Patent Application
20230272411
The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. The ST.26 copy, created on Feb. 2, 2023, is named Sequence Listing.xml and is 6 kilobytes in size.
The present disclosure relates to the field of molecular biology, and in particular relates to a heat-shock related gene ZmHsf11, isolation and identification of a protein encoded by the heat-shock related gene ZmHsf11, a method for obtaining a genomic sequence of the heat-shock related gene ZmHsf11, and an application of the heat-shock related gene ZmHsf11 in regulating heat-resistance of plants.
Maizes serves as one of the important food-feed crops in China. A slight change in an environment can reduce yield and quality of the maizes. Currently, environmental factors, mainly including biotic and abiotic stresses, can influence growth and development of the maizes. Problems caused by the biotic stresses can be solved by applying herbicides and pesticides, and so on. The abiotic stresses include high temperatures, droughts, frost damages, and so on, and cannot be solved effectively. Especially for crops, each of these stresses can cause low or extinction yields of the crop (Liu et al., 2016).
A responsive molecular mechanism of the crops under the abiotic stresses is explored to find an advantageous trait gene to improve the yield and the quality of the crops. In addition, as the modern molecular technology and molecular genetic breeding develop rapidly, the advantageous gene may be transfected into and stably expressed in an advantageous strain. In this way, tolerance of the crops under adverse stresses may be improved. Therefore, the study of resistance genes and breeding of new species having advantageous traits have become one of hot topics in plant resistance genetic breeding research.
Plants are immobilized organisms, and the high-temperature stress has a great impact on their growth, development and reproduction. For no beneficial tropism, plants can only achieve homeostasis by regulating an internal environment to resist an external environment. When the plant is subjected to a high temperature transiently, expression of some heat-shock genes is activated in vivo so that the plant may generate acquired heat-resistance once growth is resumed. For basal heat-resistance, when the plant is subjected to the high temperature stress, some in vivo genes may regulate the homeostasis of the internal environment of the plant to be resistant to the external environment. The above process involves expression of certain genes, such as genes of heat shock proteins. The genes that have been reported to affect the heat-resistance of plants include genes related to the phytohormone or signaling pathways, genes related to Ca2+ signaling pathways, genes related to reactive oxygens, and genes of heat-shock transcription factors and heat-shock protein genes that play a major role (Mittler et al., 2012).
The heat shock transcription factor (HSF) is a major transduction element in a heat stress signaling pathway. The HSF activates expression of related genes under the heat stress and plays a critical role in regulation of heat stress response processes in plants (Kotak et al., 2007). The HSF family has three major sub-families: A, B, and C. Proteins expressed by the three major sub-families have similar structures, but these structures may differ from each other across the three major sub-families. For example, some members of the B sub-family may further have a repressor region (RD) (Nover et al., 2001). Therefore, functions of different HSF members may be involved in a wide variety of adverse stresses. Current studies have shown that the functional studies of the HSF are mainly focused on stresses such as droughts, salts, and high temperatures.
The present disclosure aims to screen and identify ZmHsf11, a heat-shock related gene capable of responding to high temperatures and an application of the ZmHsf11 in regulating heat resistance of plants.
In order to achieve the above aim, the present disclosure provides the following technical solutions.
The present disclosure provides a heat-stress related gene ZmHsf11. A nucleotide sequence of the heat-stress related gene ZmHsf11 is shown as SEQ ID No: 1, and the heat-stress related gene ZmHsf11 has function of reducing heat resistance in plants.
The present disclosure provides a protein encoded by the heat-stress related gene ZmHsf11. The protein is the following (1) and (2) proteins.
(1) A protein consists of an amino acid sequence as shown in SEQ ID No: 2 in the sequence listing.
(2) A protein is derived from the protein (1) by substituting and/or adding one to ten amino acid residues to the amino acid sequence of SEQ ID No: 2 in the sequence listing, and has the function of the heat-shock related gene ZmHsf11.
The present disclosure provides a plant expression vector for the heat-shock related gene ZmHsf11.
Further, a nucleic acid molecule of the heat-shock related gene ZmHsf11 is inserted into an expression vector p1301a, and a vector for overexpressing the ZmHsf11, p1301a-ZmHsf11, is obtained.
The present disclosure provides a host bacterium of the plant expression vector.
The present disclosure provides a plant cell including the heat-shock related gene ZmHsf11, a plant including the plant cell, and a seed of the plant, and the plant is preferably rice or maize.
The present disclosure provides a primer pair for cloning the heat-shock related gene ZmHsf11. The primer pair includes an upstream primer and a downstream primer. A nucleotide sequence of the upstream primer is shown in SEQ ID No: 3, and a nucleotide sequence of the downstream primer is shown in SEQ ID No: 4.
