Sweet potato ibexpa11 gene and application thereof in regulating stem growth of sweet potato

Abstract

The application provides a sweet potato IbEXPA11 gene and application thereof in regulating stem growth of sweet potato, and belongs to the technical field of sweet potato molecular breeding. IbEXPA11 The 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. IbEXPA11 The sweet potato IbEXPA11 gene can be applied to regulate stem growth of sweet potato, improve the expression level of the gene, promote stem growth of sweet potato, reduce the expression level of the gene, and inhibit stem growth of sweet potato. IbEXPA11 The application also establishes a feasible method for cultivating a new short-stem sweet potato variety by using RNA interference to express the gene. IbEXPA11 IbEXPA11 The application provides important gene resources for effectively regulating stem length of sweet potato, preventing overgrowth, improving mechanical harvesting efficiency and yield of sweet potato.

Description

Technical Field

[0001] This invention belongs to the field of sweet potato molecular breeding technology, and particularly relates to a sweet potato IbEXPA11 Genes and their application in regulating sweet potato stem and vine growth. Background Technology

[0002] sweet potato( Ipomoea batatas Sweet potato (L.) Lam. is an important multi-purpose (food, cash crop, and forage) specialty economic crop, possessing advantages such as stable and high yields, wide adaptability, strong resistance to adverse conditions, and high nutritional value. It has irreplaceable industrial value in ensuring food security, optimizing agricultural planting structure, boosting rural income, and in starch processing and functional food processing. Its planting scale ranks first in the world. In recent years, with the continuous advancement of agricultural modernization, large-scale, intensive, and mechanized sweet potato planting models have gradually become widespread, placing higher standards on plant uniformity, field growth balance, and harvest suitability.

[0003] Sweet potatoes are root crops with a creeping growth habit, a well-developed root system, and a strong ability to absorb water and fertilizer. In actual field production, factors such as hot and rainy climates and excessive application of nitrogen fertilizer can easily lead to excessive physiological growth of the above-ground stems and vines, which is a common and core problem in the current large-scale cultivation of sweet potatoes.

[0004] Currently, the methods for controlling excessive vine growth in sweet potato production are relatively traditional, relying mainly on manual vine turning or spraying chemical growth regulators such as paclobutrazol. Manual growth control is labor-intensive, inefficient, and costly. Furthermore, vine turning can easily damage the plant's stems, leaves, and roots, disrupting the normal transport of photosynthetic products and potentially leading to yield reduction. Chemical growth regulators have problems such as difficulty in controlling dosage, short control period, premature plant aging, and soil residue. Their control accuracy and safety are insufficient, making them unsuitable for the needs of large-scale, standardized, and green modern sweet potato cultivation.

[0005] In summary, how to accurately regulate the growth of sweet potato vines, inhibit ineffective excessive growth, and thus adapt to the needs of mechanized harvesting operations, and break through the technical bottlenecks of high and stable yield and simplified cultivation of sweet potatoes, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a sweet potato IbEXPA11 Genes and their application in regulating sweet potato vine growth; This invention confirms the overexpression of... IbEXPA11 The gene significantly promotes sweet potato vine growth, while reducing its expression using RNA interference significantly inhibits vine growth. This finding provides a new genetic resource for improving the vine growth trait of sweet potato.

[0007] This invention provides a sweet potato IbEXPA11Genes, the ones mentioned IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] This invention provides the aforementioned sweet potato IbEXPA11 A gene-encoded protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] This invention provides the aforementioned sweet potato IbEXPA11 The application of genes in regulating sweet potato vine growth, improving the... IbEXPA11 The gene expression level promotes sweet potato vine growth and reduces the aforementioned IbEXPA11 The gene expression level inhibits the growth of sweet potato vines.

[0010] This invention provides biomaterials for controlling the growth of sweet potato vines, including one or more of the following biomaterials: 1) Interference IbEXPA11 Homologous arm primers for gene expression; 2) Interference IbEXPA11 Recombinant vectors for gene expression; 3) Interference IbEXPA11 Recombinant Agrobacterium gene expression.

[0011] Preferably, the sequences of the homologous arm primers are shown in SEQ ID NO.7~8, SEQ ID NO.9~10 and SEQ ID NO.11~12.

[0012] Preferably, the recombinant vector comprises an initial vector and a DNA fragment amplified by the homologous arm primer.

[0013] This invention provides a method for regulating the growth of sweet potato vines, comprising: (a) Overexpression in sweet potato IbEXPA11 Genes that promote the growth of sweet potato vines; or (b) Reduced in sweet potatoes IbEXPA11 Gene expression is used to suppress the growth of sweet potato vines; The IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Preferred, overexpression IbEXPA11 Genes include the said IbEXPA11 Genes were cloned into the initial vector to construct a recombinant vector, which was then transformed into Agrobacterium tumefaciens and used to infect sweet potatoes. reduce IbEXPA11 Gene expression includes interfering with IbEXPA11 The recombinant vector for gene expression was transformed into Agrobacterium and then used to infect sweet potatoes.

