Silencing system of potato transcription factor StLRP1 gene and application thereof

Abstract

The invention discloses a silence system of a potato transcription factor StLRP1 gene and application of the silence system, and aims to regulate formation and development of potato tubers by silence of the StLRP1 gene so as to improve the yield and quality of the potato tubers. By silencing the StLRP1 gene, the tuberization efficiency of potatoes is remarkably improved, the tuberization time is advanced, early maturing is promoted, and the planting period is shortened. Meanwhile, the growth period is shortened due to gene silencing, the planting efficiency is enhanced, and the potatoes are more suitable for short growth seasons. In addition, the tubers of the plants are changed from an ovulate shape to a long cylinder shape, and the weight of a single potato plant is reduced, so that the storage and transportation efficiency can be improved, the market preference can be met, more choices are provided for breeding, and the cultivation of potato varieties adapting to different conditions is facilitated. Generally, the present invention enhances the production and adaptability of potatoes and brings advantages to the entire production chain of potatoes by improving the form and weight of tubers.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a silencing system of a potato transcription factor StLRP1 gene and an application thereof. Background Art

[0002] potato( Solanum tuberosum L.) is the world's fourth largest food crop, second only to rice, wheat and corn in terms of output. Potato tubers provide the main nutrients and carbohydrates as food, playing an important role in ensuring my country's food security. In-depth exploration of candidate genes involved in regulating potato tuber development is of great significance for accelerating the breeding of high-yield potato varieties.

[0003] The SHORT INTERNODES / STYLISH (SHI / STY) protein family is an important transcription factor in plant growth and development, playing an important role in all aspects of plant growth and development (Fridborg et al., 1999). The transcription factors of this protein family are involved in the auxin regulatory pathway and affect the development of lateral branch organs.

[0004] The SHI / STY protein family is a family unique to plants, encoding a specific transcription factor with two different conserved regions. The first conserved region is located at the N-terminus of the protein, which contains a RING-like zinc finger motif, known as the RING domain; the motif is described as C-X2-CXI2-C-X2-C-X7-C (Zhao et al., 2020). The second conserved region is located at the C-terminus and is a protein domain unique to the SHI family. This domain contains four highly conserved residues and is therefore named IGGH. The IGGH domain contains acidic amino acids unique to the SHI / STY family and acts as a transcriptional activator (Fridborg et al, 2001; Singh et al, 2020). In addition to these two domains, there are one or two Gln-rich regions in the SHI / STY protein, which have been shown to be transcriptional activation domains, revealing that SHI / STY may play the role of a transcriptional activation domain. (Eklund et al, 2010). Apart from these two conserved structures, the remaining protein sequences are highly divergent (Zhu et al, 2022; Fridborg et al, 2001).

[0005] SHI / STY family members mainly affect the development of plant lateral organs by participating in the auxin regulatory pathway. As a member of the SHI / STY family, LRP1 is involved in the modification of chromatin and auxin signals during lateral root formation and development (Kuusk et al., 2006; Singh et al., 2020). Studies have shown that LRP1 plays a key role in plant root development and has a conserved function in poplar adventitious root formation by promoting taproot elongation and negatively regulating lateral root development. (Yu et al., 2022). LRP1 is a direct target of PuMYB40 and PuWRKY75, and is involved in regulation through auxin signaling and chromatin modification. (Wang et al., 2022). It works synergistically with the SLR / IAA14 / ARF7 / ARF19 module and genes such as LBD16, LBD18 and LBD29, and is affected by histone deacetylation. (Singh et al., 2020). In addition, factors such as SRS5 and SHI / STYs also affect root development by directly or indirectly regulating LRP1 and its downstream targets (De Rybel et al., 2012; De Smet et al., 2008).

