold-resistant gene OsICE2, and an agrobacterium-mediated method is utilized to create OsICE2 transgenic rice; the method specifically comprises the following steps:

s1 cloning rice cold tolerance gene OsICE2

S1.1 extraction of Rice RNA

Treating rice seeds with rice roots and buds after 5 days of culture at 4 ℃ for 2 hours, shearing 0.2 g of buds, and extracting RNA by using a Trizol method;

s1.2 reverse transcription to obtain rice cDNA

Removing residual genome DNA in the RNA and carrying out reverse transcription to obtain cDNA, wherein the reverse transcription system comprises the following steps: total RNA 2.5. mu.g; 0.5 μ L of Anchored; oligo (dt)18Primer (0.5. mu.g/. mu.L) 5. mu.L; 2 × Ts reaction mix 0.5 μ L; DNA Remover 0.5 μ L; RNase-Free Water was made up to 10. mu.L; the reaction conditions are as follows: PCR was incubated at 42 ℃ for 30 min and heated at 85 ℃ for 5 seconds to inactivate the TransScript RT/RI Enzyme Mix and the gDNA Remover;

s1.3 acquisition of OsICE2 Gene

Primer design is carried out by using Primer Premier5.0 software, the TM value is controlled to be kept at 58%, the GC content is controlled to be 40% -60%, and cloning primers OsICE2-clone-F and OsICE2-clone-R of OsICE2 are designed; according to the restriction enzyme cutting sites on pCsV1300, XbaI and EcoRI restriction enzyme cutting sites are introduced to the flanks of the OsICE2 gene, and the coding region of the rice OsICE2 gene is prepared by PCR amplification, wherein, the cloning primer sequence of OsICE 2:

OsICE2-clone-F: 5' ATGGACGAGGCGGAGG 3'

OsICE2-clone-R: 5' TACATTGCGTTCTGAAGGCCA 3'

the PCR reaction system is as follows: 2 mu L of template; primer 1 (10. mu.M) 0.4. mu.L; primer 2 (10. mu.M) 0.4. mu.L; 2 × TransScript Goldpfu PCR Supermix 10 μ L; ddH2O 7.2.2 μ L;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 20 s, annealing at 58 ℃ for 32 s, and extension at 72 ℃ for 45 s, and circulating for 30 times; extending for 5 min at 72 ℃, and performing 1% agarose gel electrophoresis;

obtaining the coding region of OsICE2 with the enzyme cutting site at the end by PCR amplification: carrying out PCR amplification by using the rice OsICE2 full-length coding region product amplified in the last step as a template and using OsICE2-clone-XbaI-F and OsICE2-clone-EcoRI-R primers, wherein the side of OsICE2 is introduced with a primer sequence related to the restriction enzyme site:

OsICE2-clone-XbaI-F: 5' TGCTCTAGAATGGACGAGGCGGAGG 3'

OsICE2-clone-EcoRI-R: 5'CCGGAATTCTACATTGCGTTCTGAAGGCCA 3'

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 20 s, annealing at 58 ℃ for 32 s, and extension at 72 ℃ for 45 s, and circulating for 30 times; extending for 5 min at 72 ℃;

carrying out PCR by using the specific primer of the coding region of OsICE2 by using cDNA as a template to obtain a coding region fragment of the OsICE2 gene;

construction of expression vectors of S2 and pCsV1300-OsICE2

The pCsV1300 plasmid was digested with EcoRI, xbaI, and 50. mu.L of the following reaction system solution was prepared in a sterilized 0.2mL centrifuge tube: 1 μ L of pCsV1300 plasmid; XBa 11 μ L; EcoR 11 μ L; cutsmart 5. mu.L; water 42. mu.L; 37^oC reacting for 15 min, detecting the obtained product by electrophoresis, and storing in 4^oC, keeping the refrigerator for later use;

the obtained coding region fragment of the OsICE2 gene is cut by EcoRI and xbaI, and the following reaction system solution is prepared: the recovered target gene OsICE 11 mu L; XBa 11 μ L; EcoR 11 μ L; cutsmart 5. mu.L; water 42. mu.L; 37^oC reacting for 15 min, and storing the obtained product in 4^oAnd C, the refrigerator is reserved.

The plasmid and the product of OsICE2 were ligated by DNA ligase, and the following reaction solution 15. mu.L was prepared in a sterilized 0.2mL centrifuge tube: 0.2 mu L of T4DNA ligase; 2 XT 4 Buffer 1.5 μ L; the restriction enzyme digestion product of pCsV1300 plasmid is 0.2 mu L; the restriction enzyme product Water of the OsICE2 fragment is 12.9 mu L; the reaction condition is 16 ℃ for 8 h; s3, creating OsICE2 transgenic rice by using an agrobacterium-mediated method.

