Application of SbTCP2 gene in regulation and control of tiller number of rice

Abstract

The invention relates to the technical field of agriculture, and particularly discloses application of a SbTCP2 gene in regulation and control of the tiller number of rice, the nucleotide sequence of the SbTCP2 gene is shown as SEQ ID NO.1, and the amino acid sequence of protein coded by the SbTCP2 gene is shown as SEQ ID NO.2. According to the invention, the SbTCP2 gene is over-expressed in a rice plant, so that the tiller number of rice is reduced, and a new rice plant type is constructed.

Description

TECHNICAL FIELD

[0001] The application relates to the field of agricultural technology, in particular to SbTCP2 application of a gene in regulation of rice tiller number. BACKGROUND

[0002] Tillering is a key agronomic trait of gramineous crops, directly affecting effective panicle number and final yield. Ideal tiller number is a key agronomic trait for realizing high yield and stable yield, and too many or too few tillers will have adverse effects on the growth and yield of crops. The tillering capacity of plants determines the population structure, and further affects light utilization efficiency and lodging resistance. Therefore, it is of great significance to excavate key genes for regulating tillering for optimizing plant type and improving crop yield potential, thereby realizing high yield and stable yield and providing important support for sustainable agricultural development.

[0003] Rice, Oryza sativa as an important food crop, the molecular mechanism of tiller regulation is very important. Tiller number is a main factor for plant type formation of rice. Therefore, it is of great significance to excavate the application of existing genes in regulation of rice tiller number for plant type formation of rice. SUMMARY

[0004] To develop an application of a gene in regulation of rice tiller number, the application provides SbTCP2 application of a gene in regulation of rice tiller number. The application reduces the rice tiller number by overexpressing the SbTCP2 gene in the rice plant, and constructs a new rice plant type.

[0005] The application provides SbTCP2 application of a gene in regulation of rice tiller number, wherein the SbTCP2 nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0006] The application reduces the rice tiller number by overexpressing the SbTCP2 gene in the rice plant, and constructs a new rice plant type. The constructed SbTCP2 gene overexpression vector constructs a transgenic rice with reduced plant height and tiller number. SbTCP2 The transgenic rice overexpresses the gene.

[0007] Further, the rice tiller number is reduced by overexpressing the SbTCP2 gene.

[0008] Further, the rice overexpresses the SbTCP2 gene by transforming the rice with the gene overexpression vector. SbTCP2 The gene overexpression vector is constructed.

[0009] Further, the gene overexpression vector is constructed. SbTCP2The steps of the gene overexpression vector are as follows: SbTCP2 The gene fragment is recombined with the pOX vector after enzyme cutting through seamless cloning to obtain a recombinant vector, and the Escherichia coli is transformed, and the plasmid is extracted after culture to obtain SbTCP2 The gene overexpression vector.

[0010] Further, SbTCP2 The gene fragment cloning step is that the cDNA of Sorghum halepense tillering bud is taken as a template, and the nucleotide fragments shown in SEQ ID NO. 9 and SEQ ID NO. 10 are taken as primers to perform PCR amplification to obtain SbTCP2 The gene fragment.

[0011] Further, the overexpression vector is transformed into rice by using the agrobacterium infection method.

[0012] The application provides SbTCP2 The application of the gene in rice plant type building, and the SbTCP2 Gene overexpression vector is used to build a rice plant type with reduced tiller number.

[0013] The application provides SbTCP2 The application of the gene in creating new germplasm resources, and the SbTCP2 Gene overexpression vector is used to build a transgenic rice with reduced plant height and reduced tiller number. SbTCP2 Overexpression.

[0014] Compared with the prior art, the beneficial effects of the application are as follows: The application reduces the tiller number of rice and reduces the plant height of rice by overexpressing the SbTCP2 Gene in rice plants. SbTCP2 The gene is used for regulating the tiller number and plant height of rice plants, which is beneficial to rice plant type building and lays a foundation for creating new rice germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 The SbTCP2 Gene expression quantity analysis of different parts of sorghum roots, stems, tillering buds and leaves.