[0006] At the same time, the SHI / STY gene family plays an important role in plant growth and development, hormone regulation and stress resistance. It affects stem elongation, leaf morphology, floral organ development, carpel fusion and marginal tissue formation of pistils by regulating cell elongation and division. In crops such as rice and barley, SHI genes are also involved in the development of stem strength, panicle branching and awn. For example, in rice, the SHI1 mutant is characterized by a sharp decrease in the number of tillers, increased culm strength, and increased panicle branching, while the plant height of the rice SHI1 overexpression line is significantly reduced (Duan et al., 2019). SHI affects the apical-basal pattern and elongation of plant organs by regulating auxin biosynthesis (such as activating YUC4 and YUC8 genes). For example, SHI / STY family member STY1 can increase the transcription level of YUCCA family member YUC4, increase auxin levels and auxin biosynthesis in Arabidopsis seedlings, including cell expansion and nodule vascular bundle formation (Zhao et al., 2020). At the same time, some members of the SHI family may play an indispensable role in the development and photomorphogenesis of female flowers and leaves in Arabidopsis by regulating the homeostasis of auxin or the expression of HY5, BBX21 and BBX22 (Smith and Fedoroff, 1995; Eklund et al., 2010; Fridborg et al. 1999, 2001; Kuusk et al. 2002, 2006; Baylis et al., 2013; Yuan et al., 2018; Sohlberg et al., 2006). In addition, SHI mutants exhibit a phenotype similar to GA deficiency, indicating its role in the gibberellin signaling pathway. There is significant redundancy in the functions of the SHI gene family, and different members are involved in multiple developmental processes. Their regulation depends on specific elements (such as GCC boxes) and their binding to transcription factors. More importantly, this gene family shows significant responses under environmental stresses such as salt stress, drought, and low temperature, indicating that it plays a key role in plant resistance.

[0007] However, the function of SHI / STY transcription factors in regulating potato tuber formation and development has not been reported. Summary of the invention

[0008] The present invention aims to regulate the formation and development process of tubers by silencing the StLRP1 gene in potatoes, thereby optimizing the tuber morphology of potatoes and improving their production efficiency and adaptability.

[0009] To achieve the above objectives, the present invention provides the following technical solutions: The present invention relates to a recombinant expression vector, which comprises a pHELLSGATE-8 vector and a StLRP1 gene interference fragment inserted into the vector. The StLRP1 gene interference fragment comprises a nucleotide sequence shown in SEQ ID NO.1 or a reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO.1.

[0010] The application of the recombinant expression vector in the gene silencing system for regulating potato tuber formation and development can effectively reduce the expression level of the StLRP1 gene.

[0011] The present invention further relates to a gene silencing system, comprising: Agrobacterium liquid containing the above-mentioned recombinant expression vector; An Agrobacterium bacterial liquid containing the auxiliary vector pHELLSGATE-8; the Agrobacterium is a GV3101 strain.

[0012] The present invention also relates to a method for constructing the above-mentioned recombinant expression vector, comprising: RNA was extracted from potato tuber samples and reverse transcribed to generate cDNA; The cDNA was amplified by PCR using the designed specific primers StLRP1-F and StLRP1-R to obtain the StLRP1 gene interference fragment; The interference fragment was inserted into the pHELLSGATE-8 vector through homologous recombination technology to construct a recombinant expression vector.

[0013] Based on the above vector and gene silencing system, the present invention provides a method for silencing potato StLRP1 gene, comprising: Constructing the above-mentioned recombinant expression vector; The vector was transformed into Agrobacterium GV3101; Infecting potato chips using the gene silencing system; The transgenic plants were obtained by cultivation.

[0014] The present invention has the following beneficial effects: The present invention significantly improves the efficiency of potato tuber formation and advances the tuber formation time by silencing the transcription factor StLRP1 gene, and this improvement directly contributes to the earlier formation of tubers in plants. This early-maturing characteristic helps to shorten the planting cycle and increase the yield per unit area. At the same time, the silencing of the StLRP1 gene also leads to a shortened growth period, which enhances the planting efficiency and enables potatoes to better adapt to a shorter growing season. In addition, the potato plants with the gene silenced show significant changes in tuber morphology, from the traditional obovate to the long cylindrical shape, and the weight of the single plant tuber is significantly reduced, which may not only improve the storage and transportation efficiency of the tubers, but also provide the market with a more popular tuber morphology, while providing breeders with more choices, which helps to cultivate potato varieties that meet specific needs. These changes in morphology and weight may also be related to the increased resistance of plants to environmental stress, so that crops can better adapt to different growth conditions. In general, the present invention not only enhances the production efficiency and adaptability of potatoes, but also brings additional advantages to the storage, transportation, processing and market sales of potatoes by improving the morphology and weight of tubers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : PCR amplification results of StLRP1 gene; wherein M represents molecular marker; 1 represents StLRP1 gene PCR product.

