Potato StTRG gene, RNAi silencing vector and application

Abstract

The invention provides a potato StTRG gene, an RNAi silencing vector and application, and belongs to the technical field of gene engineering. According to the invention, by cloning and silencing the potato StTRG gene, the function of the StTRG gene in development regulation of potato plants and tubers is found. Specifically, an RNAi interference vector of the potato StTRG gene is constructed for genetic transformation, so that early flowering and early tuberization of potato plants are promoted, the tuberization quantity is increased, and the negative regulation effect of the StTRG gene on important agronomic traits of potatoes is disclosed.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a potato StTRG gene, an RNAi silencing vector, and its applications. Background Technology

[0002] The potato (Solanum tuberosum L.) is an important dual-purpose crop, used for both food and vegetables. The potato tuber is both a crucial commercial organ and its reproductive organ. The formation and development of potato tubers mainly involves three stages: stolon formation, stolon apex swelling, and tuber formation and development, accompanied by the accumulation of nutrients such as starch, protein, and vitamins within the tuber.

[0003] Tuber number is one of the important traits affecting potato yield, and the timing of tuber formation has practical guiding significance for breeding specialized varieties suitable for different climatic regions. Tuber number and tuber formation time are synergistically regulated by genotype and environmental factors, but the genes that have been reported in potatoes that affect tuber formation time and number are very limited, and there are no reports of genes that can simultaneously promote early tuber formation and increase the number of tubers in potatoes. Summary of the Invention

[0004] The purpose of this invention is to address the scarcity of gene resources for regulating tuber formation time and tuber quantity in existing technologies, achieving the technical effect of promoting earlier tuber formation and increasing tuber quantity in potatoes, and providing new gene targets and theoretical basis for breeding high-yielding and early-maturing potato varieties. To this end, this invention provides a potato StTRG gene, a StTRG-RNAi silencing vector, and its applications.

[0005] This invention provides a potato StTRG gene, wherein the nucleotide sequence of the CDS includes at least one of the following:

[0006] ①The nucleotide sequence shown in SEQ ID NO.1;

[0007] ②The nucleotide sequence shown in SEQ ID NO.2;

[0008] ③The nucleotide sequence shown in SEQ ID NO.3;

[0009] ④ A nucleotide sequence that has at least 98% identity with any of the sequences in ① to ③.

[0010] The present invention also provides an RNAi silencing vector for the potato StTRG gene described in the above technical solution, wherein the RNAi silencing vector contains an interfering fragment sequence of StTRG as shown in SEQ ID NO.4.

[0011] Preferably, the introductory vector for constructing the RNAi silencing vector includes pENTR; and the backbone vector for constructing the RNAi silencing vector includes pHELLSGATE 12.

[0012] Preferably, the nucleotide sequence of the upstream primer of the primer pair for constructing the RNAi silencing vector is shown in SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 6.

[0013] This invention also provides the application of the potato StTRG gene or the RNAi silencing vector described in the above-mentioned technical solutions in improving potato yield and / or cultivating high-yield potatoes.

[0014] Preferably, the increase in potato yield is achieved by increasing the number of potato tubers.

[0015] This invention also provides the application of the potato StTRG gene or the RNAi silencing vector described in the above-mentioned technical solutions in promoting early tuber formation and / or cultivating early-maturing potatoes.

[0016] Preferably, promoting early potato tuber formation includes promoting early potato flowering.

[0017] The present invention also provides a method for increasing potato yield and / or promoting early tuber formation in potatoes, the method comprising the following steps:

[0018] The expression level of the StTRG gene is reduced using the RNAi silencing vector described in the above technical solution.

[0019] The present invention also provides a method for cultivating high-yield and / or early-maturing potatoes, the method comprising the following steps:

[0020] Potatoes with reduced StTRG gene expression levels as described in the above technical solution were used as parents for breeding.

[0021] Beneficial effects:

[0022] This invention provides a potato StTRG gene, whose CDS nucleotide sequence includes at least one of the following: ① the nucleotide sequence shown in SEQ ID NO.1; ② the nucleotide sequence shown in SEQ ID NO.2; ③ the nucleotide sequence shown in SEQ ID NO.3; ④ a nucleotide sequence having at least 98% identity with any of ① to ③. Tissue expression specificity analysis revealed that the StTRG gene is specifically highly expressed in stolons. Further cloning of the potato StTRG gene, since the StTRG cultivar Atlantic has three alleles with sequence similarity exceeding 98%, a conserved segment was selected to construct its RNAi interference vector for genetic transformation, revealing the negative regulatory effect of the StTRG gene on important agronomic traits of potato. Specifically, reducing the expression level of the StTRG gene significantly promotes earlier flowering, earlier tuber formation, and increased tuber quantity in potato plants. This discovery provides an important theoretical basis for improving key agronomic traits such as tuber quantity and tuber formation period in potatoes. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0024] Figure 1 Figure showing the expression levels of the StTRG gene in potato DM and RH;

[0025] Figure 2 Figure showing the tuber formation phenotype of the StTRG silent line after four weeks of growth;