[0017] Figure 2 The figure of the pOX vector, and the resistance is KAN.

[0018] Figure 3 For SbTCP2 Effects of overexpression on the number of tillers of rice; In the figure, A is the phenotype of rice SbTCP2 Overexpression of rice phenotype; B is the overexpression of rice SbTCP2 Positive seedlings SbTCP2 Identify the electrophoretogram, and the positive seedling OsUbiq as the internal reference; C is the wild type and SbTCP2 Statistical chart of overexpression of rice plant height; D is the wild type and SbTCP2 Statistical chart of overexpression of rice tiller number. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0020] The unknown function gene in the genome of sorghum SbTCP2 has been studied, and the SbTCP2 CDS nucleotide sequence of the gene is 1143 bp in length, and the specific sequence information is shown as SEQ ID NO. 1.

[0021] SEQ ID NO. 1:

[0022] The SbTCP2 The full-length amino acid sequence of the protein encoded by the gene is 380 aa, and the specific sequence information is shown as SEQ ID NO. 2.

[0023] SEQ ID NO. 2: MFPFCDSSSPMDLPLYQQLQLSPPSPKPDQSSSFYCCYPCSPPFAAAAADASFHLSYQIGSAAAAIPPQAVINSPEDLPVQPLMEQAPAPPTELVACASGGAQGAGVSVSLDRAAAAAAARKDRHSKICTAGGMRDRRMRLSLDVARKFFALQDMLGFDKASKTVQWLLNTSKAAIQEIMADDVDASSECVEDGSSSLSVDGKHNPAEQLGDQKPKGNGRSEGKKPAKSRKAATTPKPPRKSGNNAHPVPDKETRAKARERARERTKEKHRMRWVKLASAIDVEAAAASVASDRPSSNHLNHHHHSSSSMNMPRAAEAELEERERCSSTLNNRGRMQEITGASEVVLGFGNGGGYGGGNYYCQEQWELGGVVFQQNSRFY*.

[0024] The present application carries out biological analysis on the rice SbTCP2 gene, and the expression mode analysis result shows that SbTCP2 the expression amount of the gene in the root stem tillering place is the highest, followed by the stem and the root, and the expression content in the leaf is relatively low. SbTCP2 In order to explore the function of the gene, the SbTCP2 overexpression vector is constructed, the callus of the wild type ZH11 rice is used as the material, the genetic transformation is carried out by using the agrobacterium mediation method to obtain the overexpression transgenic plant, and the phenotype of the transgenic plant is analyzed. SbTCP2 It is found that, compared with the wild type, SbTCP2 some important yield traits of the overexpression strain change beneficially, including the tiller number reduction and the plant height lower than the wild type.

[0025] The present application proves by a series of experiments that: SbTCP2 the gene is derived from the rice tiller number related gene, and the overexpression of the gene can reduce the tiller number of the plant, and finally can cultivate the ideal plant type. SbTCP2 The above results can be used in the transgenic breeding field, and are helpful to lay the ideal foundation for the rice ideal plant type and trait improvement.

[0026] The rice material used in this invention is wild-type Zhonghua 11, namely ZH11.

[0027] Example 1: SbTCP2 Gene cloning.

[0028] 1. SbTCP2 Cloning of genes The sorghum variety used in this experiment was Hercules. Using the tillering sites of the sorghum rhizomes as templates, the nucleotide fragment shown in SEQ ID NO.3 was used as the upstream primer SbTCP2-fl-F, and the nucleotide fragment shown in SEQ ID NO.4 was used as the downstream primer. SbTCP2 PCR amplification was performed using -fl-R, and the product was collected. SbTCP2 The target gene was identified, and the amplified products were sequenced. The sequencing results showed... SbTCP2 The full-length CDS of the gene is 1143 bp, encoding 380 amino acid residues. The CDS nucleotide sequence is shown in SEQ ID NO.1. SbTCP2 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0029] SEQ ID NO.3:TTGGAGTCCCCATCAGTAAAGC; SEQ ID NO. 4: TCATGCGATGACCAAACGA.