[0016] Figure 2 : Construction and verification of StLRP1 gene silencing vector; wherein M represents a molecular marker; 1 represents a Gate8-StLRP1 plasmid control, and 2 represents an XhoI digestion product.

[0017] Figure 3 :Detection of StLRP1 gene silencing efficiency; Figure 3 A: DNA electrophoresis diagram of StLRP1 gene silencing strain; Figure 3 B: Real-time fluorescence quantitative PCR results of StLRP1 gene in silenced strain and wild-type strain.

[0018] Figure 4 :Comparison of phenotypes of early tuber setting caused by silencing of StLRP1 gene; Figure 4 A: Phenotypic diagram of early tuber setting caused by silencing of StLRP1 gene. Figure 4 B: The proportion of plants that formed tubers in the StLRP1 gene silenced line and the wild-type line on the 35th day of medium daylight, n=15.

[0019] Figure 5 :Phenotype comparison of shortened growth period caused by StLRP1 gene silencing.

[0020] Figure 6:Comparison of the changes in tuber length-width ratio and tuber weight per plant caused by StLRP1 gene silencing; Figure 6 A: Phenotypic diagram of tuber morphology of StLRP1 gene silenced strain; Figure 6 B: Statistics of single-plant tuber yield of StLRP1 gene silenced strains and wild-type strains, n=13; Figure 6 C: Statistics of the length-to-width ratio of tubers of StLRP1 gene silenced strains and wild-type strains, n=20. DETAILED DESCRIPTION

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Example 1: Acquisition of StLRP1 gene The gene sequence and amino acid sequence information of StLRP1 (Soltu.DM.11G018850.1) were obtained through the potato database (http: / / spuddb.uga.edu / index.shtml). The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0023] StLRP1 gene sequence:

[0024] Amino acid sequence encoded by the StLRP1 gene: MWSASRQMQINYGLQPTEMGMFVVHPASFQHHHQHHENTINFDPHGNNNSNLSNTCTTNTSLGVGVIPLLTATPLTNMVTFDDQDLVTRNNRGGNNDGTSGFQFFSNQQPQNSTTNYTKNSTSSNNIILGGGGGGSISTTTCQDCGNQAKKDCTHRRCRTCCKSRGYDCNTHVRSTWVPAS RRRERQLLGGATTTNVNVVAAGSSSQSTSSAKKPRLVNSQTTTTASHTSTSNNTPPRSFDTSSSHQDASFKGSLPGQVRAPAVFKCVRVTSVDEGEDEYAYQAVVKIGGHLFKGFLYDQGLDEPKDNNNNNNNNNFPNLSDLHLGAVATNEHNSHLYAASTSGGLLGGGSNYGNQIN;SEQ ID NO.2.

[0025] Primers StLRP1-F (SEQ ID NO.3) and StLRP1-R (SEQ ID NO.4) were designed based on the CDS sequence of the StLRP1 gene using Premier 5.0 as shown in Table 1 and synthesized by Qingke Biotechnology Co., Ltd. The cDNA of the tuber sample was used as a template for PCR amplification of the full length of the StLRP1 gene. The amplification conditions were: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 72°C for 6 s, 35 cycles (denaturation-extension); extension at 72°C for 5 min, and storage at 4°C. The PCR amplification results of the CDS fragment of the StLRP1 gene are shown in Figure 1. Figure 1 The amplified product was recovered by gel excision and purified, connected to the pMD18-T vector, transformed into DH5α competent cells, PCR screened positive clones, and the positive single clones were picked and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid of the positive clone was extracted, and the StLRP1 gene sequence was confirmed by sequencing.