[0015] Preferably, the receptor for infecting sweet potatoes is sweet potato embryonic callus.

[0016] This invention also provides sweet potato IbEXPA11 The application of genes in short-vine sweet potato assisted breeding reduces IbEXPA11 Gene expression levels, and the development of short-vine sweet potato varieties.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a sweet potato IbEXPA11 The gene and its application in regulating sweet potato vine growth, the IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.1; this invention has discovered... IbEXPA11 The gene is a key gene regulating the growth of sweet potato vines, thus providing a way to regulate... IbEXPA11 Effective techniques for directionally altering sweet potato vine growth by increasing gene expression levels (overexpression or repression): IbEXPA11 The gene expression level promotes sweet potato vine growth and reduces the aforementioned IbEXPA11 The gene expression level inhibits the growth of sweet potato vines.

[0018] Furthermore, this invention also establishes a system for expressing RNA interference. IbEXPA11 This invention provides an important genetic resource for effectively regulating sweet potato vine length, preventing excessive growth, and thus improving the efficiency and yield of mechanized sweet potato harvesting. Attached Figure Description

[0019] Figure 1 sweet potato IbEXPA11 Gene clone agarose gel electrophoresis image.

[0020] Figure 2 for IbEXPA11 Schematic diagram of the overexpression vector structure.

[0021] Figure 3 for IbEXPA11 Schematic diagram of the interference expression vector structure.

[0022] Figure 4 for IbEXPA11 PCR detection diagram of overexpressing transgenic sweet potato.

[0023] Figure 5 for IbEXPA11 PCR detection diagram of transgenic sweet potato with interference expression.

[0024] Figure 6 for IbEXPA11 Expression level analysis in transgenic sweet potatoes, where A represents overexpression. IbEXPA11 Analysis of expression levels in sweet potato plants, B represents interference expression. IbEXPA11Analysis of expression levels in sweet potato plants.

[0025] Figure 7 Phenotypic and length measurements of wild-type and transgenic sweet potato vines, where A represents overexpression and interference expression. IbEXPA11 Field phenotype of sweet potato plants, B indicates overexpression IbEXPA11 Sweet potato vine length statistics, C represents interference expression. IbEXPA11 Statistics on the vine length of sweet potato plants. Detailed Implementation

[0026] This invention provides a sweet potato IbEXPA11 Genes, the ones mentioned IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.1, as follows: ATGGCTCATTATGGTGGTCTGGGACTAGCATTCATGCTTGGATTACTTCATTTCCTTTGTTTATTCATCAATGTCAATGCTTTCACAGCTTCTGGGTGGCAAAGGGCTCATGCAACCTTCTATGGAGGCAGTGATGCTTCTGGAACTATGGGAGGGGCATGTGGGTATGGAAACCTGTATACCACAAGGTATGGGACTAGGACTGCGGCACTGAGCACAGCATTGTTCAACGATGGTGCCTCTTGTGGTCAATGCTACAAGATCATATGTGACTTCAAAGCAGAGCCTCGATGGTGCAGGAAGGGAGTTTCGGTCACCATCACGGCCACTAATTTCTGTCCACCTAATTACGCTCTTCCTAGTGATAATGGAGGTTGGTGCAATCCTCCTCGTCCCCACTTCGACATGGCCCAACCCGCTTGGGAAAAGATCGGAATTTACAGTGGTGGTATCGTCCCTGTTATCTACCAAAGGGTACCATGCAAGAAACGTGGTGGGGTGAGATTTACGATCAACGGTCATGATTACTTTGAGCTAGTGCTAGTCACAAATGTGGGTGGAGCTGGATCCATTAGATCAGTGCAAATCAAGGGTTCAAAAACTAACTGGATGACAATGTCAAGAAATTGGGGAGTCAATTGGCAATCCAACGCATACCTAACGGGTCAATCAATCTCTTTCCGGGTCACATCCAGTGATGGCGTCACCATGACTTTCTTGAACGTGGCCCCATCCAATTGGAGATTTGGGCAGACTTTCGCCAGCCCACTCCAGTTCTCATAA。

[0027] The present invention also provides the sweet potato as described IbEXPA11 protein encoded by the gene, wherein the amino acid sequence of the protein is as shown in SEQ ID NO. 2, specifically as follows: MAHYGGLGLAFMLGLLHFLCLFINVNAFTASGWQRAHATFYGGSDASGTMGGACGYGNLYTTRYGTRTAALSTALFNDGASCGQCYKIICDFKAEPRWCRKGVSVTITATNFCPPNYALPSDNGGWCNPP RPHFDMAQPAWEKIGIYSGGIVPVIYQRVPCKKRGGVRFTINGHDYFELVLVTNVGGAGSIRSVQIKGSKTNWMTMSRNWGVNWQSNAYLTGQSISFRVTSSDGVTMTFLNVAPSNWRFGQTFASPLQFS

[0028] This invention provides the aforementioned sweet potato IbEXPA11 The application of genes in regulating sweet potato vine growth, improving the... IbEXPA11 The gene expression level promotes sweet potato vine growth and reduces the aforementioned IbEXPA11 The gene expression level inhibits the growth of sweet potato vines.

