Application of stg5.2 gene in regulating salt tolerance of rice

Abstract

The application provides application of a STG5.2 gene in regulating salt tolerance of rice, and relates to the technical field of biology. The application clones rice LOC_Os05g07260 (STG5.2, Salt Tolerance and Grain Yield-related Gene 5.2), which is annotated as a phosphatidylinositol glycosylation related gene. The inventors find that the STG5.2 gene has the function of positively regulating salt tolerance and grain yield of rice. After the gene is knocked out, the growth of the plant is hindered in a saline-alkali environment, and the grain yield is seriously reduced. The application has potential application value for mining salt-tolerant genes of rice and cultivating new salt-tolerant rice varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of an STG5.2 gene in regulating salt tolerance in rice. Background Technology

[0002] Rice is one of the main food crops for improving coastal tidal flats and saline-alkali lands. Discovering and breeding new salt-tolerant rice germplasm is an important measure to promote the high-quality development of modern rice breeding technology and ensure food security. Rice salt tolerance has a complex genetic basis and physiological characteristics, and is a quantitative trait controlled by multiple genes. In recent years, scientists at home and abroad have conducted extensive work on salt tolerance at different developmental stages of rice based on different mapping populations, identifying more than 1,000 QTLs related to salt tolerance and discovering more than 200 salt tolerance-related genes.

[0003] The development of functional genomics has driven the discovery of major genes: SKC1 is a major salt-tolerant gene based on the cloning of rice genetic segregation populations, which regulates the excessive Na+ in the aboveground parts. + Transported to the roots, regulating intracellular Na+ + / K + Maintaining homeostasis enhances rice salt tolerance. The STH1 gene negatively regulates rice salt tolerance and positively regulates flowering time, enabling it to maintain vegetative growth to withstand salt stress. qSE3 encodes K... + The transport gene OsHAK21 increases the resistance of rice to K under salt stress. + and Na + It promotes seed germination and seedling growth by increasing absorption and reducing ROS accumulation. The STG5 gene is mainly involved in regulating the expression of multiple members of the HKT gene family, thereby regulating Na+. + / K + Homeostasis endows rice with strong tolerance to salt stress. Furthermore, salt-tolerant genes such as DST (encoding a C2H2-type zinc finger domain protein), RR22 (encoding a type B response regulatory protein), and RST1 (encoding an auxin response factor) have been screened and cloned through mutant libraries. Although salt-tolerant genes such as SKC1 have been cloned, many existing genes suffer from weak effects, low yield despite salt tolerance, high yield but low salt tolerance, and the synergistic regulatory mechanism between salt tolerance and yield traits remains unclear.

[0004] GPI-anchored proteins (GPI-APs) are widely anchored on the outer side of the cell membrane and participate in various key biological processes such as cell wall construction, cell signal recognition, and extracellular environment perception. Phosphatidylinositol glycosylation genes are mainly involved in the biosynthesis of glycosylphosphatidylinositol-anchored proteins. Studies in barley have found that salt stress induces responses in multiple proteins in the phosphatidylinositol (PI) signaling pathway. For example, PI signaling-related proteins (such as EF1α, annexin 6, and 14-3-3-like protein A) show significant changes in expression under salt stress, and their expression is related to calcium. 2+ It is closely related to early stress responses such as signals and ROS bursts.

[0005] Although existing research supports the important role of phosphatidylinositol signaling and its metabolic processes in the salt stress response of crops such as rice, no direct functional studies of phosphatidylinositol glycosylation-related genes (such as the PIG gene family) in rice salt stress have been found. This direction may still be a research gap and deserves further exploration.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide the application of the STG5.2 gene in regulating salt tolerance in rice, thereby providing direction for the regulation of salt tolerance in rice.

[0008] A second objective of this invention is to provide a carrier.

[0009] A third objective of this invention is to provide a genetically engineered bacterium.

[0010] The fourth objective of this invention is to provide a marker related to salt tolerance traits in rice.