The invention also provides a cold resistance analysis method of the T2 generation plant of OsICE2 transgenic rice, and the cold stress resistance expression of the 0sICE2 gene is observed from the aspect of the cold stress phenotype of the transgenic rice plant.

In the scheme, the rice cold-resistant gene OsICE2 is cloned, the pCsV1300-OsICE2 overexpression vector is constructed by an enzyme digestion connection method, the transformation of rice is completed by an agrobacterium-mediated method, the transgenic positive mycelium is obtained by PCR identification, and a foundation is laid for the creation of a new germplasm of cold-resistant rice by a transgenic method.

Drawings

FIG. 1 shows the identification of total RNA extracted from rice after cold treatment;

FIG. 2 shows the amplification of the coding region of OsICE2 gene of rice;

FIG. 3 map of pCsV1300-OsICE2 expression vector;

FIG. 4 shows the restriction enzyme digestion detection of the expression vector pCsV1300-OsICE 2;

FIG. 5 shows the survival rates of transgenic and control rice under cold conditions;

FIG. 6 shows the dry weight of transgenic and control rice under cold conditions.

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Cloning of the target Gene

1.1 Rice Germination

Firstly, selecting plump Japanese fine rice seeds, putting the seeds into a sterile culture dish, sterilizing the seeds with raw mercury, soaking the seeds for 15 min, washing the sterilized seeds on gauze, and putting the washed seeds back into the culture dish for later use. Spreading the upper and lower layers of the culture dish with ultrapure water-wetted filter paper, placing the sterilized seeds on the lower layer of filter paper, covering the upper layer with the culture dish with wetted filter paper, and culturing at 25 deg.C in the absence of light. Note that the water content on the filter paper was constantly observed, the filter paper was kept wet, and rice roots and shoots appeared after 5 days of culture.

1.2 extraction of Rice RNA

The rice seeds cultured in 1.1 were treated at a low temperature (4 ℃) for 2 hours, then 0.2 g of shoots were cut off, and RNA extraction was performed using Trizol method.

1.3 RNA integrity analysis

And (3) carrying out agarose gel electrophoresis detection on the rice RNA obtained in the step 1.2. As shown in FIG. 1, the RNA of the rice shoots subjected to cold stress 5 days after germination was obtained in all three lanes of FIG. 1, and 28S bands were significantly brighter than 18S bands, and 5S RNA bands were the weakest. The above results indicate that the extracted rice RNA has better integrity.

1.4 reverse transcription to obtain Rice cDNA

The residual genomic DNA in the RNA was removed and cDNA was obtained by reverse transcription, which was performed according to the instructions of TransScript One-Step gRNA Removal and cDNA Synthesis SuperMix of all-Kabushiki Co. The system is as follows:

TABLE 1 reverse transcription System Table

The reaction conditions are as follows: PCR was incubated at 42 ℃ for 30 min and heated at 85 ℃ for 5 seconds to inactivate the TransScript RT/RI Enzyme Mix and the gDNA Remover.

1.5 acquisition of OsICE2 Gene

Primer design is carried out by using Primer Premier5.0 software, the TM value is controlled to be kept at 58%, the GC content is controlled to be 40% -60%, and cloning primers OsICE2-clone-F and OsICE2-clone-R of OsICE2 are designed; XbaI and EcoRI restriction sites are introduced to flanks of the OsICE2 gene according to the restriction sites above pCsV1300, and primers used are OsICE2-clone-xbaI-F and OsICE 2-clone-EcoRI-R.

Cloning primer sequence of OsICE 2:

OsICE2-clone-F: 5' ATGGACGAGGCGGAGG 3'

OsICE2-clone-R: 5' TACATTGCGTTCTGAAGGCCA 3'

the primers related to the restriction sites are introduced into the flanks of OsICE 2:

OsICE2-clone-XbaI-F: 5'TGCTCTAGAATGGACGAGGCGGAGG 3'

OsICE2-clone-EcoRI-R: 5'CCGGAATTCTACATTGCGTTCTGAAGGCCA 3'

the primers were synthesized by Jilin province, Kuumei, Biotechnology, Inc., and the primers synthesized above were diluted to a concentration of 10. mu.M for use.

The coding region of the OsICE2 gene of rice is prepared by PCR amplification, and the PCR reaction system is shown in Table 2.

TABLE 2 PCR reaction System Table

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 20 s, annealing at 58 ℃ for 32 s, and extension at 72 ℃ for 45 s, and circulating for 30 times; extension at 72 ℃ for 5 min. Electrophoresis on a 1% agarose gel.

Obtaining the coding region of OsICE2 with the enzyme cutting site at the end by PCR amplification: the rice OsICE2 full-length coding region product amplified in the last step is used as a template, and primers OsICE2-clone-XbaI-F and OsICE2-clone-EcoRI-R are used for carrying out PCR amplification, and the reaction conditions are the same as those in the last step.

