Sweet potato IbSOC1 gene, protein, recombinant expression vector, recombinant bacterium and application

Abstract

The invention provides a sweet potato IbSOC1 gene, a protein, a recombinant expression vector, a recombinant bacterium and application, and belongs to the technical field of genetic engineering, and the nucleotide sequence of the sweet potato IbSOC1 gene is as shown in SEQ ID No.1. According to the invention, the IbSOC1 gene is transferred into a sweet potato variety Taizhong No.6 with poor natural flowering capability and late flowering, so that an IbSOC1 gene overexpressed sweet potato plant with obviously advanced flowering time is obtained. A research shows that compared with a wild sweet potato plant, the sweet potato plant OE-3 with the overexpressed IbSOC1 gene blooms 11.7 days earlier.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a sweet potato IbSOC1 gene, protein, recombinant expression vector, recombinant bacteria and applications. Background Art

[0002] Sweet potato is an important crop for food, feed, industrial raw material and biomass energy. Sexual hybridization is the main way to breed new sweet potato varieties. However, most sweet potato varieties cannot bloom naturally in areas north of 23°N. Although some sweet potato varieties can bloom naturally in areas south of 23°N, their natural flowering ability is poor and they bloom late, which is not conducive to sweet potato hybridization and seriously restricts the efficiency of sweet potato hybrid breeding.

[0003] SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) is an important integrator in the flowering pathway of plants. Currently, there is no research on SOC1 in sweet potato. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a sweet potato IbSOC1 gene, protein, recombinant expression vector, recombinant bacteria and applications, wherein the sweet potato IbSOC1 gene can promote sweet potato flowering.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a sweet potato IbSOC1 gene, and the nucleotide sequence of the sweet potato IbSOC1 gene is shown as SEQ ID No.1.

[0007] The present invention also provides a protein encoded by the sweet potato IbSOC1 gene, and the amino acid sequence of the protein is shown as SEQ ID No.2.

[0008] The present invention also provides the use of overexpressing the sweet potato IbSOC1 gene in regulating the flowering of sweet potatoes.

[0009] The present invention also provides the use of overexpressing the sweet potato IbSOC1 gene in cultivating early flowering sweet potato germplasm.

[0010] The present invention also provides a primer pair for amplifying the sweet potato IbSOC1 gene, and the nucleotide sequences of the primer pair are shown in SEQ ID No.3 and SEQ ID No.4.

[0011] The present invention also provides a recombinant expression vector for overexpressing the sweet potato IbSOC1 gene, comprising the sweet potato IbSOC1 gene and an overexpression vector.

[0012] Preferably, the overexpression vector is pCAMBIA1301S.

[0013] The present invention also provides a recombinant bacterium for over-expressing the sweet potato IbSOC1 gene, which is obtained by transferring the recombinant expression vector into Agrobacterium.

[0014] Preferably, the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0015] The present invention also provides the application of the recombinant expression vector and the recombinant bacteria in regulating the flowering of sweet potatoes.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a sweet potato IbSOC1 gene, the nucleotide sequence of which is shown in SEQ ID No. 1, and the gene can be used to regulate the flowering of sweet potatoes and promote the flowering of sweet potatoes. The present invention clones the sweet potato IbSOC1 gene and constructs a recombinant expression vector, transfers the IbSOC1 gene into the sweet potato variety Taizhong No. 6, which has poor natural flowering ability and blooms late, and the obtained IbSOC1 gene overexpression plant has a significantly earlier flowering time than the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the amplified band of sweet potato IbSOC1 gene; where M is Marker;

[0019] Figure 2 The PCR test results of sweet potato overexpressing IbSOC1 gene; M is Marker; CK1 is the positive control pCAMBIA1301S-IbSOC1 recombinant vector; CK2 is the negative control ddH2O; OE-1, OE-3, OE-7 are sweet potato plants overexpressing IbSOC1 gene; WT is the wild-type sweet potato variety Taizhong No. 6 plant;