[0026] Figure 3 A statistical chart showing the percentage of tuber formation initiated in StTRG silent lines after four weeks of growth;

[0027] Figure 4 Phenotypic results of tuber number in StTRG silent lines after seven weeks of growth;

[0028] Figure 5 A graph showing the statistical results of tuber formation in StTRG silent lines after seven weeks of growth;

[0029] Figure 6 Phenotypic results of tuber number in StTRG silent lines after 15 weeks of growth;

[0030] Figure 7 A graph showing the statistical results of tuber count in StTRG silent lines after 15 weeks of growth;

[0031] Figure 8 A graph showing the percentage of flowering plants in the StTRG silent lines;

[0032] Figure 9The figure shows the results of the determination of the relative expression level of the StTRG gene in the StTRG silencing line. Detailed Implementation

[0033] This invention provides a potato StTRG gene, the CDS nucleotide sequence of which includes at least one of the following:

[0034] ①The nucleotide sequence shown in SEQ ID NO.1;

[0035] ②The nucleotide sequence shown in SEQ ID NO.2;

[0036] ③The nucleotide sequence shown in SEQ ID NO.3;

[0037] ④ A nucleotide sequence having at least 98% sequence identity with any of sequences ① to ③. The three CDS sequences of the potato StTRG gene described in this invention are three alleles of the Atlantic StTRG gene, possessing the same silencing-related segment. As one embodiment, this invention can influence and control StTRG gene-related traits through the aforementioned identical silencing-related segment. The nucleotide sequence shown in SEQ ID NO.1 of this invention is specifically as follows:

[0038] ATGGAATCATCATCATCATCATCGTCATCAAAGAGGGCCAAGGCACCAGGGAATATAGCTCATTGCTTGGTTGATGGGTGTAATGCAGACCTCAGTGAATGCAGAGAGTATCATCGCCGGCATAAAGTTTGTGAGGTGCATTCAAAGACTGCCAAAGTCACCATTGCAGGTCGAGACCAACGCTTCTGTCAGCAATGCAGCAGGTTTCATTCATTGGTAGAATTTGATGACGGAAAGAGAAGCTGTCGGAAACGTCTTGATGGACATAACAGGCGTCGAAGGAAGCCTCAGCCAGATTCTATGGCAAAAAATTCTGGATTACTTTTTGGCCAACAAGGAACGAAACTCCTGTCATTTAGCAGTCAACAAATATTTCCAAGTGCAGTTGTGAGCTCTGCATGGGCTGGCGTTGTCAAAACAGACAGTGATATGGTATTATACAACAACCAATCACATATGAATGGTATGGACAGTCAAAACTCGTTCCCTGATTCTTCGGGTCATAGCTATACAGGAGGAAGCCAATTCCAGTTCATGCAAGGCAGTGATCATAGTCTGACTGAATCTTCTTCAATCTGCCAGCCACTTTTTGAACATCCCACTTCTGCAGCAGGAATTTCTAGCAGCGGACAAAAGATCTTCTCCAGTGGATTAAATGATATTGTTGATTCTGATCGTGCTCTCTCTCTTCTGTCATCAGCACCCGCTGTAACTAGGGAGATTGGTTTGAGCCACATGGTGCAGCAGCCTGCTTCTATCCCACGTTCCCAGTCACAGGGCCTGCAGTATGATGGTCTAAGCCATTTCCCTTTTACTCAAGATTTCAATAGCAAACCTCAAGATTCACATGTTAGCAACAGCAGCAACCCTCTCCATTTCCATGATATGTTGCAAAATGCACAAGATGGATCATCTACAAGTGGTGGCTCTCAGCAAACACTAGCCTTTATGTGGGACTAA, length 960 bp.

[0039] The amino acid sequence of the protein corresponding to the nucleotide sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.14:

[0040] MESSSSSSSSKRAKAAPGNIAHCLVDGCNADLSECREYHRRHKVCEVHSKTAKVTIARGRDQRFCQQCSRFHSLVEFDDGKRSCRKRLDGHNRRRRKPQPDSMAKNSGLLFGQQGTKLLSFSSQQIFPSAVVSSAWAGVVKTDSDMVLYNNQSHMNGMDSQN SFPDSSGHSYTGGSQFQFMQGSDHSLTESSSICQPLFEHPTSAAGISSSGQKIFSSGLNDIVDSDRALSLLSSAPAVTREIGLSHMVQQPASIPRSQSQGLQYDGLSHFPFTQDFNSKPQDSHVSNSSNPLHFHDMLQNAQDGSSTSGGSQQTLAFMWD.