[0026] Table 1

[0027] Example 2: Construction of StLRP1 gene silencing expression vector The plasmid was extracted from the pMD19T recombinant vector containing the StLRP1 gene. The plasmid was used as a template to amplify the StLRP1 silencing fragment using the primers StLRP1-Ri-1F (SEQ ID NO.5) and StLRP1-Ri-1R (SEQ ID NO.6) in Table 2. Then, the reverse complementary fragment of the StLRP1 silencing fragment was amplified using the primers StLRP1-Ri-2F (SEQ ID NO.7) and StLRP1-Ri-2R (SEQ ID NO.8). The pHELLSGATE-8 vector was cut with XhoI, and the amplified fragment was homologously recombined with the cut vector. After transformation, bacterial inspection, and correct sequencing, the plasmid of the correct bacterial solution was extracted, the plasmid was cut with Xbal, and the reverse complementary fragment of the StLRP1 silencing fragment was homologously recombined with it, and finally the plasmid with correct sequencing was obtained, completing the construction of the StLRP1 gene silencing expression vector Gate8-StLRP1. The vector was verified by XhoI digestion, and the results are as follows. Figure 2 shown.

[0028] Table 2

[0029] Example 3: Genetic transformation of StLRP1 gene silencing expression vector The Gate8-StLRP1 plasmid was transferred into the Agrobacterium strain GV3101, and the GV3101 Agrobacterium transferred with the Gate8-StLRP1 plasmid was used to infect sterile potato slices with a thickness of about 0.2 cm. After the infection was completed, the slices were cultured on MS30 plates containing antibiotics until differentiated resistant buds grew on the potato slices. The resistant buds were cut off and cultured in MS culture medium containing antibiotics to obtain resistant buds that could root.

[0030] Example 4: Planting of potatoes The wild-type potato and the obtained StLRP1 interference strain were preserved in the form of sterile potato tissue culture seedlings. The tissue culture subculture method was as follows: the stem tips and stem segments of the potato tissue culture seedlings were cut and cultured in ordinary MS medium in a 22°C tissue culture room. After 14 days of subculture, they were taken out of the MS medium, and after two days of hydroponic training, they were planted in plastic pots (about 24 cm in diameter).

[0031] Example 5: Gene silencing efficiency detection Leaves of the silenced strains obtained by genetic transformation and the wild-type strain tissue culture seedlings were collected, and the genomic DNA and total RNA of the samples were extracted respectively, and the total RNA was reverse transcribed into cDNA. The qRT-PCR primers StLRP1-QF (SEQ ID NO. 9) and StLRP1-QR (SEQ ID NO. 10) were designed according to the cloned StLRP1 gene sequence as shown in Table 3, and the BLASTn test was used in NCBI to ensure the specificity of the primers. The DNA was PCR-treated with the interference fragment primer LRP1 Ri-F (SEQ ID NO. 11) and the vector primer PDK intron-R (SEQ ID NO. 12) to detect whether the target fragment was inserted into the genome of the StLRP1 interference strain. The results are shown in Table 3. Figure 3 As shown in A, it shows that the Ri-LRP1-4 / 15 / 37 / 44 strain is a transgenic positive strain. The potato StEF1a gene was used as the internal reference gene for real-time quantitative PCR, and the 2-ΔCt method was used to analyze the silencing efficiency of the StLRP1 gene. The results are shown in Figure 3 As shown in B, the interference efficiencies are 74.31%, 42.93%, and 57.23%, respectively.