[0029] This invention provides biomaterials for controlling the growth of sweet potato vines, including one or more of the following biomaterials: 1) Interference IbEXPA11 Homologous arm primers for gene expression; 2) Interference IbEXPA11 Recombinant vectors for gene expression; 3) Interference IbEXPA11 Recombinant Agrobacterium gene expression.

[0030] In this invention, the sequences of the homologous arm primers are shown in SEQ ID NO.7~8, SEQ ID NO.9~10 and SEQ ID NO.11~12; specifically as follows: IbEXPA11 -RNAi-F1 (SEQ ID NO.7): 5'-TTACAATTACAATTAGGATCCATGGCTCATTATGGTGGTCTGG-3'; IbEXPA11 -RNAi-R1 (SEQ ID NO.8): 5'-AAATTCTTACCCTGCACCATCGAGGCT-3'; IbEXPA11 -RNAi-F2 (SEQ ID NO.9): 5'-ATGGTGCAGGGTAAGAATTTCTTATGTTACATTATTACATTCAACGTTTTATCTTAAT-3'; IbEXPA11 -RNAi-R2 (SEQ ID NO.10): 5'-ATGGTGCAGGCTGCAAATTGACCAAAAAAGATGTGAAGAAAAC-3'; IbEXPA11 -RNAi-F3 (SEQ ID NO.11): 5'-CAATTTGCAGCCTGCACCATCGAGGCT-3'; IbEXPA11 -RNAi-R3 (SEQ ID NO.12): 5'-AGATCTGGTCGACGGCGCTGGTACCATGGCTCATTATGGTGGTCTGG-3'.

[0031] This invention utilizes selectable restriction enzyme sites on the interference expression vector pFGC5941-DsRed ( Bam HI and Kpn I), Design the above IbEXPA11 Homologous arm primers for constructing interference expression vectors.

[0032] This invention is based on IbEXPA11 Using the gene coding region sequence and the pFGC5941-DsRed vector sequence as templates, PCR amplification was performed using the aforementioned homologous arm primers to obtain samples containing... IbEXPA11 The first 300 bp coding sequence and its complementary DNA fragment, as well as the non-coding DNA fragment inserted between them; as shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 respectively; specifically as follows: SEQ ID NO.19: TTACAATTACAATTAGGATCCATGGCTCATTATGGTGGTCTGGGACTAGCATTCATGCTTGGATTACTTCATTTCCTTTGTTTATTCATCAATGTCAATGCTTTCACAGCTTCTGGGTGGCAAAGGGCTCATGCAACCTTCTATGGAGGCAGTGATGCTTCTGGAACTATGGGAGGGGCATGTGGGTATGGAAACCTGTATACCACAAGGTATGGGACTAGGACTGCGGCACTGAGCACAGCATTGTTCAACGATGGTGCCTCTTGTGGTCAATGCTACAAGATCATATGTGACTTCAAAGCAGAGCCTCGATGGTGCAGGGTAAGAATTT; SEQ ID NO.20: SEQ ID NO.21: CAATTTGCAGCCTGCACCATCGAGGCTCTGCTTTGAAGTCACATATGATCTTGTAGCATTGACCACAAGAGGCACCATCGTTGAACAATGCTGTGCTCAGTGCCGCAGTCCTAGTCCCATACCTTGTGGTATACAGGTTTCCATACCCACATGCCCCTCCCATAGTTC CAGAAGCATCACTGCCTCCATAGAAGGTTGCATGAGCCCTTTGCCACCCAGAAGCTGTGAAAGCATTGACATTGATGAATAAACAAAGGAAATGAAGTAATCCAAGCATGAATGCTAGTCCCAGACCACCATAATGAGCCATGGTACCAGCGCCGTCGACCAGATCT.

[0033] Preferably, the recombinant vector comprises an initial vector and a DNA fragment amplified by the homologous arm primers. In this invention, the amplified DNA fragments shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21 are sequentially tandem and then introduced into the initial vector. In this invention, the initial vector is preferably the pFGC5941-DsRed plasmid; the cloning site of the DNA fragment is preferably... Bam HI and Kpn Between I enzyme digestion; after the DNA fragment is purified by gel recovery, the amplified DNA fragment is ligated with enzyme digestion. Bam HI and Kpn The empty vector pFGC5941-DsRed plasmid was digested with enzyme I and ligated. The recombinant plasmid was then transformed into E. coli DH5α competent cells and sequenced. The sequencing yielded the constructed interference. IbEXPA11 Recombinant vectors for gene expression.

