Rice alanine aminotransferase Qsd1 (PHS10) gene as well as encoding protein and application thereof

Abstract

The invention discloses a rice alanine aminotransferase Qsd1 (PHS10) gene as well as an encoding protein and application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence of the rice alanine aminotransferase Qsd1 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the encoding protein of the rice alanine aminotransferase Qsd1 gene is shown as SEQ ID NO: 2. According to the qsd1 mutant obtained through a knockout technology, seeds of the qsd1 mutant have high dormancy, and the treatment sensitivity of exogenous ABA in the seed germination period and the seedling period is high. The invention provides a solid theoretical basis for transforming a new variety of pre-harvest sprouting resistant rice and increasing the crop yield, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a rice alanine aminotransferase Qsd1 (PHS10) gene and a coded protein and application thereof. Background Art

[0002] Seed dormancy is a complex quantitative trait that is regulated by factors such as the external environment and the internal gene network. It is of great significance for plants to adapt to unfavorable environmental conditions and ensure the reproduction and evolution of their own populations. Shortening seed dormancy can enhance the adaptability of crops and promote crop rotation in agriculture. However, too weak seed dormancy of cereal crops will directly lead to seed germination before harvest, namely pre-harvest sprouting. As one of the biggest hazards in the grain production of cereal crops, pre-harvest sprouting seriously harms the yield and quality of grains. Therefore, exploring the genes related to rice seed dormancy and clarifying their molecular mechanisms for regulating rice seed dormancy are of great significance for improving the rice seed dormancy regulatory network and cultivating rice varieties with moderate dormancy. In barley and wheat, the Qsd1 gene has been preliminarily identified to be related to seed dormancy, but the function of the rice Qsd1 gene in regulating seed dormancy and germination remains to be studied. It is of great significance to develop new genes for regulating ABA signals and improving the dormancy ability of rice seeds. Summary of the invention

[0003] One of the purposes of the present invention is to provide an application of a rice alanine aminotransferase Qsd1 (PHS10) gene in regulating rice seed dormancy. The amino acid sequence of the protein encoded by the Qsd1 (PHS10) gene is shown in SEQ ID NO.2.

[0004] Preferably, the nucleotide sequence of the Qsd1 (PHS10) gene is as shown in SEQ ID NO.1.

[0005] A second object of the present invention is to provide a method for prolonging the dormancy period of rice seeds, the method comprising: weakening or knocking out the Qsd1 (PHS10) gene in rice by genetic engineering means, wherein the Qsd1 (PHS10) gene is the same as described above.

[0006] Preferably, the method comprises: taking the Qsd1 (PHS10) gene as the target gene, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas9, transforming rice, and thereby obtaining transgenic rice with loss of gene function.

[0007] More preferably, the nucleotide sequence of the sgRNA action site is: ATGTACCTCTTCCCA CGGCTG (SEQ ID NO.9).

[0008] The third object of the present invention is to provide a method for shortening the dormancy period of rice seeds, the method comprising: using genetic engineering means to overexpress the Qsd1 (PHS10) gene in the plant, wherein the Qsd1 (PHS10) gene is the same as described above.

[0009] More preferably, the overexpression method is selected from the following 1)-5), or an optional combination:

[0010] 1) by introducing a plasmid carrying the gene;

[0011] 2) by increasing the copy number of the gene on the plant chromosome;

[0012] 3) by changing the promoter sequence of the gene on the plant chromosome;

[0013] 4) by operably linking a strong promoter to the gene;

[0014] 5) By introducing enhancers.

[0015] A fourth object of the present invention is to provide application of the transgenic rice obtained by the above method in rice breeding.

[0016] Preferably, the breeding method comprises transgenic, hybridization, backcrossing, selfing or asexual propagation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention clones the rice Qsd1 (PHS10) gene from rice using a PCR method. The full-length sequence of the Qsd1 (PHS10) gene coding region obtained by the present invention corresponds to Os09g26380 published in The Rice Annotation Project. It is also discovered for the first time that Qsd1 can regulate ABA signals and seed dormancy.