[0011] The fifth objective of this invention is to provide the application of reagents for detecting the above-mentioned markers in the detection of salt tolerance in rice.

[0012] The sixth objective of this invention is to provide a reagent for detecting the salt tolerance of rice.

[0013] The seventh objective of this invention is to provide a method for regulating the salt tolerance of rice.

[0014] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of the STG5.2 gene in regulating salt tolerance in rice; the amino acid sequence of the protein encoded by the STG5.2 gene is shown in SEQ ID NO:2.

[0015] As a further technical solution, the nucleic acid sequence of the STG5.2 gene is shown in SEQ ID NO:1.

[0016] As a further technical solution, knocking out the STG5.2 gene reduces the salt tolerance of rice and decreases rice yield under salt stress (including the number of effective panicles, main panicle length, seed setting rate, and thousand-grain weight).

[0017] Secondly, the present invention provides a vector containing the STG5.2 gene.

[0018] Thirdly, the present invention provides a genetically engineered bacterium containing the aforementioned vector.

[0019] Fourthly, the present invention provides a marker related to salt tolerance in rice, wherein the marker is selected from any one of the following: a. STG5.2 gene; b. mRNA transcribed from the STG5.2 gene; c. Protein encoded by the STG5.2 gene.

[0020] Fifthly, the present invention provides the application of reagents for detecting the above-mentioned markers in the detection of salt tolerance in rice.

[0021] In a sixth aspect, the present invention provides a reagent for detecting salt tolerance in rice, the reagent being used to detect mRNA transcribed from the STG5.2 gene; The reagents include primer pairs for amplifying the mRNA transcribed from the STG5.2 gene, the nucleic acid sequences of which are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0022] In a seventh aspect, the present invention provides a method for regulating the salt tolerance of rice, including knocking out the STG5.2 gene to reduce the salt tolerance of rice.

[0023] As a further technical solution, methods for knocking out the STG5.2 gene include CRISPR / Cas9 technology.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention cloned rice gene LOC_Os05g07260 (STG5.2, Salt Tolerance and Grain Yield-related Gene 5.2), annotated as a phosphatidylinositol glycosylation-related gene. The inventors discovered that the STG5.2 gene positively regulates rice salt tolerance and grain yield. Knockout of this gene leads to inhibited plant growth and a severe decrease in grain yield under saline-alkali stress. This invention has potential application value for identifying rice salt tolerance genes and breeding new salt-tolerant rice varieties. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 : Expression levels of STG5.2 in untreated and salt-treated samples; Figure 2 Schematic diagram of the STG5.2 gene structure and CRISPR / Cas9-induced mutations in the stg5.2 mutant; Figure 3 STG5.2 positively regulates salt tolerance in rice; Figure 4 Phenotype of stg5.2 mutant under saline-alkali soil conditions. Detailed Implementation

[0027] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] In a first aspect, the present invention provides the application of the STG5.2 gene in regulating salt tolerance in rice; the amino acid sequence of the protein encoded by the STG5.2 gene is shown in SEQ ID NO:2: (SEQ ID NO:2).

[0029] This invention cloned rice gene LOC_Os05g07260 (STG5.2, Salt Tolerance and Grain Yield-related Gene 5.2), which was annotated as a phosphatidylinositol glycosylation-related gene and may be involved in the GPI-anchored biosynthetic pathway. The inventors' research found that the STG5.2 gene has a positive regulatory function on rice salt tolerance and grain yield, and has potential application value for breeding new salt-tolerant rice varieties.

[0030] In some optional embodiments, the primer sequences for cloning the STG5.2 gene are as follows: STG5.2-F: ATGCCAGGCAGGCAGCAG (SEQ ID NO:3); STG5.2-R: TGTTTCCGAACGGTTCGGT (SEQ ID NO:4).