[0034] In this invention, interference IbEXPA11 The recombinant Agrobacterium gene expression is generated by the interference constructed above. IbEXPA11 The gene expression recombinant vector is obtained by transforming Agrobacterium, preferably Agrobacterium EHA105. The present invention does not have any particular limitation on the transformation method, and conventional Agrobacterium transformation methods in the art can be used.

[0035] The present invention also provides a method for regulating the growth of sweet potato vines, comprising: (a) overexpressing in sweet potato IbEXPA11 (a) Genes that promote the growth of sweet potato vines; or (b) genes that reduce the growth of sweet potato vines. IbEXPA11Gene expression to suppress the growth of sweet potato vines; IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0036] In this invention, overexpression IbEXPA11 Genes include the said IbEXPA11 Genes were cloned into the initial vector to construct a recombinant vector, which was then transformed into Agrobacterium and used to infect sweet potatoes.

[0037] In this invention, the initial carrier is preferably pCAMBIA1301s-DsRed. IbEXPA11 The preferred site for gene cloning is the initial vector. Bam HI and Kpn Between I restriction sites, based on this design IbEXPA11 The homologous arm primers for the overexpression vector are as follows: IbEXPA11 -OE-F (SEQ ID NO.5): 5'-TTACAATTACAATTAGGATCCATGGCTCATTATGGTGGTCTGG -3'; IbEXPA11 -OE-R (SEQ ID NO.6): 5'-AGATCTGGTCGACGGCGCTGGTACCTTATGAGAACTGGAGTGGGCTGG-3'.

[0038] The present invention preferably uses sweet potato genomic DNA as a template and performs PCR amplification using the above-mentioned overexpression homologous arm primers to obtain a sample containing... IbEXPA11 The complete coding sequence DNA fragment was obtained, and the amplification product was purified and recovered; simultaneously, the pCAMBIA1301s-DsRed empty vector plasmid was double-digested with enzymes. Bam HI and Kpn (I. Thermo Scientific); The purified DNA fragment and the digested empty vector plasmid were ligated using ABclonal's 2x MultiFSeamless Assembly Mix seamless ligase. The recombinant plasmid was then transformed into *E. coli* DH5α competent cells and sequenced. Correct sequencing yielded accurate results. IbEXPA11 -OE overexpression recombinant vector.

[0039] In this invention, the recombinant vector obtained is transformed into Agrobacterium and then used to infect sweet potatoes. In this invention, Agrobacterium is preferably Agrobacterium EHA105. This invention does not specifically limit the transformation method; any conventional Agrobacterium transformation method in the art can be used.

[0040] In this invention, the recipient for infecting sweet potato is preferably sweet potato embryogenic callus. Preferably, the sweet potato embryogenic callus is obtained by sterilizing and peeling the shoot tip meristem from the sweet potato shoot tip as an explant, followed by induction culture. The induction culture is preferably carried out in MS solid medium containing 2.0 mg / L 2,4-D, at a temperature of 26-30°C, for a duration of 3-5 weeks, and in the dark. After induction culture, subculture is preferably performed. Specifically, the callus obtained from the induction culture is transferred to a new culture medium for subculture. Every 15-20 days, well-formed callus clusters are screened, non-embryonic tissues are removed, and the callus is divided into uniformly sized tissue blocks for continued culture to achieve continuous propagation.

[0041] In this invention, before infecting sweet potatoes, sweet potato embryogenic callus tissue is preferably ground and sieved to a particle size of 0.8-1.2 mm, and then pre-cultured in MS liquid medium containing 2.0 mg / L 2,4-D. The successfully transformed Agrobacterium is mixed with acetylsuccinyl syringone to obtain an Agrobacterium-infected bacterial solution, wherein the OD of the Agrobacterium-infected bacterial solution is... 600 The concentration is 0.4-0.6 mg / L; the final concentration of acetylsuccinone is 25-35 mg / L, preferably 28-32 mg / L, and more preferably 30 mg / L. In this invention, pre-cultured sweet potato embryogenic callus is mixed with Agrobacterium infection solution, incubated in the dark for 20-40 min, and then sonicated for 10-20 s; transferred to MS solid medium containing 2.0 mg / L 2,4-D for co-culture to achieve infection; the co-culture temperature is preferably 26-30℃, more preferably 28℃; the co-culture is preferably carried out in the dark, and the co-culture time is preferably 2-4 days, more preferably 3 days.

[0042] In this invention, the reduction IbEXPA11 Gene expression includes interfering with IbEXPA11 The recombinant vector for gene expression was transformed into Agrobacterium and then used to infect sweet potatoes. The methods for transforming Agrobacterium and infecting sweet potatoes are described above and will not be repeated here.