[0030] 2. Sorghum SbTCP2 expression level study Tissues from different parts of the fresh sorghum cultivar, including roots, stems, tillers, and leaves, were collected and ground separately in liquid nitrogen. Total RNA was extracted using the Novizum RNA column extraction kit. Quantitative PCR was performed using Novizum's SuperReal PreMix Plus SYBR Green reagent. The reaction system preparation process was carried out in the dark. The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2. Three biological replicates and three technical replicates were set up for each sample to ensure the reliability of the experimental data. Samples were placed in a real-time quantitative PCR instrument. Target gene upstream primers were used. SbTCP2 -qPCR-F, as shown in SEQ ID NO.5, downstream primers for the target gene. OsUbiq -qPCR-R1-2, as shown in SEQ ID NO.6, is the upstream primer of the internal reference gene. OsUbiq -qRT-F, as shown in SEQ ID NO.7, is the downstream primer for the internal reference gene. SbTCP2 -qRT-R is shown in SEQ ID NO.8.

[0031] Primer pairs for quantitative real-time PCR of the target gene: SEQ ID NO.5: TTGGCAACGGAGGAGGATAC; SEQ ID NO. 6: TGAGTTCTGCTGAAAGACGACTC.

[0032] Primer pairs for quantitative real-time PCR of internal reference gene: SEQ ID NO.7: AGGGTTCACAAGTCTGCCTATTT; SEQ ID NO. 8: TCTTCCATGCTGCTCTACCACA.

[0033] For testing SbTCP2 This invention investigated the expression levels in different parts of sorghum plants, using tillers, roots, stems, and leaves of the sorghum Hercules variety as experimental materials. Figure 1 The expression level of [the substance] was quantitatively detected. The results are as follows: SbTCP2 As shown, SbTCP2 The gene is expressed in tillers, roots, stems and leaves. The expression level is relatively low in roots and leaves, and higher in stems and tillers. The expression level in tillers is significantly higher than in other parts.

[0034] Example 2: Creation of SbTCP2 transgenic rice.

[0035] In order to investigate SbTCP2 The function of genes, this invention constructs SbTCP2 Overexpression vectors were used, with protoplasts of wild-type ZH11 rice as material, and genetic transformation was performed using Agrobacterium-mediated transformation. The specific steps included:

[0036] 1. Target gene amplification According to sorghum SbTCP2 The CDS sequence of the gene was used to design a sequence as shown in SEQ ID NO. 9 using DNAMAN software. SbTCP2 -pOX-F primers and SEQ ID NO. 10 SbTCP2 -pOX-R primers.

[0037] Using cDNA from sorghum tiller shoots as a template, as shown in SEQ ID NO.9 SbTCP2 -pOX-F and SEQ ID NO. 10 pn-pOX-R is a primer, PCR amplification is carried out according to the reaction procedure in Table 3, the product is collected and purified: the collected product of PCR amplification is electrophoresed under the condition of 130v 25min, the target band is cut, and the product is purified according to the method recorded in the Novozyme FastPure® Gel DNA Extraction Mini Kit instruction book, to obtain the target gene fragment.

[0038] SEQ ID NO. 9: ATGTTTCCTTTCTGTGATTC; SEQ ID NO. 10: TCAGTAGAAGCGTGAGTTC.

[0039] 2. Linearization of the vector construction The pOX vector is double digested and the product is purified. The specific steps are as follows:

[0040] (1) Take 10 μL pOX vector bacterial solution into 10 mL LB containing working concentration of 50 mg / L Kan antibiotic, shake at 28°C overnight, and extract plasmid according to the detailed steps in the Novozyme FastPure Plasmid Mini Kit instruction book.