[0020] Figure 3 The qRT-PCR results of IbSOC1 gene in sweet potato plants overexpressing IbSOC1 gene and wild-type sweet potato plants; OE-1, OE-3, and OE-7 are sweet potato plants overexpressing IbSOC1 gene, and WT is wild-type sweet potato Taizhong No. 6 plant; the data shown in the figure are mean ± standard deviation, the number of replicates is 3, and different lowercase letters represent significant differences at the P < 0.05 level;

[0021] Figure 4 The flowering phenotypes of the wild-type and IbSOC1-overexpressing sweet potato plants OE-3. DETAILED DESCRIPTION

[0022] The invention provides a sweet potato IbSOC1 gene, and the nucleotide sequence of the sweet potato IbSOC1 gene is shown as SEQ ID No.1.

[0023] In the present invention, the nucleotide sequence of the sweet potato IbSOC1 gene is shown in Table 1.

[0024] The present invention also provides a protein encoded by the sweet potato IbSOC1 gene, and the amino acid sequence of the protein is shown as SEQ ID No.2.

[0025] In the present invention, the amino acid sequence of the protein is shown in Table 1.

[0026] The present invention also provides the use of overexpressing the sweet potato IbSOC1 gene in regulating the flowering of sweet potatoes.

[0027] In the present invention, the regulating sweet potato flowering preferably includes promoting early flowering of sweet potato.

[0028] The present invention also provides the use of overexpressing the sweet potato IbSOC1 gene in cultivating early flowering sweet potato germplasm.

[0029] The present invention also provides a primer pair for amplifying the sweet potato IbSOC1 gene, and the nucleotide sequences of the primer pair are shown in SEQ ID No.3 and SEQ ID No.4.

[0030] In the present invention, the nucleotide sequences of the primer pairs are shown in Table 1.

[0031] The present invention also provides a recombinant expression vector for overexpressing the sweet potato IbSOC1 gene, comprising the sweet potato IbSOC1 gene and an overexpression vector.

[0032] In the present invention, the overexpression vector is preferably pCAMBIA1301S.

[0033] The present invention also provides a recombinant bacterium for over-expressing the sweet potato IbSOC1 gene, which is obtained by transferring the recombinant expression vector into Agrobacterium.

[0034] In the present invention, the Agrobacterium is preferably Agrobacterium tumefaciens GV3101.

[0035] The present invention also provides the application of the recombinant expression vector and the recombinant bacteria in regulating the flowering of sweet potatoes.

[0036] In the present invention, the regulating sweet potato flowering preferably includes promoting early flowering of sweet potato.

[0037] Table 1 Sequence information

[0038]

[0039]

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1 Extraction of total RNA from sweet potato

[0042] Young leaves of sweet potato variety Eshu 17 (provided by the Institute of Food Crops, Hubei Academy of Agricultural Sciences) were quickly ground into powder in liquid nitrogen, and total RNA was extracted using a total RNA extraction kit (catalog number: RC411-01, purchased from Nanjing Novozyme Biotechnology Co., Ltd.). The quality of RNA was tested by 1% agarose gel electrophoresis, and the results showed that the quality of RNA was good and could be used for subsequent experiments.

[0043] Example 2 Cloning of sweet potato IbSOC1 gene

[0044] Primer 5.0 software was used to design specific primers such as the primer pair shown in SEQ ID No. 3 (5'-ATGGTGAGGGGGAAGACGCAGA-3') and SEQ ID No. 4 (5'-TTATGCCAATGGAGGACAACAG-3').

[0045] use The total RNA extracted in Example 1 was reverse transcribed into cDNA using the All-in-One First-Strand cDNA Synthesis SuperMix for qPCR test kit (Catalog No.: AT341-01, purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

[0046] Using cDNA as a template, La Taq (catalog number: RR52A, purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.) was used for PCR amplification. The primers used in the PCR amplification are shown in SEQ ID No. 3 and SEQ ID No. 4. The amplified product was subjected to 1% agarose gel electrophoresis to obtain a 663 bp target amplified band (as shown in Figure 1As shown). The target band was recovered using a gel recovery kit (catalog number: D2500, purchased from Omega Bio-Tek, USA), and connected to the pMD19-T vector (purchased from Bio-Tech (Beijing) Co., Ltd.) at 16°C overnight to obtain a connection system. 10 μL of the obtained connection system was transferred into Escherichia coli DH-5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a monoclonal plaque was picked and placed in LB liquid culture medium (containing 100 μg / mL ampicillin), cultured at 37°C and 200 rpm for 16 hours, and the bacterial solution was sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing.