The present disclosure provides an application of the heat-shock related gene ZmHsf11 in reducing or increasing the heat resistance of plants.
The regulation of heat resistance of the plant is to increase the heat resistance of the plant. Deletion or inhibition of the ZmHsf11 gene expression in the plant may increase the heat resistance of the plant.
The present disclosure provides a method for breeding a plant species resistant to high temperatures. The method includes knocking out or suppressing the expression of the heat-stress related gene ZmHsf11 to improve a survival rate of the plant after a heat treatment, such that the heat resistance of the plant may be improved.
The plant is rice or maize.
The present disclosure provides a heat-resistance negative-regulation related gene ZmHsf11 and a protein of the gene ZmHsf11. Specifically, in the present disclosure, the ZmHsf11 gene is cloned from the maize. An expression level of the ZmHsf11 gene can be induced by a high temperature and other adverse stresses. The ZmHsf11 gene may be overexpressed in rice. Functional detection is performed on the ZmHsf11 gene. It is found that, after overexpression, rice plants which has the overexpressed ZmHsf11 gene and receives a heat treatment has a significantly reduced survival rate. Functional detection is performed on a ZmHsf11 mutant gene provided by the present disclosure. It is found that, rice plants which has the ZmHsf11 mutant gene has significantly increased heat resistance. The above results indicate that the ZmHsf11 gene has negative regulation on the heat resistance of the plant. The present disclosure provides a new technical basis for breeding maizes for heat resistance.
Technical solutions of the present disclosure are illustrated in details by referring to the embodiments. However, these embodiments are used for illustrative purposes only and do not limit the scope of the present disclosure.
It should be understood that the experimental methods used in the following embodiments are conventional methods if not specifically described.
It should also be understood that the experimental materials, reagents, and so on, used in the following embodiments are commercially available, if not otherwise specified.
A full length of a CDS sequence (SEQ ID No: 1) of the maize ZmHsf11 gene and an amino acid sequence (SEQ ID No: 2) of a protein encoded by the maize ZmHsf11 gene are obtained from the Plant Genome Database web site (https://phytozome.jgi.doe.gov/pz/portal.html).
A heat-shock related gene ZmHsf11 is provided by the present disclosure, and a nucleotide sequence of the heat-shock related gene ZmHsf11 is shown as SEQ ID NO: 1.
The present disclosure provides a protein encoded by the heat-shock related gene ZmHsf11, and the protein is described in (1) or (2) as follows.
(1) A protein consists of an amino acid sequence as shown in SEQ ID No: 2 in the sequence listing.
(2) A protein is derived from the protein (1) by substituting and/or adding one to ten amino acid residues to the amino acid sequence of SEQ ID No: 2 in the sequence listing, and has the function of the heat-shock related gene ZmHsf11.
The present disclosure provides a plant expression vector for the heat-shock related gene ZmHsf11. A nucleic acid molecule of the heat-shock related gene ZmHsf11 is inserted into an expression vector p1301a, and a vector for overexpressing the ZmHsf11, p1301a-ZmHsf11, is obtained.
The present disclosure provides a host bacterium of the plant expression vector.
The present disclosure provides a plant cell including the heat-shock related gene ZmHsf11, a plant including the plant cell, and a seed of the plant, and the plant is preferably rice or maize.
The present disclosure provides a primer pair for cloning the heat-shock related gene ZmHsf11. The primer pair includes an upstream primer and a downstream primer. A nucleotide sequence of the upstream primer is shown in SEQ ID No: 3, and a nucleotide sequence of the downstream primer is shown in SEQ ID No: 4.
The present disclosure provides an application of the heat-shock related gene ZmHsf11 in reducing or increasing the heat resistance of plants.
The regulation of heat resistance of the plant is to increase the heat resistance of the plant. Deletion or inhibition of the ZmHsf11 gene expression in the plant may increase the heat resistance of the plant.
The present disclosure provides a method for breeding a plant species resistant to high temperatures. The method includes knocking out or suppressing the expression of the heat-stress related gene ZmHsf11 to improve a survival rate of the plant after a heat treatment, such that the heat resistance of the plant may be improved.
The plant is rice or maize.