[0041] (2) Use K ind I and H SbTCP2 III enzyme to linearize the pOX vector by double digestion, and the reaction system is configured according to the following Table 4.

[0042] (3) Then slowly suck and hit the reaction solution, and centrifuge momentarily.

[0043] (4) Incubate at 37°C for 1h.

[0044] (5) Electrophoresis is carried out at 130v for 25min, and the target band is cut.

[0045] (6) The product is purified to obtain the double-digested vector, i.e. the linearized vector.

[0046] 3. Homologous recombination The obtained SbTCP2 target gene fragment and the linearized enzyme-digested vector are used for homologous recombination to construct a recombinant vector by using the Novozyme Seamless Cloning Kit, and the detailed steps are recorded in the Novozyme ClonExpress Ultra One Step Cloning Kit C115 instruction book.

[0047] (1) The use amount of linearized vector and inserted target fragment Optimal amount of cloning vector used = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol); Optimal amount of insert used = [0.04 x number of base pairs of insert] ng (0.06 pmol); (2) Recombination reaction To ensure the accuracy of the sample, the linearized vector and the insert can be appropriately diluted before the preparation of the recombination reaction system, and the sample volume of each component should not be less than 1 μL.

[0048] Prepare the following reaction system on ice: Gently mix with a pipette.

[0049] Single fragment recombination reaction, 37°C, 30 min. Obtain the product after homologous recombination, i.e. the recombination product.

[0050] 4, Transformation of recombination product (1) Thaw the E. coli DH5α chemically competent cells from Invitrogen on ice.

[0051] (2) Add 10 μL of the recombination product to 100 μL of competent cells, mix gently by flicking the tube wall, and incubate on ice for 30 min.

[0052] (3) After 42°C water bath heat shock for 45 sec, immediately cool on ice for 3 min.

[0053] (4) Add 900 μL of LB liquid medium without antibiotics, and shake at 37°C, 200 rpm for 1 h.

[0054] (5) Preheat the LB solid medium containing a working concentration of 50 mg / L Kan antibiotic in a 37°C incubator.

[0055] (5) Centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant. Resuspend the bacterial cells with the remaining medium, and gently spread them on the LB plate containing Kan antibiotic using a sterile spreader.

[0056] (6) Incubate in a 37°C incubator for 16 h.

[0057] 5, Identification of recombination product (1) Pick a single colony of E. coli DH5α from the Kan antibiotic LB plate after overnight incubation, and transfer it to 1 mL of LB medium containing a working concentration of 50 mg / L Kan antibiotic. Shake at 37°C, 200 rpm until turbidity.

[0058] (2) Take 10 μL of the recombination product and add it to 100 μL of competent cells, mix gently, and then sequentially place on ice for 5 min, in liquid nitrogen for 5 min, in a 28°C water bath for 5 min, and on ice for 5 min. SbTCP2 - pOX-F and NOS-R2 as shown in SEQ ID NO. 11 as primers, the reaction system is shown in Table 6, and the reaction procedure is shown in Table 7, to perform PCR amplification, and the collected PCR amplification product is electrophoresed under the condition of 130v for 25 min.

[0059] (3) Select the bacterial liquid with the target band, and send the bacterial strain with the target band for sequencing. Extract the plasmid from the bacterial liquid with correct sequencing results to obtain a positive plasmid.

[0060] SEQ ID NO. 9: ATGTTTCCTTTCTGTGATTC; SEQ ID NO. 11: ATTGCGGGACTCTAATCATA.

[0061] 6. Agrobacterium transformation (1) Place the chemically competent E. coli EHA105 cells from Unique Company on ice to thaw.

[0062] (2) Take 10 μL of the recombination product and add it to 100 μL of competent cells, mix gently, and then sequentially place on ice for 5 min, in liquid nitrogen for 5 min, in a 28°C water bath for 5 min, and on ice for 5 min.

[0063] (3) After 42°C water bath heat shock for 45 sec, immediately place on ice to cool for 3 min.