[0047] After sequencing, the PCR target product has a nucleotide sequence as shown in SEQ ID No.1, and the gene shown in the sequence is named IbSOC1 gene. The coding region of the gene is nucleotides 1-663 from the 5' end. The protein encoded by the gene is named IbSOC1 protein or protein IbSOC1, and its amino acid sequence is shown in SEQ ID No.2, which consists of 220 amino acid residues.

[0048] Example 3 Construction of overexpression vector

[0049] Primers were designed based on the CDS sequence of the sweet potato IbSOC1 gene, and KpnI and XbaI restriction sites were introduced. The nucleotide sequence of the forward primer was:

[0050] The bold underlined part is the KpnI restriction site sequence), and the nucleotide sequence of the reverse primer is:

[0051] (SEQ ID No. 6, the bold underlined part is the XbaI restriction site sequence).

[0052] PCR amplification was performed using the nucleotide sequence shown in SEQ ID No. 1 as a template. The amplified product and the overexpression vector pCAMBIA1301S (purchased from Jiangsu Sanshu Biotechnology Co., Ltd.) were digested with KpnI and XbaI, and then the target gene and the overexpression vector pCAMBIA1301S were connected and sequenced for verification.

[0053] The sequencing results showed that the sequence shown in SEQ ID No. 1 from position 1 to position 663 at the 5' end was inserted between the KpnI and XbaI restriction recognition sites of the vector pCAMBIA1301S, and the other nucleotide sequences of the vector pCAMBIA1301S remained unchanged, indicating that the recombinant vector was constructed correctly. The recombinant vector was named pCAMBIA1301S-IbSOC1.

[0054] Example 4 Obtaining sweet potato plants overexpressing the IbSOC1 gene

[0055] The recombinant vector pCAMBIA1301S-IbSOC1 constructed in Example 3 was transformed into Agrobacterium tumefaciens GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium.

[0056] The shoot apical meristem of sweet potato Taizhong No. 6 (provided by the Tuber Biotechnology Research Group of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) was peeled and placed in MS solid medium (containing 2.0 mg / L2,4-D) and cultured in the dark at 27±1℃ to induce embryonic callus growth; the clustered callus was broken by suction and blowing with a pipette, and the broken callus was transferred to MS liquid medium (containing 2.0 mg / L 2,4-D) and suspended cultured for 13 hours at 27±1℃, 100rpm, and 3000lx light per day for 10 weeks. Grind the embryonic suspension cells 3 days before genetic transformation to obtain embryonic cell clusters with wounds.

[0057] The recombinant Agrobacterium liquid obtained above was used to infect the pre-cultured embryonic cell mass with wounds. After sufficient contact, the bacterial liquid was discarded, and the infected embryonic cell mass was placed in MS solid medium (containing 30 mg / L acetosyringone + 2.0 mg / L 2,4-D) and cultured in the dark for 3 days. The co-cultured embryonic cell mass was transferred to MS liquid medium (containing 400 mg / L cephalosporin and 2.0 mg / L 2,4-D), washed twice, and then the cell mass was transferred to MS liquid medium (containing 2.0 mg / L 2,4-D) again, and delayed cultured for 1 week. The embryonic cell mass after delayed culture was evenly spread on MS solid medium (containing 5 mg / L hygromycin + 150 mg / L cephalosporin + 2.0 mg / L 2,4-D), and cultured in the dark at 27±1°C for 8 weeks to obtain resistant callus. The resistant callus was transferred to MS solid medium (containing 150 mg / L cephalosporin + 1 mg / L abscisic acid), cultured at 27±1°C and 3000 lx light for 13 h per day for 3 weeks to induce somatic embryos. The green and mature somatic embryos were transferred to MS solid medium, cultured at 27±1°C and 3000 lx light for 13 h per day for 6 weeks, and three sweet potato plants overexpressing the IbSOC1 gene were regenerated and named OE-1, OE-3, and OE-7.