PCR was performed using the cDNA as a template and primers specific to the coding region of OsICE2 to obtain a fragment of the coding region of OsICE2 gene. 2 bands of expected length were obtained in this study, and as shown in FIG. 2, lane 1 and lane 3 were obtained as positive clones. Sequencing results show that the coding region fragment of the OsICE2 gene is 1146bp, and the sequence is consistent with the sequence of the OsICE2 coding region annotated in rice genome data.

2. Expression vector construction

Construction of expression vector pCsV1300-OsICE2

Cutting pCsV1300 (vector map is shown in figure 3) by X-BaI and EcoRI, cutting gel and recovering a 9755bp fragment; a1627 bp long fragment of the coding region of the OsICE2 gene is recovered. After connection, transformation and identification, the positive clone colony is selected for amplification culture, and the recombinant plasmid is extracted for enzyme digestion identification. As shown in FIG. 4, two DNA bands on lane 2 can be clearly seen, and the length of the two DNA bands matches the expected size, namely: the fragment of pCsV1300 after the enzyme digestion of X-BaI and EcoRI is 9755bp, and the smaller fragment is 1627bp OsICE2 gene. The nucleotide sequence and the protein sequence of the gene are shown as SEQ ID NO. 1.

2.1 PCR product recovery

After the electrophoresis is finished, the amplified product is rapidly cut off a gel block containing a DNA fragment by a clean blade rubbed with alcohol cotton under an ultraviolet lamp, and the gel block is put into a 1.5 mL sterile centrifuge tube, and then a PCR product is recovered by using a Coolaber's kit to obtain a desired target gene fragment.

2.2 double digestion

For the pCsV1300 plasmid, digestion was performed with EcoRI, xbaI, and the following reaction system solution (50. mu.L) was prepared in a sterilized 0.2mL centrifuge tube:

TABLE 3 Table of the enzyme digestion reaction system

37 ^oC reacting for 15 min, detecting the obtained product by electrophoresis, and storing in 4^oAnd C, the refrigerator is reserved.

The OsICE2 fragment of the target gene recovered in 1.5 is selected and digested with EcoRI and xbaI, and the reaction system is shown in Table 4.

TABLE 4 Table of the enzyme digestion reaction system

The mixture is placed in 37^oC reacting for 15 min, and storing the obtained product in 4^oAnd C, the refrigerator is reserved.

2.3 DNA ligation

The plasmid and the double-digested product of OsICE2 were ligated with DNA ligase, and the following reaction solution (15. mu.L) was prepared in a sterilized 0.2mL centrifuge tube:

TABLE 5 Table of the enzyme digestion reaction system

3 transformation of

3.1 transformation of E.coli

The ligation product was transformed into e.coli DH5 α using a heat shock method.

3.2 transformation of Agrobacterium

The rice transformation process refers to the agrobacterium-mediated rice genetic transformation system and identification (see specifically the following documents: treying, cai chard, lin champion, chenhao. (2018). agrobacterium-mediated rapid transformation of rice: Bio-101: e 1010176.).

Selecting mature and full rice seeds, and shelling; sterilizing with 75% alcohol for 1-2 min, and pouring out the alcohol; adding 0.15% mercuric chloride (containing 0.1% Tween 20) and sterilizing for 10 min; mercury mercuric oxide was poured off, and washed with sterilized distilled water 6 times. Inoculating the sterilized seeds into an induction callus culture medium, and culturing for 20 days at 28 ℃ under illumination.

Agrobacterium EHA105 (transformed with pCsV1300-OsICE2 vector) was streaked on LB plate medium containing (20 mg/L Rif,50 mg/L Kan) and cultured at 28 ℃ for 2 d; selecting single colony, and performing activation culture in LB plate culture medium at 28 deg.C and 200 r/min for 2 d; before infection, scraping the activated agrobacterium into a suspension culture medium, performing shake culture at 28 ℃ and 180 rpm for 3-3.5 h, and then adjusting the concentration of a bacterial liquid to OD600=0.1-0.15 by using the suspension culture medium; soaking rice callus with diameter of 2-4mm in Agrobacterium suspension for 20 min. The bacterial solution was decanted and the wound surface was blotted dry with sterile filter paper. Covering sterilized filter paper on the surface of the callus, and drying for 30 min by an ultra-clean bench. After drying, transferring the callus into a co-culture medium with a layer of sterilized filter paper covered on the surface, performing dark culture at 20 ℃ for overnight, and then transferring into an incubator at 25 ℃ for continuous dark culture for 2 d. After the co-culture was completed, the callus was transferred to an empty sterilized container with forceps. Repeatedly washing the callus with sterilized distilled water for 7-8 times, and finally soaking the callus with sterilized distilled water containing 500 mg/l Carbenicillin (Cn) for 30 min. Pouring the Cn solution, sucking the water on the surface of the callus with sterile filter paper as much as possible, covering a layer of sterile filter paper on the surface of the callus, and drying for 1 hour by using an ultra-clean bench. Placing the cleared callus on a screening culture medium containing hygromycin for 32 ℃, and culturing for 14 d by illumination.