[0064] (4) Add 700 μL of LB liquid medium without antibiotics, and shake the bacteria at 28°C and 200 rpm for 3 h.

[0065] (5) Centrifuge at 6000 rpm for 1 min, collect the bacterial bodies, take 100 μL of the supernatant, blow and mix the bacterial bodies, and then coat on an LB plate containing working concentrations of 50 mg / L Kan and 25 mg / L Rif antibiotics, and place in a 28°C incubator for culture for 3 d.

[0066] 7. Agrobacterium identification (1) Pick the Agrobacterium EHA105 single colony on the LB plate containing Kan and Rif antibiotics and cultured overnight, and place in 1 mL of LB medium with working concentrations of 50 mg / L Kan and 25 mg / L Rif antibiotics, and shake the bacteria at 28°C and 200 rpm until turbidity.

[0067] (2) Take the agrobacterium with the target gene after shaking as the template, and the nucleotide fragments shown in SEQ ID NO. 9 and SEQ ID NO. 11 as the primers, and the reaction system as shown in Table 8 and the reaction procedure as shown in Table 9, to perform PCR amplification, and the collected product is electrophoresed under the condition of 130v for 25 min.

[0068] (3) Select the bacterial liquid with the target band, and send the bacterial strain with the target band for sequencing.

[0069] (4) Preserve the bacterial liquid with correct sequencing: mix 400 μL of 50% glycerol and the bacterial liquid 1:1 in a cryogenic tube, and store at -80°C for standby.

[0070] 8, rice SbTCP2 Obtaining of overexpression strain (1) Induction of rice callus 1) Callus induction: select mature and full rice seeds, disinfect with 75% alcohol for 2 min, rinse twice with sterile water, soak in 0.15% mercury for 16 min, shake several times during the period, pour out the mercury, and rinse five times with sterile water. The disinfected seeds are inoculated on the callus induction medium, and cultured at 32°C under light for 8 d.

[0071] 2) Suspension, infection and co-culture of agrobacterium: before infection, scrape the activated agrobacterium into the suspension medium, shake culture at 28°C and 180 rpm for 3.5 h, adjust the concentration of the bacterial liquid to OD600=0.1 with the suspension medium. Put the 8d-induced callus into the agrobacterium suspension, and infect for 1.5 min. Pour out the bacterial liquid, and absorb the callus surface bacterial liquid with sterile filter paper. Cover the callus surface with sterile filter paper, and dry in the clean bench for 30 min. After drying, transfer the callus to the co-culture medium covered with a layer of sterile filter paper, first culture overnight at 20°C, and then continue to culture in the dark at 25°C for 2 d.

[0072] 3) Clear bacteria: after co-culture, transfer the callus to a sterile container with tweezers, and repeatedly wash the callus with sterile water for 8 times. The first 3 times can be quickly washed, and the last 4 times are soaked for 4 min each time. Finally, soak the callus in sterile water containing 250 mg / L Cef for 30 min, pour out the Cef solution, and try to absorb the water on the surface of the callus with sterile filter paper. Cover the callus surface with a layer of sterile filter paper, and dry in the clean bench for 1 h.

[0073] 4) Screening: place the callus after clearing bacteria on the screening medium, and culture at 25-28°C under light for 14-21 d.

[0074] 5) Differentiation: after 14 d of screening, transfer the resistant callus to the differentiation medium, and culture at 28°C.

[0075] 6) rooting: when the resistant callus forms 3cm high regenerated seedlings on the differentiation medium, it is transferred into the rooting medium for culture until the complete plant is formed.

[0076] (2) CTAB extraction of plant DNA positive seedling identification 1) RNAse is mixed with CTAB at 1:2000, and is prepared on site, and is preheated at 65℃.

[0077] 2) 0.05g of rice leaves is taken into a 2mL centrifuge tube, is frozen with liquid nitrogen, is ground into homogenate, and 700μL of CTAB is added.

[0078] 3) 65℃ constant temperature preservation for 30min, and is inverted for 3 times in the middle.