[0019] The present invention uses CRISPR / Cas9 knockout technology and genetic transformation to obtain Qsd1 (PHS10) gene knockout mutants, and finds that qsd1 mutants show strong seed dormancy, and are highly sensitive to exogenous ABA treatment during seed germination and seedling growth. Germination experiments and exogenous ABA sensitivity analysis experiments have fully demonstrated that Qsd1 can negatively regulate ABA signals to affect seed dormancy.

[0020] Studying the expression pattern of rice alanine aminotransferase OsQsd1 (PHS10) gene and its biological function under adverse stress to analyze the regulatory mechanism of OsQsd1 on seed dormancy provides a solid theoretical basis for transforming new rice varieties resistant to panicle germination and increasing crop yields, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a picture of the expression pattern of the Qsd1 gene in different tissues in Example 2.

[0022] Figure 2 These are pictures of the expression levels of the Qsd1 gene in grains 7, 14, 21, and 30 days after heading in Example 2.

[0023] Figure 3 This is the gene sequence and mutation type picture of T0 generation qsd1 in Example 2.

[0024] Figure 4 This is a picture of identifying the Qsd1 gene overexpression material in Example 2.

[0025] Figure 5 These are phenotypic pictures of the Qsd1 gene mutant and overexpression strain in Example 2.

[0026] Figure 6 These are the statistical results of the germination rates of the Qsd1 gene mutants and overexpression strains in Example 2.

[0027] Figure 7 This is a picture of the sensitivity analysis experiment of Qsd1 gene expression to exogenous ABA in Example 3.

[0028] Figure 8 This is the expression analysis of ABA-responsive genes in the qsd1 mutant in Example 4.

[0029] Fig. 9 These are the statistical results of the effect of 0 μM exogenous ABA on the germination rates of the qsd1 mutant and the wild type in Example 5.

[0030] Fig.10 These are the statistical results of the effect of 1 μM exogenous ABA on the germination rates of the qsd1 mutant and the wild type in Example 5.

[0031] Fig.11 These are the statistical results of the effect of 3 μM exogenous ABA on the germination rates of the qsd1 mutant and the wild type in Example 5.

[0032] Fig.12 It is the inhibition rate of ABA on germination of qsd1 mutant, overexpression strain and wild type under the condition of 1 μM ABA treatment in Example 5.

[0033] Fig.13 This is the inhibition rate of ABA on germination of qsd1 mutant, overexpression strain and wild type under the treatment condition of 3 μM ABA in Example 5. DETAILED DESCRIPTION

[0034] Example 1 Cloning of Rice Alanine Aminotransferase Qsd1 (PHS10) Gene

[0035] First, using the cold-region early-maturing japonica rice variety Hejiang No. 19 (HJ19) as the experimental material, the total RNA of the leaves was extracted according to the operation manual of the TRIzol kit of Invitrogen;

[0036] Second, the total RNA extracted in step 1 was treated with DNase I;

[0037] 3. Take 1 μg of the total RNA treated in step 2 for cDNA synthesis. The cDNA synthesis operation is carried out according to the instructions of the reverse transcription kit purchased from TOYOBO Company to obtain cDNA;

[0038] 4. Using the cDNA obtained above as a template, refer to TaKaRa's HSDNA Polymerase operating instructions, forward primer F1 and reverse primer R1 were used to amplify the OsQsd1 gene. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 60s, a total of 38 cycles; 72℃ final extension for 10min. Finally, the PCR product was sequenced on an ABI3130 sequencer (ABI Company). The sequencing results showed that the rice alanine aminotransferase OsQsd1 gene consisted of 486bp bases, its CDS nucleotide sequence was shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene was shown in SEQ ID NO.2.