[0031] In some alternative embodiments, the nucleic acid sequence of the STG5.2 gene is shown in SEQ ID NO:1: MPGRQQAASETMEQERKGEVSIGMYTYNHNSDSGFDVHEIYVKRSRFWVLLSYIGMVFLLASVCQLYLSKEGLSTGSVWSIAFGILVAKCLQYKPVKKESVVIMPTFGVQLEIHF WSGRVHRRFVPIGKILKPLLNECVTPVTCYWSLALLLRDEEELRLVFQVKFICSNLLLRDQRKIQSPIELNLQKSHPPVKMLVPVWKALCKFLNSGCQTSSAVSEPNRSET (SEQ ID NO:1).

[0032] Secondly, the present invention provides a vector containing the STG5.2 gene.

[0033] When this vector is introduced into recipient cells, it can express the STG5.2 protein.

[0034] Thirdly, the present invention provides a genetically engineered bacterium containing the aforementioned vector.

[0035] For example, the above-mentioned vector can be introduced into Agrobacterium to transfect rice to improve the salt tolerance of rice; or the above-mentioned vector can be introduced into recipient cells for the preparation of the STG5.2 gene or a vector containing the gene.

[0036] Fourthly, the present invention provides a marker related to salt tolerance in rice, wherein the marker is selected from any one of the following: a. STG5.2 gene; b. mRNA transcribed from the STG5.2 gene; c. Protein encoded by the STG5.2 gene.

[0037] Since the STG5.2 gene has the function of positively regulating rice salt tolerance, the salt tolerance of rice can be judged based on the STG5.2 gene or its mRNA and protein.

[0038] Fifthly, the present invention provides the application of reagents for detecting the above-mentioned markers in the detection of salt tolerance in rice.

[0039] Since STG5.2 is related to the salt tolerance of rice, the salt tolerance of rice can be detected by testing STG5.2.

[0040] In a sixth aspect, the present invention provides a reagent for detecting salt tolerance in rice, the reagent being used to detect mRNA transcribed from the STG5.2 gene; The reagents include primer pairs for amplifying the mRNA transcribed from the STG5.2 gene, and the nucleic acid sequences of the primer pairs are shown in SEQ ID NO:5 and SEQ ID NO:6: STG5.2-qPCR-F: TGCGTGATGAAGAGGAACTCAGG (SEQ ID NO: 5); STG5.2-qPCR-R: TTTGCAGAGAGCCTTCCAGACAG (SEQ ID NO: 6).

[0041] By designing specific primers to detect the mRNA transcribed from the STG5.2 gene, the salt tolerance of rice can be inferred to a certain extent.

[0042] In a seventh aspect, the present invention provides a method for regulating the salt tolerance of rice, including knocking out the STG5.2 gene to reduce the salt tolerance of rice.

[0043] In some alternative implementations, methods for knocking out the STG5.2 gene include, but are not limited to, CRISPR / Cas9 technology.

[0044] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0045] Example 1: Cloning of the STG5.2 gene Total RNA was extracted from leaves of rice 'Nipponbare' using the Trizol method, and the first strand of cDNA was synthesized after reverse transcription using a kit. Using the reverse-transcribed cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase KOD-Plus-Polymerase with primers STG5.2-F (SEQ ID NO:3) and STG5.2-R (SEQ ID NO:4). The full-length CDS region of the STG5.2 gene was amplified by PCR. The PCR product was run on a 1% agarose gel, excised, and sent to the company for sequencing. The sequence of the STG5.2 gene is shown in SEQ ID NO:2.

[0046] Example 2: STG5.2 gene expression was significantly downregulated at 2, 6, and 24 h of salt treatment. Two-week-old 'Zhonghua 11' rice varieties with uniform growth were selected and treated with 0 mM NaCl and 150 mM NaCl. Samples were taken at 2, 6, 12, and 24 h after treatment. RNA was extracted from rice leaves using the Trizol method. Using cDNA as a template and the ACTIN gene as an internal control, RT-qPCR was performed using primers STG5.2-qPCR-F (SEQ ID NO:5) and STG5.2-qPCR-R (SEQ ID NO:6). The results showed that the expression level of the STG5.2 gene decreased significantly at 2, 6, and 24 h after salt treatment. Figure 1 ).