[0032] Table 3

[0033] Example 6: Silencing of the StLRP1 gene leads to early tuber formation To clarify the effect of StLRP1 expression on potato plants, the interference strains were planted in a medium-day growth chamber (set conditions were 12 h light, 12 h dark, and temperature was 22 ± 2°C). In the fifth week after planting in the medium-day growth chamber, the number of plants that formed tubers in wild-type plants and interference strains was photographed and counted ( Figure 4 A), the results showed that the proportion of plants that formed tubers in the wild-type strain was 41%, while the proportions of the three StLRP1 interference strains were 87%, 86%, and 93%, respectively ( Figure 4 B). The number of tubers formed in the interference transgenic lines was significantly higher than that in the control. The experiment was repeated in a long-day growth chamber (set at 16 h light, 8 h dark, and 22 ± 2°C). Since long-day light inhibits tuber formation, observations and statistics were made in the seventh week, and it was found that the number of tubers formed in the interference lines was still earlier than that in the wild type ( Figure 4 A). These results suggest that silencing of StLRP1 may lead to an early tuber formation.

[0034] Example 7: Silencing of the StLRP1 gene leads to a shortened reproductive period The interference strain and the wild-type strain were planted in a medium-day growth chamber. In the ninth week after planting, it was found that the bottom leaves of the interference strain turned yellow earlier than those of the wild-type plants, showing signs of senescence. As the planting time increased, the bottom leaves of the interference strain aged and fell off earlier. These results suggest that the silencing of StLRP1 may lead to a shortened growth period ( Figure 5 ).

[0035] Example 8: Silencing of the StLRP1 gene resulted in a significant increase in the tuber length-width ratio and a significant decrease in the weight of a single tuber The interference strains and wild-type strains were planted in a medium-day growth chamber and harvested four months after planting. The harvested tubers were observed ( Figure 6 A). After statistical analysis of the tuber weight per plant, it was found that the tuber weight per plant of Ri-LRP1-15 / 37 / 44 was significantly lower than that of the wild type, decreasing by 22.1%, 11.8%, and 27.0%, respectively ( Figure 6 B). Compared with the wild-type plants, the tuber shape of the interference strains changed from obovate to long cylindrical. The statistical analysis of the length-to-width ratio of the tubers showed that the average length-to-width ratio of the wild-type strains was 1.226, while the average length-to-width ratios of Ri-LRP1-15 / 37 / 44 were 2.047, 1.706, and 1.600, respectively, which were increased by 67.0%, 39.2%, and 30.5%, respectively. Figure 6 C).

Claims

1. A recombinant expression vector for silencing potato StLRP1 gene, characterized in that: The recombinant expression vector comprises a pHELLSGATE-8 vector and a StLRP1 gene interference fragment inserted into the vector; The interference fragment includes the nucleotide sequence shown in SEQ ID NO.1 or the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO.

1.

2. Use of the recombinant expression vector according to claim 1 in preparing a gene silencing system for regulating potato tuber formation and development.

3. A gene silencing system for silencing the StLRP1 gene, characterized in that: The gene silencing system comprises: An Agrobacterium bacterial solution containing the recombinant expression vector according to claim 1; Agrobacterium culture containing the helper vector pHELLSGATE-8.

4. The gene silencing system according to claim 3, characterized in that: The Agrobacterium is a GV3101 strain.

5. A method for constructing the recombinant expression vector according to claim 1, characterized in that: The following steps are involved: RNA was extracted from potato tuber samples and reverse transcribed to obtain cDNA; The cDNA is amplified by PCR using an upstream primer StLRP1-F (SEQ ID NO. 3) and a downstream primer StLRP1-R (SEQ ID NO. 4) to obtain a StLRP1 gene interference fragment; The interference fragment is inserted into the pHELLSGATE-8 vector, and a recombinant expression vector is constructed using homologous recombination technology.

6. A method for silencing potato StLRP1 gene, characterized in that: The following steps are involved: Constructing the recombinant expression vector according to claim 1; The recombinant expression vector is transformed into Agrobacterium GV3101; Infecting potato chips using the gene silencing system described in claim 3; Infected potato tubers are cultured until plant growth occurs.

7. The method according to claim 6, characterized in that By silencing the StLRP1 gene, the plant exhibits phenotypic characteristics of early tuber setting, shortened growth period, increased tuber length-to-width ratio and reduced tuber weight per plant.

8. Application of the technical solution according to any one of claims 1 to 7 in increasing potato yield and optimizing tuber morphology.

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