Maize B73 is planted in a pot having a diameter of 40 cm and a height of 50 cm. 3 maize seeds are planted in one pot, and the seeds are planted in 6 pots in total. The 6 pots are placed in a greenhouse (28° C. approximately) and grown for 14 days. Roots, stems and leaves of the maizes are sampled respectively from the 6 pots, snap frozen in liquid nitrogen, and stored at −80° C. When the maize is grown to reach a flowering stage, filaments, bracts and stamens of the maizes are sampled respectively, snap frozen in liquid nitrogen, and stored at −80° C. RNAs of all samples are extracted, reverse transcribed into cDNA samples, and stored in a refrigerator at −20° C.
In addition, the maize B73 is planted in a pot having a diameter of 15 cm and a height of 10 cm. 4 maize seeds are planted in one pot, and the seeds are planted in 24 pots in total. The 24 pots are placed in a greenhouse (28° C. approximately) and grown for 14 days. A treatment of 42° C., 200 Mm NaCl, 20% PEG6000 and 100 μM ABA is performed on each of the 24 pots. Samples are taken at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after the treatment. Each sample is mixed from three plants. All samples are snap frozen in liquid nitrogen and stored at −80° C. RNAs of all samples are extracted, reverse transcribed into cDNA samples, and stored at −20° C. in a refrigerator.
Based on a full length of a cDNA sequence of the obtained ZmHsf11 gene, RT-qPCR quantification primers are designed by using the Primer5 software.
A primer qPCR-ZmHsf11-F: 5′-CTGGGAGCGACCACGACG-3′.
A primer qPCR-ZmHsf11-R: 5′-AAACACTGGAGATTTTTACATAGG-3′.
A gene GAPDH of the maize is taken as an internal reference gene, serving as a control, and primers are designed.
A primer of Zm-GAPDH-F: 5′-CCTCTGGAAAA TTGTGGCGTG-3′.
A primer of Zm-GAPDH-R: 5′-GCCCAAACGAACAGTCAAGTC-3′.
The qPCR is performed by diluting a concentration of the cDNA templates for 10 times
Amplification signals and data are processed by applying a comparative Ct method (ΔΔCt method). A relative expression value Cq=2(−ΔΔCt). For each fluorescence quantification, three biological replicates and three technical replicates are performed.
As shown in
By analyzing the CDS sequence of ZmHsf11 and a sequence of a subcellular localization vector p1305-GFP, following primers are designed.
A primer ZmHsf11-F (XbaI): 5′-gctctagaATGGCCGCCGAGCATGCCA-3′.
A primer ZmHsf11-R (SmaI): 5′-cccccgggCCTCGAGTCGTTGGACCC-3′.
(1) The cDNA obtained in the Example 1 is taken as a template for PCR cloning and a reaction system is as follows.
At the end of the PCR reaction, 3 μL of 10× loading buffer is added. Later, agarose gel electrophoresis is performed. A gel extraction kit is applied to extract a target fragment.
(2) The PCR product extracted from (1) and an empty p1305-GFP vector are digested. A digestion system is as follows.
The reaction is carried out in a metal bath at 37° C. for 3 h. After the digestion is completed, 3 μL of loading buffer is added to terminate the reaction. Further, agarose gel electrophoresis is performed, and the digested product is extracted.
(3) Linkage reaction is performed, and a linkage reaction system is as follows:
The above linkage reaction system is mixed evenly, and placed into a PCR instrument. The reaction is performed at 16° C. for 1 h.
(4) A ligated product is transfected into E. coli and amplified to obtain a recombinant plasmid.
Protoplasts of the maize B73 are obtained. The p1305-GFP-ZmHsf11 recombinant plasmids and a nuclear localization signal plasmid NLS-RFP are co-transfected into the protoplasts. The transfected protoplasts are incubated at dark for 18 h and then observed by a laser confocal microscopy.
The results are shown in
A primer ZmHsf11-F(KpnI): 5′-gggtaccATGGCCGCCGAGCATGCCA-3′.
A primer ZmHsf11-R(PstI): 5′-gctctagaTCACCTCGAGTCGTTGGACCC-3′.
Sterilized seeds are inoculated into an inoculation flask containing an induction medium. No more than 8 seeds are inoculated in one flask. The flask is incubated in a tissue culturing room for 14-20 days.
An Agrobacterium solution containing the target gene is scribed on a resistant YEP solid medium and incubated at 28° C. for 2 days. A single colony is picked and incubated in a 15 mL shaking tube containing 5 mL of media for 36 h. 3 mL of glucose, 800-900 μL of bacterial solution, and 150 μL of AS are added to 150 mL of suspension medium for expanding the culture.