[0043] This invention also provides sweet potato IbEXPA11 The application of genes in short-vine sweet potato assisted breeding reduces IbEXPA11 Gene expression levels, and the development of short-vine sweet potato varieties.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] sweet potato IbEXPA11 Cloning of genes

[0047] 1. Extraction of total RNA from sweet potato leaves

[0048] Total RNA was extracted using the Trizol-centrifugation column method (Total RNA Rapid Extraction Kit, GK3016, Shanghai Jierui Biotechnology Co., Ltd.). The specific steps are as follows: (1) Grind the fresh sweet potato leaf sample into powder using liquid nitrogen, transfer it to a 1.5 ml RNase-Free centrifuge tube (50-100 mg), and add 1 ml RnaEX (Trizol). (2) Vortex for 30 s, then let stand at room temperature for 5 min to fully lyse the sample; (3) Add 200 μl of chloroform to a fume hood, vortex for 30 s, and let stand at room temperature for 5 min; (4) Centrifuge at 12000 rpm and 4℃ for 10 min to allow the solution to separate into layers. (5) Pipette 450-500 μl of supernatant into a centrifuge column, add 200 μl of anhydrous ethanol, mix thoroughly, and let stand at room temperature for 2 min (flocculated precipitate may form). (6) Centrifuge at 8000 rpm for 1 min at room temperature; (7) Discard the filtrate, add 600 μl of Buffer RWA, centrifuge at 8000 rpm at room temperature for 30 s, and discard the filtrate; (8) Repeat step (7), discard the filtrate, and centrifuge at 12000 rpm at room temperature for 1 min; (9) Place the centrifuge column into a new 1.5 ml RNase-Free centrifuge tube, add 50~100 μl of DEPC-H2O to the center of the membrane, and incubate at 55~80℃ for 2 min; (10) Centrifuge at 12000 rpm for 1 min to obtain RNA sample and store at -80℃.

[0049] 2. cDNA was generated by reverse transcription of total RNA from sweet potato leaves.

[0050] The extracted total RNA was reverse transcribed into cDNA using the First-Strand cDNA Synthesis Kit (Toyobo, FSQ301). The specific steps are as follows: (1) Take 1 μg of total RNA into a sterile RNAase-free PCR tube, heat denature it at 65℃ for 5 min, and then immediately place it on ice to cool.

[0051] (2) Add 2 μL of 4x DN Master Mix (with gDNA Remover) to ice, and bring the volume to 8 μL with RNase-FreeH2O. Gently mix the reaction solution and incubate at 37°C for 5 min.

[0052] (3) Next, add 2 μL of 5x RT Master Mix II to ice. After gently mixing the reaction solution, perform the reverse transcription reaction at the following temperatures: 37℃ for 15 min, 50℃ for 5 min, 98℃ for 5 min, and store at 4℃.

[0053] (4) The obtained product is diluted 10 times with RNase-free water before being used in subsequent experiments or stored at -20℃.

[0054] 3. IbEXPA11 CDS sequence amplification of the gene

[0055] Sweet potatoes were obtained based on the sweet potato genome (Beauregard V1.0). IbEXPA11 The full-length CDS sequence of the gene was obtained, and upstream and downstream primers for CDS amplification were designed using Primer3Plus online software. IbEXPA11 -F: 5'-ATGGCTCATTATGGTGGTCTGGGA-3' (SEQ ID NO.3); IbEXPA11 -R: 5'-TTATGAGAACTGGAGTGGGCTGG-3' (SEQ ID NO. 4).

[0056] Using the 10-fold diluted cDNA obtained above as a template, a 50 μL reaction mixture (Takara, R045A) was prepared on ice, comprising 2 μL template cDNA, 25 μL PrimeSTAR Max Premix (2×), 1 μL each of forward and reverse primers, and 21 μL ddH2O. After adding the samples, the mixture was thoroughly mixed and slightly centrifuged before being placed on a PCR instrument for amplification. C 10 s, C 15s, C 30 s, 32 cycles; C 5 min; Save as C.

[0057] The PCR product was subjected to agarose gel electrophoresis, and the gel was used for recovery (Novizan, DC221-01) to obtain the target fragment. Figure 1 The sequencing vector was ligated, positive single clones were selected and sent for sequencing to obtain... IbEXPA11The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0058] Example 2

[0059] Construction of recombinant expression vectors

[0060] 1. IbEXPA11 Construction of overexpression vectors

[0061] Based on the available restriction enzyme sites of the overexpression vector pCAMBIA1301s-DsRed ( Bam HI and Kpn I), Design IbEXPA11 Homologous arm primers constructed from overexpression vectors: IbEXPA11 -OE-F (SEQ ID NO.5): 5'-TTACAATTACAATTAGGATCCATGGCTCATTATGGTGGTCTGG -3'; IbEXPA11 -OE-R (SEQ ID NO.6): 5'-AGATCTGGTCGACGGCGCTGGTACCTTATGAGAACTGGAGTGGGCTGG-3'.