[0079] 4) 700μL of DNA extraction solution is added, and is fully shaken to completely mix.

[0080] 5) 13000rpm centrifugation for 15min at room temperature.

[0081] 6) 650μL of supernatant is taken into a new centrifuge tube, 2 / 3 volume of isopropanol is added, and is placed at -20℃ for 15min.

[0082] 7) 4℃ 13000rpm centrifugation for 15min, the supernatant is removed, and 75% ethanol is added for washing for 3 times.

[0083] 8) 4℃ 13000rpm centrifugation for 5min to remove the upper isopropanol, is dried in a clean bench, and after drying, an appropriate amount of sterile water is added.

[0084] 9) DNA is obtained and is stored at -20℃ for standby.

[0085] The rice obtained by the above method SbTCP2 The overexpression lines are denoted as OE1 and OE2.

[0086] Example 3: SbTCP2 Transgenic plant phenotype analysis.

[0087] The obtained SbTCP2 -OE genetic transformation lines are identified, and overexpression lines are selected Figure 3 -OE, and the two overexpression lines OE1 and OE2 are used for phenotype observation in the container, and the agronomic traits are counted.

[0088] The results are shown in SbTCP2 The average plant height of the wild type is 55.58cm, SbTCP2 The average plant height of OE1 is 37.29cm, SbTCP2 The average plant height of OE2 is 36.67cm, and the average tiller number of the wild type is 4.25,SbTCP2 The average tiller number of OE1 is 2.11, SbTCP2 The average tiller number of OE2 is 1.37. The above results show that overexpression of the gene can reduce the plant height of rice and decrease the tiller occurrence of rice. SbTCP2 The gene can reduce the plant height of rice and decrease the tiller occurrence of rice.

[0089] Therefore, the present application reduces the tiller number of rice, reduces the plant height of rice and constructs a new rice plant type by overexpressing the gene in rice plants. SbTCP2 The gene is used for regulating the tiller number and plant height of rice plants, which is beneficial to the construction of rice plant type and lays a foundation for creating new resources of rice germplasm. ​ The gene is used for regulating the tiller number and plant height of rice plants, which is beneficial to the construction of rice plant type and lays a foundation for creating new resources of rice germplasm.

[0090] Although the preferred embodiments of the present application have been described, those skilled in the art who know the basic inventive concept can make additional changes and modifications to the embodiments.

[0091] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, The SbTCP2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

2. As described in claim 1 SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, Through overexpression SbTCP2 Genes reduce the number of tillers in rice.

3. As described in claim 2 SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, By building SbTCP2 Transformation of rice with gene overexpression vector SbTCP2 Gene.

4. The method according to claim 3 SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, The construction SbTCP2 The steps for using a gene overexpression vector are: to... SbTCP2 The gene fragment was homologously recombinated with a pOX vector linearized by restriction enzyme digestion to obtain a recombinant vector, which was then transformed into E. coli. After culturing, plasmids were extracted and transformed into Agrobacterium to obtain the desired result. SbTCP2 Engineered strains that overexpress genes.

5. The method according to claim 4 SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, SbTCP2 The gene fragment cloning steps were as follows: using cDNA from the tillers of Sorghum Hercules as a template, and using the nucleotide fragments shown in SEQ ID NO. 9 and SEQ ID NO. 10 as primers, PCR amplification was performed to obtain... SbTCP2 Gene fragments.

6. The method according to claim 3 SbTCP2 The application of genes in regulating the number of rice tillers is characterized by, Rice overexpression vector transformation was achieved using Agrobacterium infection.

7. SbTCP2 The application of genes in rice plant architecture is characterized by, use SbTCP2 Gene overexpression vectors were used to construct rice plant types with reduced tiller numbers.

8. SbTCP2 The application of genes in the creation of new germplasm resources is characterized by, use SbTCP2 Gene overexpression vectors resulted in reduced plant height and fewer tillers. SbTCP2 Transgenic rice with overexpression.

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