[0047] Example 3: Identification of STG5.2 knockout mutants To verify the role of STG5.2 in salt stress tolerance, we constructed an STG5.2 gene knockout mutant (stg5.2) in the genetic background of 'Zhonghua 11' using CRISPR / Cas9 technology and performed editing type and homozygosity tests. DNA was extracted from stg5.2 plants using a DNA extraction kit. PCR was performed using STG5.2-KO-F (SEQ ID NO:7) and STG5.2-KO-R (SEQ ID NO:8) verification primers. The PCR products were run on a 1% agarose gel, excised, and sent to the company for sequencing. Sequencing results compared with SEQ ID NO:2 revealed that the deletion of 4 bases (ACAT) in the second exon led to a frameshift mutation, producing a premature stop codon and forming a non-functional truncated protein, thus resulting in the loss of function of STG5.2. Figure 2 ).

[0048] Example 4: The stg2 mutant reduced rice salt tolerance. Two-week-old seedlings of wild-type (WT) and stg5.2 mutant were treated with 150 mM NaCl solution for 7 days, followed by a 12-day recovery period. Survival rate, ion leakage rate, and MDA index of each plant were measured. Results showed that before treatment, the growth of WT and stg5.2 plants was consistent; after salt stress, the growth of stg5.2 mutant was significantly worse than that of WT. Figure 3 (A) Morphological and physiological data were consistent with the phenotype. The survival rate of the stg5.2 mutant (50%-63%) was significantly lower than that of the WT mutant (75%-83%). Figure 3 (B in the text). Conversely, ion leakage rate and MDA were not significantly different from WT before treatment, but were significantly higher than WT after salt treatment. Figure 3 C and Figure 3 (D in the text). These results indicate that STG5.2 positively regulates rice salt tolerance during the seedling stage.

[0049] Example 5: Loss of STG5.2 function led to increased salt sensitivity and impaired field performance in rice. To comprehensively evaluate the field application value of STG5.2, we observed the phenotypes of WT and stg2 mutants throughout their entire growth period in moderately saline-alkali fields (3%-5%). Compared with WT, the stg5.2 mutant exhibited stronger salt sensitivity, with significantly lower plant height (WT: 101.0 ± 6.05 cm; stg5.2: 72.5 ± 8.47 cm, P<0.0001) and tiller number (WT: 12 ± 2.21; stg5.2: 9 ± 2.62, P<0.0001) than WT. Figure 4 The AC value in stg5.2 was significantly underdeveloped, with the number of effective panicles (WT: 11±1.43; stg5.2: 8±1.52, P<0.001) and the length of the main panicle (WT: 22.8±2.51; stg5.2: 18.6±1.48, P<0.0001) significantly lower than that of WT ( ). Figure 4 In addition, under this environment, the seed setting rate and yield per plant of the stg5.2 mutant were significantly reduced, and the grain width was also narrower than that of the wild type. Figure 4 The GL in the middle. Correspondingly, the thousand-grain weight of the stg5.2 mutant was also significantly reduced, while the grain length did not show a significant change (GL). Figure 4 (M and N in the text). Overall, these observations confirm that the STG5.2 gene has a positive regulatory effect on rice salt tolerance, and its loss of function reduces rice tolerance and grain yield in saline-alkali soil.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the STG5.2 gene in regulating salt tolerance in rice; the amino acid sequence of the protein encoded by the STG5.2 gene is shown in SEQ ID NO:1; Knocking out the STG5.2 gene reduces the salt tolerance of rice and decreases rice yield under salt stress.

2. The application according to claim 1, characterized in that, The nucleic acid sequence of the STG5.2 gene is shown in SEQ ID NO:

2.

3. A method for reducing salt tolerance in rice, characterized in that, This includes knocking out the STG5.2 gene as described in claim 1 or 2 to reduce the salt tolerance of rice.

4. The method according to claim 3, characterized in that, Methods for knocking out the STG5.2 gene include CRISPR / Cas9 technology.

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