[0062] PCR amplification was performed using primers with the above-mentioned enzyme restriction sites to obtain [the desired product]. IbEXPA11 The complete coding sequence DNA fragment was obtained, and the amplification product was purified and recovered; simultaneously, the pCAMBIA1301s-DsRed empty vector plasmid was double-digested with enzymes. Bam HI and Kpn (I. Thermo Scientific); The purified DNA fragment and the digested empty vector plasmid were ligated using ABclonal's 2x MultiF Seamless Assembly Mix ligase. The recombinant plasmid was then transformed into *E. coli* DH5α competent cells and sent to Shanghai Qingke Sequencing Co., Ltd. for sequencing to obtain accurate results. IbEXPA11 -OE overexpression recombinant plasmid ( Figure 2 ).

[0063] 2. IbEXPA11 Construction of interference expression vectors

[0064] Based on the available restriction enzyme sites of the interference expression vector pFGC5941-DsRed ( Bam HI and Kpn I), Design IbEXPA11 Homologous arm primers for constructing interference expression vectors: IbEXPA11 -RNAi-F1 (SEQ ID NO.7): 5'-TTACAATTACAATTAGGATCCATGGCTCATTATGGTGGTCTGG-3'; IbEXPA11 -RNAi-R1 (SEQ ID NO.8): 5'-AAATTCTTACCCTGCACCATCGAGGCT-3'; IbEXPA11 -RNAi-F2 (SEQ ID NO.9): 5'-ATGGTGCAGGGTAAGAATTTCTTATGTTACATTATTACATTCAACGTTTTATCTTAAT-3'; IbEXPA11 -RNAi-R2 (SEQ ID NO.10): 5'-ATGGTGCAGGCTGCAAATTGACCAAAAAAGATGTGAAGAAAAC-3'; IbEXPA11 -RNAi-F3 (SEQ ID NO.11): 5'-CAATTTGCAGCCTGCACCATCGAGGCT-3'; IbEXPA11 -RNAi-R3 (SEQ ID NO.12): 5'-AGATCTGGTCGACGGCGCTGGTACCATGGCTCATTATGGTGGTCTGG-3'.

[0065] Using the primers with homologous arms described above, PCR amplification was performed to obtain [a sample containing...]. IbEXPA11 The first 300 bp coding sequence and its complementary DNA fragment, along with the non-coding DNA fragment inserted between them, were purified by gel back purification. The three fragments were then tandemly linked and ligated using enzyme digestion. Bam HI and Kpn The empty vector pFGC5941-DsRed plasmid was digested with enzyme I and ligated. The recombinant plasmid was then transformed into E. coli DH5α competent cells and sent to Qingke for sequencing to finally obtain the constructed plasmid. IbEXPA11 Interference expression vector plasmid IbEXPA11 -RNAi ( Figure 3 ).

[0066] Example 3

[0067] Agrobacterium-mediated genetic transformation of sweet potato

[0068] 1. Transformation of Agrobacterium tumefaciens with recombinant expression vector

[0069] Validated by sequencing IbEXPA11 -OE and IbEXPA11 - The RNAi plasmid was transformed into Agrobacterium EHA105 (AC1010M, Shanghai Weidi Biotechnology Co., Ltd.): 1.5 μL of plasmid and 50 μL of competent cells were incubated on ice for 30 min, flash-frozen in liquid nitrogen for 5 min, heat-shocked at 37℃ for 5 min, and then incubated on ice for 2 min; 600 μL of LB medium was added and mixed well, and the cells were thawed at 28℃ and 200 rpm for 2 h. The cells were then plated on LB plates and incubated at 28℃ for 2 days; the resulting single colonies were verified by PCR.

[0070] 2. Sweet potato embryogenic callus induction and subculture

[0071] (1) Take 1.0 cm of sweet potato stem tip, rinse repeatedly with pure water, then surface sterilize with 70% ethanol for 30 s, then soak in 0.1% mercuric chloride solution for 3 min, and finally rinse with sterile water 3-5 times to completely remove residual sterilizing agent. Under a biological microscope, peel off the stem tip meristem and inoculate it into MS solid medium containing 2.0 mg / L 2,4-D. Incubate at 28℃ in the dark for 4 weeks to induce embryogenic callus formation.

[0072] (2) During subculture, callus tissue was transferred to fresh MS medium with the same hormone concentration. Every 20 days, callus in good condition was screened, non-embryonic tissue was removed, and the tissue was divided into tissue blocks of about 2 mm in size and then cultured to achieve continuous propagation.

[0073] 3. Infection and Co-cultivation

[0074] (1) One day in advance, sweet potato embryogenic callus was ground and sieved to a particle size of 0.8~1.2 mm and precultured in MS liquid medium containing 2.0 mg / L 2,4-D as a transformation recipient.

[0075] (2) The PCR-identified positive Agrobacterium was inoculated into 15 mL of LB liquid medium containing 100 mg / L kanamycin (Kan) and 100 mg / L rifampin (Rif), and cultured at 28°C with shaking at 200 rpm until OD. 600 When the bacterial cell density reaches 0.6-0.8, centrifuge at 4000 rpm for 10 min to collect the cells, resuspend in MS liquid medium, and then centrifuge and wash again. Finally, adjust the OD of the bacterial culture with MS medium. 600 Add acetylsuccione (AS, final concentration 30 mg / L) to 0.5 to prepare Agrobacterium infection solution.