[0039] SEQ ID NO.1:

[0040]

[0041] SEQ ID NO.2:

[0042] MSYNHSPSITAETINQKVRIFTYEPCGEIVRHARRLEKEIYENPGSLPFQEIIYCNLGNPQALGQRPINFFREVLSLCDNPSLIDRDEARALFSPCALKRARKIIESLPGRDSGSYTSSQG VRGLREAVADGIAARDGFPSKPDNIFLTDGASSAINMMMQILIRSHEDGILCPLPEYPLYSASIILHGGTMVPYNLTEDSIWGLEIFEVKRCLEDARASGLTIRAMVVINPGNPTGQVLSIT NQEEIVEFCRKEGLVILADEVYQENVYTENKRFNSFKKVARSLGYDHHDLSIVSFHSVSMGYYGECGRRGGYMEICGFGDDVIDEMYKLASLTICPNIAGQILISLVMDPPKLGDEAFEIFM VEKEETYSSLLKRAKALQKAFNGLEGVSCNKFEGAMYLFPRLRLPQAAIKAAQLEGVSPDVFYAHRLLDATGIAVVPGSGFHPVSGTSHIRCTILPGEETITAMVPSLQAFHEAFMDEFRG.

[0043] Forward primer F1 (SEQ ID NO. 3): 5′-ATGTCGTACAACCATTCGCC-3′;

[0044] Reverse primer R1 (SEQ ID NO. 4): 5'-TCAGCCGCGAAACTCGTCCA-3'.

[0045] Example 2 Obtaining qsd1 mutants

[0046] 1. Input the CDS sequence of the OsQsd1 (PHS10) gene into the CRISPR Primer Designer software and design two pairs of target site primers (F2, R2 and F3, R3) for the subsequent construction of the knockout vector.

[0047] Forward primer F2 (SEQ ID NO. 5): 5′-ATCTTCACCGACGACACCG-3′;

[0048] Reverse primer R2 (SEQ ID NO. 6): 5′-ATTGTGACAGGGCCTAACCG-3′;

[0049] Forward primer F3 (SEQ ID NO. 7): 5′-ACCGGCAAGCTCATATCTCG-3′;

[0050] Reverse primer R3 (SEQ ID NO. 8): 5'-CAAGCCAGACGACAAGCAAC-3'.

[0051] 2. Primer annealing: dilute the dry powder primer to 100 μM, mix 1 μL of each upstream and downstream primer of each gene with 98 μL 0.5×TE solution, heat shock at 90℃ for 30s and then cool to room temperature to make a connector; (0.5×TE: dilute with 10×TE, 25 μL 10×TE + 475 μL ddH 2 (0 = 500 μL 0.5×TE).

[0052] 3. gRNA expression cassette ligation reaction:

[0053]

[0054] PCR reaction procedure:

[0055] 37℃5min (cut), 20℃5min (continuous), 5 cycles (can be paused and stored at 4℃)

[0056] 4. The first round of PCR amplification, the system is as follows:

[0057]

[0058]

[0059] PCR reaction procedure:

[0060]

[0061] 5. The second round of PCR amplification, the system is as follows:

[0062] First, dilute the first round of PCR amplification products by 30 times and 60 times respectively.

[0063]

[0064] Bn′ / Bn+1 refers to the first pair of primers F + the second pair of primers R, and the other group is the second pair of primers F + BL.

[0065] PCR reaction procedure:

[0066]

[0067] 6. Detect the second round of PCR amplification products by agarose gel electrophoresis, and use a gel recovery kit to recover the PCR products. The concentration after recovery should be around 50ng / μL;

[0068] VII. Ligation reaction, system, PCR reaction procedures are as follows: (In this step, mix the target products of different dilution multiples: 30x mix together, 60x mix together)

[0069]

[0070]

[0071] PCR reaction program: 37°C for 15 min;

[0072] Then add:

[0073] T4 ligase buffer 1.5μL

[0074] T4 ligase 0.3μL

[0075] PCR reaction procedure:

[0076]

[0077] 8. Transformation into E. coli

[0078] (1) Add 5-10 μL of the ligation product to 100 μL of competent cells DH5α / TOP10 and incubate on ice for 30 min;

[0079] (2) Heat shock at 42°C for 2 min, followed by ice bath for 2 min;

[0080] (3) Add 700 μL LB (no resistance) liquid medium and shake at 37°C, 180 rpm, for 1 h.