3.3 differentiation and regeneration of callus

After 14 days of selection, the resistant calli were transferred to differentiation medium and cultured at 28 ℃ (photoperiod 14 h light/10 h dark). And when the resistance callus forms a regenerated seedling with the height of 3-4 cm on a differentiation culture medium, transferring the regenerated seedling into a rooting culture medium for culture until a complete transgenic rice plant is formed. The inbred progeny of the transgenic rice can adopt hygromycin to screen homozygous transgenic plants.

4 identification of Cold tolerance of transgenic Rice

Wild type rice grown to the three-leaf one-heart stage and homozygous OsICE2 overexpressing transgenic rice (OsICE 2)OX-1、OsICE2OX-2And OsICE2OX-3) The cells were transferred to 4 ℃ and treated for 5 days, and then recovered (28 ℃) for 2 days, and then the survival rate and dry weight were counted.

The results show that compared with wild rice, the OsICE2 overexpression rice has enhanced cold stress tolerance, and the survival rate and the dry weight of the OsICE2 overexpression rice are significantly higher than those of the wild rice (as shown in figures 5 to 6).

According to the above-mentioned technology, the invention clones the cold-resistant gene of riceOsICE2. By an Agrobacterium-mediated transformation method, the method comprisingOsICE2The rice is successfully transformed by the overexpression vector, and a homozygous T2 generation transgenic plant is obtained. It was found that under the stress of cold salt,OsICE2over-expression plantOsICE2OX-1、OsICE2OX-2AndOsICE2OX-3the survival rate and dry weight of (a) is significantly higher than that of wild type rice. The above results indicate that in rice overexpressingOsICE2The gene can improve the cold stress tolerance of rice.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Sequence listing

<110> Jilin agriculture science and technology institute

Application of rice cold-resistant gene OsICE2 in improving rice cold resistance

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<213> Artificial Sequence (atggacgagg cggagg 16)

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<213> Artificial Sequence (tacattgcgt tctgaaggcca 21)

<400> 4

<210> 5

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<213> Artificial Sequence (tgctctagaa tggacgaggc ggagg 25)

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Claims

1. The application of the rice cold-resistant gene OsICE2 in improving the cold resistance of rice is characterized in that: through an enzyme cutting method, a pCsV1300-OsICE2 overexpression vector is constructed based on a cloned rice cold-resistant gene OsICE2, and an agrobacterium-mediated method is utilized to create OsICE2 transgenic rice.

2. The use of claim 1, wherein: the method specifically comprises the following steps:

s1 cloning rice cold tolerance gene OsICE2

S1.1 extraction of Rice RNA

s1.2 reverse transcription to obtain rice cDNA

s1.3 acquisition of OsICE2 Gene

OsICE2-clone-F: 5' ATGGACGAGGCGGAGG 3'

OsICE2-clone-R: 5' TACATTGCGTTCTGAAGGCCA 3'

OsICE2-clone-XbaI-F: 5' TGCTCTAGAATGGACGAGGCGGAGG 3'

OsICE2-clone-EcoRI-R: 5'CCGGAATTCTACATTGCGTTCTGAAGGCCA 3'

construction of expression vectors of S2 and pCsV1300-OsICE2

the obtained coding region fragment of the OsICE2 gene is cut by EcoRI and xbaI, and the following reaction system solution is prepared: the recovered target gene OsICE 11 mu L; XBa 11 μ L; EcoR 11 μ L; cutsmart 5. mu.L; water 42. mu.L; 37^oC reacting for 15 min, and storing the obtained product in 4^oC, keeping the refrigerator for later use;

the plasmid and the product of OsICE2 were ligated by DNA ligase, and the following reaction solution 15. mu.L was prepared in a sterilized 0.2mL centrifuge tube: 0.2 mu L of T4DNA ligase; 2 XT 4 Buffer 1.5 μ L; the restriction enzyme digestion product of pCsV1300 plasmid is 0.2 mu L; the restriction enzyme product Water of the OsICE2 fragment is 12.9 mu L; the reaction conditions were 16^oC, reacting for 8 hours;

s3, creating OsICE2 transgenic rice by using an agrobacterium-mediated method.

3. The use of claim 1, wherein: the resistance expression of the 0sICE2 gene to cold stress is observed from the aspect of cold stress phenotype of transgenic rice plants.