[0076] (3) Add the pre-cultured sweet potato embryogenic callus to Agrobacterium infection solution (1 g: 5 ml), wrap it in tin foil and shake it slowly for 30 min in the dark, then sonicate it for 15 s; remove the bacterial solution, wait for the surface of the cell cluster to dry slightly, and gently transfer it to the surface of MS solid medium (containing 30 mg / L AS + 2.0 mg / L 2,4-D) covered with sterile filter paper using a spatula and forceps, and co-culture at 28℃ in the dark for 3 days to complete the co-culture.

[0077] 4. Sterilization, selection culture and regeneration of transgenic plants

[0078] (1) After co-culture, scrape the embryonic cell mass into a sterile Erlenmeyer flask, rinse repeatedly with sterile water 5-8 times (with pipette blowing assistance) until the liquid is clear, and blot dry with sterile filter paper; transfer to MSD medium containing 200 mg / L cephalosporin (Cef), and strictly delay culture for 1 week to eliminate Agrobacterium and promote tissue recovery.

[0079] (2) The callus tissue after delayed culture was transferred to MSD + 10 mg / L hygromycin (Hyg) + 200 mg / L Cef selection medium, and subcultured every 3 weeks to remove non-embryonic tissues. Two rounds of screening were conducted.

[0080] (3) The resistant callus was transferred to MS medium with 1 mg / L abscisic acid (ABA) and 200 mg / L Cef medium and cultured under light for 1-2 weeks to induce embryoid formation.

[0081] (4) Mature somatic embryos were transferred into MS medium with 10 mg / L Hyg and 200 mg / L Cef medium and cultured for 2 months to obtain complete regenerated plants.

[0082] Example 4

[0083] Molecular identification of positive plants

[0084] 1. DNA extraction from genetically modified sweet potatoes

[0085] Leaves from regenerated sweet potato tissue culture seedlings were taken, and sweet potato genomic DNA was extracted using a modified CTAB method. The specific steps are as follows: (1) Take 20 mg of leaves and steel balls and put them into a 2 ml centrifuge tube. Add PVPP, freeze quickly in liquid nitrogen, and then quickly shake into powder using a vortex mixer. (2) Add 600 μl of preheated CTAB buffer, shake to mix, and place in a 60℃ water bath for 1 h; (3) Add 600 μl of a mixture of chloroform and isoamyl alcohol (24:1), mix well, and centrifuge at 13000 rpm for 10 min; (4) Take 400 μl of supernatant, add 300 μl of pre-cooled isopropanol and 10 μl of sodium acetate, mix well, and centrifuge at 13000 rpm for 5 min. (5) Discard the supernatant, add 800 μl of 70% ethanol, and centrifuge at 13000 rpm for 1 min; (6) Discard the supernatant, repeat step (5) once, and place it on the clean bench to dry; (7) Add 100 μl of TE buffer, and after the DNA is dissolved, add 1 μl of 10 mg / ml RNase A and incubate at 37°C for 1 h to degrade the RNA.

[0086] 2. Identification of overexpression lines by PCR amplification

[0087] by IbEXPA11 Using transgenic sweet potato DNA as a template, vector-specific detection primer pairs were used respectively. IbEXPA11 -OE-tF / tR (overexpression) was amplified by PCR and confirmed by gel electrophoresis. The results are as follows. Figure 4 As shown, the same fragment as the positive control (overexpression vector) could be detected in sweet potato plants overexpressing IbEXPA11, while the target fragment could not be detected in wild-type plants and pure water (both negative controls), indicating that the IbEXPA11 gene was successfully overexpressed in sweet potato. Positive plants were selected. IbEXPA11 -OE10, 16, and 19 (overexpression) will be used for further studies. The vector-specific detection primer sequences are as follows: IbEXPA11 -OE-tF (SEQ ID NO. 13): 5'-AGGACACGCTCGAGTATAAGAGC-3'; IbEXPA11 -OE-tR (SEQ ID NO. 14): 5'-GGACTCTAGGGACTAGTCCCG-3'.

[0088] 3. Identification of interfering expression lines by PCR amplification

[0089] by IbEXPA11 Using sweet potato DNA as a template, vector-specific detection primer pairs were employed to interfere with expression. IbEXPA11 RNAi-tF / tR was amplified by PCR and confirmed by gel electrophoresis. Figure 5 As shown, interference expression IbEXPA11 Fragments identical to those in the positive control (interference expression vector) could be detected in sweet potato plants, while the target fragment could not be detected in wild-type plants and pure water (both of which were negative controls), indicating that... IbEXPA11The gene was successfully expressed in sweet potato with interference. Positive plants were selected. IbEXPA11 -R3, 4, and 6 (interference expression) will be used for further research. The vector-specific detection primer sequences are as follows: IbEXPA11 -RNAi-tF (SEQ ID NO. 15): 5'-TTTCATTTGGAGAGGACACGC-3'; IbEXPA11 -RNAi-tR (SEQ ID NO. 16): 5'-ACAAAAGTCCTCCCTCTCTTACC-3'.