[0081] (4) Centrifuge at 8000 rpm for 3 min at room temperature, discard 500 μL of supernatant, resuspend the precipitate and spread it on LB solid medium of the corresponding resistance;

[0082] (5) After sealing with a sealing film, invert and culture in a 37°C incubator overnight.

[0083] IX. Monoclonal PCR

[0084] (1) Prepare a sterilized 2 mL EP tube, a 10 μL pipette tip, and LB liquid culture medium of corresponding resistance. Use the pipette tip to pick up a single colony and place it in an EP tube. Add 1 mL of culture medium and shake at 37°C and 180 rpm overnight.

[0085] (2) The PCR identification system is as follows:

[0086]

[0087] PCR reaction procedure:

[0088]

[0089] 10. Extract plasmid: Use a plasmid extraction kit to further digest and identify the extracted plasmid. The system is as follows:

[0090]

[0091] Enzyme digestion reaction: 37°C, 2-3h

[0092] 11. Detect the digestion products by agarose gel electrophoresis. The fragments with the correct size are transformed into Agrobacterium EHA105:

[0093] (1) Take out the EHA105 competent cells from the -80°C freezer and place them on ice to thaw;

[0094] (2) Add 0.01-1 μg of plasmid to 100 μL of EHA105 competent medium, place on ice for 20 min, and freeze in liquid nitrogen for 5 min;

[0095] (3) Rapidly place the sample in a 37°C water bath, warm for 5 min, and then cool on ice for 5 min.

[0096] (4) Add 700 μL of antibiotic-free LB liquid medium and incubate in a shaker at 28°C, 130 rpm, for 3-5 h;

[0097] (5) Centrifuge at 8000 rpm for 3 min, discard most of the supernatant, spread an appropriate amount of bacterial solution on a medium containing the corresponding resistance and rifampicin resistance (50 μg / ml), invert in a 28°C incubator, and culture for about 3 days;

[0098] (6) Pick the bacteria, shake the bacteria, and use the bacterial solution for PCR identification to determine the positive clones;

[0099] (7) Pick the positive clones and transfer them to liquid LB medium containing the corresponding antibiotics and rifampicin resistance. Incubate them in a shaker at 28°C and 180 rpm for 12-16 h. Preserve the bacterial solution with sterilized 30% glycerol (ratio 1:1) and store it in a -80°C refrigerator. When genetic transformation is required, remove the bacterial solution and reactivate it.

[0100] 12. Agrobacterium infection of rice callus

[0101] (1) Activation and cultivation of Agrobacterium: Take out Agrobacterium and add it into LB liquid culture medium containing corresponding resistance and rifampicin (50 μg / mL) resistance at a ratio of 1:100, place it in a shaker at 28°C, 160 rpm, and culture it overnight.

[0102] (2) Agrobacterium was cultured using liquid LB containing rifampicin antibiotics. When the OD600 value of Agrobacterium became about 0.5, it indicated that the growth state of Agrobacterium was good and the subsequent infection operation could be carried out.

[0103] (3) Take 500 μL of bacterial solution, centrifuge at 5000 g for 5 min, remove the supernatant, and resuspend the bacterial solution in 15 mL of co-culture medium and 100 μmol / L acetosyringone (AS) mixture. Select callus tissue and place it in the invasion dye solution and let it stand for 3-5 min.

[0104] (4) After infection, pour off the liquid, spread the callus flat on sterile filter paper, let it air-dry for 1 min, transfer the air-dried callus to a co-culture medium covered with a layer of sterile filter paper, and culture in the dark at 28°C for 3 days.

[0105] 13. Recovery: Culture on recovery medium.

[0106] 14. Screening: After 7 days of recovery culture, transfer to screening medium for further culture.