The healing tissue is placed in a tissue culturing bottle, 50 mL of bacterial solution is added, and the bottle is shaken gently for 10 min. The operation is repeated once. The infested healing tissue is incubated on the solid YEP medium containing K+ and Rif, at 24° C., for 3 days. The amount of bacterial growth in the dish is observed. Sterilized ddH2O is used to wash the healing tissue for 5 min at a time, and the washing is repeated 5-7 times. Further, the washed healing tissue is transferred to a clean bottle and soaked in carboxybenzyl water for 30 min (performed separately twice). The healing tissue is dried on a filter paper, inoculate into a selection medium, placed in the tissue culturing room, and incubated for 2 weeks.
A new healing tissue is selected from the selection medium, inoculated to the differentiation medium, placed in the tissue culturing room, and incubated to have a differentiated seedling.
When the seedling is grown in the differentiation medium, the seedling is transferred to a rooting medium in a tissue culturing flask, and cultured for 4 weeks.
Tap water is added to a rooting bottle, and the seedling is moved to the ground after 5 days in the rooting bottle.
Rice leaves are sampled and placed in a PCR tube. 100 μL of GUS staining solution is added to the tube, and the tube is placed in a 37° C. incubator. The tube is incubated overnight and protected from light. Leaf infestation is observed (
RNAs are extracted from the transgenic rice and reverse transcribed into cDNAs as templates. A semi-quantitative assay is performed to observe whether the transgenic rice has a band, and brightness of the band.
The results are shown in
A T02 seed is harvested and placed in a pipette box. Appropriate amount of water is added to the pipette box. The pipette box is placed in a greenhouse at 28° C. for about 14 days. The plant is transplanted into a small round pot (D=14.8 cm, H=12 cm) and placed in the greenhouse at 28° C. for 15 days. The plant is then placed in the greenhouse at 45° C. for 22 h. Subsequently, the plant is restored to the room temperature and cultured in the greenhouse at 28° C. for 20 days. Phenotypes of the rice before and after the heat treatment are observed. Survival rates of wild-type rice and overexpression rice are counted respectively.
The results are shown in
The results are shown in
(2) DAB Staining and Trypan Blue Staining: The Rice Plant is Treated at 50° C. for 2.5 h. Subsequently, DAB Staining and Trypan Blue Staining are Performed on Plants Before and After the Treatment, and Staining Results are Observed.
The results are shown in
The purchased maize species W22 is taken as a background for making a Mu mutant variant (from the Maize Genetics Cooperation Stock Center). The purchased maize species W22 is taken as a T0 generation. The T0 generation plant is verified by performing PCR, and a maize containing Mu transposons is screened. The screened maize containing the Mu transposons are self-interbred to obtain a T01 generation. A T02 generation is obtained by continuing performing the method described above. In this way, a homozygous seed is identified. The RT-qPCR is performed to determine the expression level of the ZmHsf11 gene in the mutant variant.
The results are shown in
The seed of the wild-type W22 species and the Mu mutant are respectively seeded in a small round pot and grown in the greenhouse till the plants have three unfurled leaves and one new unfurled leaf. A moisture content of the pot of the wild-type W22 and a moisture content of the pot of the Mu mutant are controlled to be consistent with each other. The plants are placed in a light incubator and incubated overnight. Subsequently, the plants are placed in the greenhouse at 45° C. for 12 h. Later, the plants are placed at the room temperature and grown in the greenhouse for 2 days. Wilting of leaves of the wild-type plant and the Mu mutant are observed (as shown in
The results are shown in
The results are shown in
The wild-type plant and the Mu mutant plant are treated at 50° C. for 4 h. A 5 cm-tip of a third leaf of the wild-type plant before the 50° C. treatment, a 5 cm-tip of a third leaf of the wild-type plant after the 50° C. treatment, a 5 cm-tip of a third leaf of the Mu mutant plant before the 50° C. treatment, and a 5 cm-tip of a third leaf of the Mu mutant plant after the 50° C. treatment are obtained and stained by DAB, and staining results thereof are observed.
The results are shown in
It shall be understood that the above description only shows preferred embodiments of the present disclosure and is not intended to limit the present disclosure. It shall be understood by any ordinary skilled person in the art that the present disclosure is not limited by the above embodiments. The above embodiments and the description describe only illustrative of the principles and methods of the present disclosure. Various variations and improvements may be made to the present disclosure without departing from the spirit and scope of the present disclosure. The scope of present disclosure is defined by the appended claims, the specification and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110582029.5 | May 2021 | CN | national |
The present application is a continuation-application of the International Patent Application No. PCT/CN2022/090924, filed on May 5, 2022, which claims the priority of the Chinese Patent Application No. 202110582029.5, filed on May 27, 2021, in the China National Intellectual Property Administration, and the entire contents of which are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/090924 | May 2022 | US |
| Child | 18181561 | US |