[0090] Example 5

[0091] Real-time quantitative PCR (RT-qPCR) to detect the expression level of transformed genes

[0092] Total RNA was extracted from leaves of overexpression lines (OE10, 16, 19) and interference expression lines (R3, 4, 6), and cDNA was synthesized using a Toyobo reverse transcription kit (FSQ301). Online design was performed using Primer3Plus. IbEXPA11 Gene-specific RT-qPCR primers (F: 5′– TGGAGGTTGGTGCAATCCTC –3′, SEQ ID NO. 17; R: 5′– AGGTATGCGTTGGATTGCCA –3′, SEQ ID NO. 18); used a real-time quantitative PCR Master Mix kit (Toyobo, QPK-201) and an ABI StepOne Plus PCR system (ABI, QuantStudio). TM 6 Flex) detection IbEXPA11 The expression level was determined. RT-qPCR reaction conditions: 95℃ pre-denaturation for 3 min; 40 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, with fluorescence collected during the 72℃ extension phase). The relative gene expression level was calculated using the ΔΔCt method. IbActin Genes were standardized as internal controls, with 3 biological replicates × 3 technical replicates, and the entire process was performed on ice.

[0093] The results are as follows Figure 6 As shown, compared to wild-type sweet potato (WT), the overexpressing transgenic lines (OE10, 16, 19) showed... IbEXPA11 Expression levels were significantly upregulated ( P <0.05); in the interference expression lines (R3, 4, 6) IbEXPA11 Expression levels decreased significantly ( P <0.05).

[0094] Example 6

[0095] IbEXPA11 Measurement of transgenic sweet potato vine length

[0096] Sixty days after planting, the vine length (cm) of wild-type WT and transgenic sweet potato plants was measured using a measuring tape.

[0097] Table 1. Vine length statistics of wild-type and transgenic sweet potato plants

[0098] like Figure 7 As shown in Table 1, compared with wild-type WT, the vine length of the overexpressing transgenic lines OE10, 16, and 19 was significantly increased by 16.05%–60.64%. P <0.05); while the interference expression lines R3, 4, and 6 showed a significant reduction of 24.40%–63.45% ( P <0.05), indicating IbEXPA11 Genes positively regulate the growth of sweet potato stems and vines.

[0099] As can be seen from the above embodiments, the present invention has discovered and confirmed that IbEXPA11 The gene is significantly associated with sweet potato vine growth, and overexpression of it is crucial. IbEXPA11 The gene can significantly promote the growth of sweet potato vines, while reducing its expression using RNA interference technology can significantly inhibit the growth of sweet potato vines; this invention provides a new gene resource for improving the vine length trait of sweet potato.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of sweet potato IbEXPA11 Genes, characterized by, The IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The sweet potato according to claim 1 IbEXPA11 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The sweet potato according to claim 1 IbEXPA11 The application of genes in regulating sweet potato vine growth is characterized by, Improve the IbEXPA11 The gene expression level promotes sweet potato vine growth and reduces the aforementioned IbEXPA11 The gene expression level inhibits the growth of sweet potato vines.

4. A biomaterial for controlling the growth of sweet potato vines, characterized in that, Includes one or more of the following biological materials: 1) Interference IbEXPA11 Homologous arm primers for gene expression; 2) Interference IbEXPA11 Recombinant vectors for gene expression; 3) Interference IbEXPA11 Recombinant Agrobacterium gene expression.

5. The biomaterial according to claim 4, characterized in that, The sequences of the homologous arm primers are shown in SEQ ID NO. 7~8, SEQ ID NO. 9~10 and SEQ ID NO. 11~12.

6. The biomaterial according to claim 4, characterized in that, The recombinant vector includes an initial vector and a DNA fragment amplified by the homologous arm primer.

7. A method for regulating the growth of sweet potato vines, characterized in that, include: (a) Overexpression in sweet potato IbEXPA11 Genes that promote the growth of sweet potato vines; or (b) Reduced in sweet potatoes IbEXPA11 Gene expression is used to suppress the growth of sweet potato vines; The IbEXPA11 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, overexpression IbEXPA11 Genes include the said IbEXPA11 Genes were cloned into the initial vector to construct a recombinant vector, which was then transformed into Agrobacterium tumefaciens and used to infect sweet potatoes. reduce IbEXPA11 Gene expression includes interfering with IbEXPA11 The recombinant vector for gene expression was transformed into Agrobacterium and then used to infect sweet potatoes.

9. The method according to claim 8, characterized in that, The receptor for infection in sweet potatoes is sweet potato embryonic callus.

10. Sweet potato IbEXPA11 The application of genes in short-vine sweet potato assisted breeding is characterized by, reduce IbEXPA11 Gene expression levels, and the development of short-vine sweet potato varieties.

Images