[0107] 15. Differentiation: About one month later, callus tissue expressing resistance is selected from the screening medium and transferred to the differentiation medium. These callus tissues gradually differentiate and grow into seedlings.

[0108] 16. Identification of genetically modified seedlings:

[0109] (1) Prepare HPT screening medium and conduct positive identification of transgenic seedlings.

[0110] (2) The T0 generation transgenic rice seedlings that were identified as positive were moved to an artificial greenhouse for planting and the seeds were collected.

[0111] 17. Acquisition and identification of the overexpression strain OE-OsQsd1:

[0112] (1) Link the CDS region of the target fragment to the 1300-FLAG vector, transfer to TOP10, send to the company for sequencing, extract the plasmid after success, transfer to Agrobacterium, and infect callus;

[0113] (2) Planting T0 generation seeds and wild type seeds at the same time, extracting DNA after they grow into seedlings, and performing PCR identification using specific primers;

[0114] (3) For rice seedlings that are positive for hygromycin, their RNA is extracted and converted into cDNA using reverse transcription technology. The expression level is analyzed using qRT-PCR to determine several independent T1 transgenic seedlings with high expression levels for continued planting.

[0115] 18. Identification of CRISPR / Cas9 plants:

[0116] (1) Plant the positive rice seedlings together with the wild type, extract DNA, and perform PCR identification using target primers

[0117] (2) Perform agarose gel electrophoresis, cut the band, use a gel recovery kit to recover the product, send the product to the company for sequencing, and compare the results after they are returned. Confirm that the mutation has occurred and the homozygous T1 transgenic seedlings continue to be planted.

[0118] The sequencing results of the OsQsd1 gene knockout mutants qsd1-1 and qsd1-2 showed that the insertion and deletion of bases caused subsequent frameshift mutations, which changed the expressed amino acids and caused the OsQsd1 protein to lose its function.

[0119] Figure 1 , wild-type rice Hejiang 19 was selected as the experimental material, and its roots, stems, leaves, and embryos were taken respectively, RNA was extracted and reverse transcribed into cDNA, and fluorescent quantitative PCR primers were designed to detect the expression characteristics of the OsQsd1 gene. The expression of the OsQsd1 gene is tissue-specific, with very weak expression in the radicle and stem during the vegetative growth stage, high expression in the developing seeds after imbibition, and also a certain expression in the leaves.

[0120] Figure 2 , the temporal expression pattern of the OsQsd1 gene was analyzed. The expression of OsQsd1 gradually increased over time during seed maturation, indicating that OsQsd1 plays a role in the seed development of rice.

[0121] Figure 3-4 The gene sequence and mutation type of qsd1 in the T0 generation and the expression level and phenotypic analysis of OE-OsQsd1 were detected by qRT-PCR.

[0122] Figure 5-6 , the above homozygous mutants and overexpression positive lines were used as test materials. 30 days after heading, the mature rice ears of wild type and transgenic lines grown in the field were harvested for germination test and phenotypic observation. Compared with WT, qsd1 showed delayed germination, while OE-OsQsd1 germinated earlier. The rice seeds were tested for germination and the germination rate was statistically calculated. The germination rate of the overexpression line reached about 50% at 60h, while the two knockout mutants slowly began to germinate at 48h and 60h. At each time point, the germination rate of the wild type and transgenic lines showed significant differences. Overall, the germination rate of the overexpression line was higher than that of the wild type, while the germination rate of the knockout line was much lower than that of the wild type, indicating that the OsQsd1 gene can regulate the dormancy of rice seeds.

[0123] Example 3 Analysis of expression patterns of qsd1 mutant materials in response to ABA (abscisic acid)

[0124] 1. Select wild rice variety HJ19 as the experimental material, transfer it into two 96-well culture boxes, and add rice nutrient solution for germination;

[0125] Second, after culturing in a 25°C incubator for 2 weeks, one of the boxes of nutrient solution was treated with 50 μM ABA, and the control group was still cultured with nutrient solution;

[0126] Third, the "second leaves" at different time periods of 0h, 1h, 3h, and 6h after the two boxes of materials were treated were taken, RNA was extracted, and the expression level of the OsQsd1 gene was detected by qRT-PCR;

[0127] 4. Result analysis.