[0058] Experimental Example 1 Detection of sweet potato plants overexpressing the IbSOC1 gene

[0059] (1) PCR detection

[0060] The CTAB method was used to extract DNA from the sweet potato plants overexpressing the IbSOC1 gene and the wild-type sweet potato plant (Ganshu Taizhong No. 6), and the PCR detection method was used to identify the three sweet potato plants overexpressing the IbSOC1 gene obtained in Example 4.

[0061] The primer pair used in the PCR detection is: 5'-CTTAGCCAGACGAGCGGGTTC-3' (SEQ ID No. 7) and 5'-CGGTTTCCACTATCGGCGAGTA-3' (SEQ ID No. 8).

[0062] At the same time, 1% agarose gel electrophoresis was performed using the recombinant vector pCAMBIA1301S-IbSOC1 as a positive control and water and wild-type sweet potato plants as negative controls. Figure 2 shown.

[0063] The results showed that the target band of 663 bp was amplified from the sweet potato plants OE-1, OE-3, OE-7 and the positive control that overexpressed the IbSOC1 gene, indicating that these three plants were IbSOC1 transgenic positive plants.

[0064] (2) qRT-PCR detection

[0065] The plants OE-1, OE-3, and OE-7 identified in step (1) were propagated and RNA from wild-type and transgenic lines was extracted, and the expression level of the IbSOC1 gene was detected by qRT-PCR. The primer pair used in the qRT-PCR detection was: 5'-TGTGATGCTGAGGTTGCTCT-3' (SEQ ID No. 9) and 5'-CGCACGCTCCTAAATCTTCC-3' (SEQ ID No. 10)

[0066] The expression of IbSOC1 gene was detected using TB Green Premix Ex Taq II kit (catalog number: RR820Q, purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.), with a repeat number of 3. The test results are shown in Figure 3 shown.

[0067] The results showed that the expression level of IbSOC1 gene in sweet potato plants OE-1, OE-3 and OE-7 overexpressing IbSOC1 gene was significantly higher than that in wild type (WT).

[0068] Experimental Example 2: Flowering phenotype analysis of positive plants

[0069] The sweet potato plant OE-3 with the highest expression of the IbSOC1 gene obtained in Example 4 in Hainan and the wild-type sweet potato (Sweet Potato Taizhong No. 6) were observed for flowering time of different plants. The results are as follows Figure 4 shown.

[0070] The results showed that compared with wild-type sweet potato plants, the sweet potato plant OE-3 overexpressing the IbSOC1 gene flowered 11.7 days earlier.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sweet potato IbSOC1 gene, characterized in that: The nucleotide sequence of the sweet potato IbSOC1 gene is shown in SEQ ID No.

1.

2. The protein encoded by the sweet potato IbSOC1 gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. Use of the sweet potato IbSOC1 gene overexpressed in claim 1 in regulating sweet potato flowering.

4. Use of the sweet potato IbSOC1 gene overexpressed in claim 1 in cultivating early flowering sweet potato germplasm.

5. A primer pair for amplifying the sweet potato IbSOC1 gene according to claim 1, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID No.3 and SEQ ID No.

4.

6. A recombinant expression vector for overexpressing the sweet potato IbSOC1 gene according to claim 1, characterized in that: It comprises the sweet potato IbSOC1 gene and overexpression vector described in claim 1.

7. The recombinant expression vector according to claim 6, characterized in that: The overexpression vector is pCAMBIA1301S.

8. A recombinant bacterium overexpressing the sweet potato IbSOC1 gene according to claim 1, characterized in that: The recombinant expression vector according to claim 6 or 7 is transformed into Agrobacterium to obtain the result.

9. The recombinant bacterium according to claim 8, characterized in that The Agrobacterium is Agrobacterium tumefaciens GV3101.

10. Use of the recombinant expression vector according to claim 6 or 7, or the recombinant bacteria according to claim 8 or 9 in regulating the flowering of sweet potatoes.

Images