[0128] See Figure 7 , the expression level of OsQsd1 gene changed under exogenous ABA treatment, and the expression level reached the highest level after 9h of treatment, indicating that OsQsd1 gene was induced by ABA treatment, suggesting that OsQsd1 may regulate seed dormancy by participating in the ABA pathway. -ΔΔCT Relative quantification was performed by the method (n=3).

[0129] Example 4 Analysis of expression of genes responsive to ABA in OsQsd1 transgenic rice

[0130] First, qRT-PCR was used to analyze the expression levels of related genes regulated by ABA in plants, and two representative downstream key genes induced by ABA were detected, namely: RAB21 (Os11g26790) and OsABI5 (Os01g64000);

[0131] 2. Result analysis.

[0132] See Figure 8 , the expression level of OsQsd1 gene in OE-OsQsd1 was higher than that in WT, and the expression levels of RAB21 and OsABI5 genes were significantly downregulated; on the contrary, the expression level of OsQsd1 gene in qsd1 was lower than that in WT, and the expression levels of RAB21 and OsABI5 genes were significantly upregulated.

[0133] Example 5 Analysis of the sensitivity of OsQsd1 transgenic rice to ABA

[0134] 1. Take freshly harvested seeds of wild type, knockout mutants, and overexpression lines, shell and sterilize them, place them in culture dishes, and add 30 seeds to each dish;

[0135] Second, add 8 mL of 0 μM, 1 μM and 3 μM ABA solutions respectively, the solvent is sterile water, and carry out germination test in an artificial climate incubator at 25°C;

[0136] 3. The appearance of coleoptile was used as the germination standard. The first addition of solution was counted as 0h. The germination was counted every 12h. Finally, the germination rate was calculated and the rice germination inhibition rate after 60h of treatment was counted.

[0137] 4. Observe and calculate the germination inhibition rate.

[0138] The calculation formula of ABA inhibition rate on seed germination is:

[0139] Inhibition rate = (number of germinated seeds under 0 μM ABA treatment - number of germinated seeds under X μM ABA treatment) / number of germinated seeds under 0 μM ABA treatment × 100%.

[0140] See Figure 9-13 During the seed germination stage, compared with the wild type, the germination of qsd1 was more sensitive to ABA, while the germination of the overexpression line was less sensitive to ABA, indicating that the OsQsd1 gene is involved in regulating seed dormancy and germination through the ABA signaling pathway.

[0141] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of a rice alanine aminotransferase Qsd1 gene in regulating rice seed dormancy, characterized in that: The amino acid sequence of the protein encoded by the Qsd1 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the Qsd1 gene is shown in SEQ ID NO.

1.

3. A method for extending the dormancy period of rice seeds, characterized in that: The method comprises: using genetic engineering means to weaken or knock out the Qsd1 gene in rice, wherein the Qsd1 gene is the same as that described in claim 1 or 2.

4. The method according to claim 3, characterized in that The method comprises: taking the Qsd1 gene as a target gene, designing an sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas9, transforming rice, and thereby obtaining transgenic rice with loss of gene function.

5. The method according to claim 4, characterized in that The nucleotide sequence of the sgRNA action site is: ATGTACCTCTTCCCACGGCTG.

6. A method for shortening the dormancy period of rice seeds, characterized in that: The method comprises: using genetic engineering means to overexpress the Qsd1 gene in plants, wherein the Qsd1 gene is the same as that described in claim 1 or 2.

7. The method according to claim 6, characterized in that The overexpression method is selected from the following 1)-5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the plant chromosome; 3) by changing the promoter sequence of the gene on the plant chromosome; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.

8. Use of the transgenic rice obtained according to the method of any one of claims 3 to 7 in rice breeding.

9. The use according to claim 8, characterized in that: Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

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