[0041] The nucleotide sequence shown in SEQ ID NO.2 of this invention is specifically as follows:

[0042] ATGGAATCATCATCATCATCATCGTCATCAAAGAGGGCCAAGGCACCAGGGAATGTAGCTCATTGCTTGGTTGATGGGTGTAATGCAGACCTCAGTGAATGCAGAGAGTATCATCGCCGGCATAAAGTTTGTGAGGTGCATTCAAAGACTGCCAAAGTCACCATTGCAGGTCGAGACCAACGCTTCTGTCAGCAATGCAGCAGGTTTCATTCATTGGTAGAATTTGATGACGGAAAGAGAAGCTGTCGGAAACGTCTTGATGGACATAACAGGCGTCGAAGGAAGCCTCAGCCAGATTCTATGGCAAAAAATTCTGGATTACTTTTTGGCCAACAAGGAACGAAACTCCTGTCATTTAGCAGTCAACAAATATTTCCAAGTGCAGTTGTGAGCTCTGCATGGGCTGGCGTTGTCAAAACAGACAGTGATATGGTATTATACAACAACCAATCACATATGAATGGTATGGACAGTCAAAACTCGTTCCCTGATTCTTCGGGTCATAGCTATACAGGAGGAAGCCAATTCCAGTTCATGCAAGGCAGTGATCATAGTCTGACTGAATCTTCTTCAATCTGCCAGCCACTTTTTGAACATCCCACTTCTGCAGCAGGAATTTCTAGCAGCGGACAAAAGATCTTCTCCAGTGGATTAAATGATATTGTTGATTCTGATCGTGCTCTCTCTCTTCTGTCATCAGCACCCGCTGTAACTAGGGAGATTGGTTTGAGCCACATGGTGCAGCAGCCTGCTTCTATCCCACGTTCCCAGTCACAGGGCCTGCAGTATGATGGTCTAAGCCATTTCCCTTTTACTCAAGATTTCAATAGCAAACCTCAAGATTCACATGTTAGCAACAGCAGCAACCCTCTCCATTTCCATGATATGTTGCAAAATGCACAAGATGGATCATCTACAAGTGGTGGCTCTCAGCAAACACTAGCCTTTATGTGGGACTAA, length 960 bp.The amino acid sequence of the protein corresponding to the nucleotide sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.15.

[0043] MESSSSSSSSKRAKAAPGNVAHCLVDGCNADLSECREYHRRHKVCEVHSKTAKVTIARGRDQRFCQQCSRFHSLVEFDDGKRSCRKRLDGHNRRRRKPQPDSMAKNSGLLFGQQGTKLLSFSSQQIFPSAVVSSAWAGVVKTDSDMVLYNNQSHMNGMDSQN SFPDSSGHSYTGGSQFQFMQGSDHSLTESSSICQPLFEHPTSAAGISSSGQKIFSSGLNDIVDSDRALSLLSSAPAVTREIGLSHMVQQPASIPRSQSQGLQYDGLSHFPFTQDFNSKPQDSHVSNSSNPLHFHDMLQNAQDGSSTSGGSQQTLAFMWD.

[0044] The nucleotide sequence shown in SEQ ID NO.3 of this invention is specifically as follows:

[0045] ATGGAATCATCATCATCATCATCGTCATCAAAGAGGGCCAAGGCACCAGGGAATATAGCTCATTGCTTGGTTGATGGGTGTAATGCAGACCTCAGTGAATGCAGAGAGTATCATCGCCGTCATAAAGTTTGTGAGGTGCATTCAAAGACTGCCAAAGTCACCATTGCAGGTCGAGACCAACGCTTCTGTCAGCAATGCAGCAGGTTTCATTCATTGGTAGAATTTGATGACGGAAAGAGAAGCTGTCGGAAACGCCTTGATGGACATAACAGGCGTCGAAGGAAGCCTCAGCCAGATTCTATGGCAAAAAATTCTGGATTACTTTTTGGCCAACAAGGAACGAAACTCCTGTCATTTAGCAGTCAACAAATATTTCCAAGTGCAGTTGTGAGCTCTGCATGGGCTGGCGTTGTCAAAACAGACAGCGATATGGTATTATACAACCGATCACATATGAATGGTATGGACAGTCAAAACTCGTTCCCTGATTCTTCGGGTCATAGCTATACAGGAGGAAGCCAATTCCAGTTCATGCAAGGCAGTGATCATAGTCTGACTGAATCTTCTTCAATCTGCCAGCCACTTTTTGAACATCCCACTTCTGCAGCAGGAATTTCTAGCAGCGGACAAAAGATCTTCTCCAGTGGATTAAATGATATTGTTGATTCTGATCGTGCTCTCTCTCTTCTGTCATCAGCACCCGCTGTAACTAGGGAGATTGGTTTGAGTCACATGGTGCAGCAGCCTGCCTCTATCCCGCGTTCCCAGTCACAGGGCCTGCAGTATGATGGTCTAAGCCATTTCCCTTTTGCTCAAGATTTCAATAGCAAACCTCAAGATTCACATGTTAGCAACAGCAGCAGCCCTCTCCATTTCCATGATATGTTGCAAAATGCACAAGATGGATCATCTACAAGTGGTGGCTCTCAGCAAACACTAGCCTTTATGTGGGACTAA, length 957 bp.The amino acid sequence of the protein corresponding to the nucleotide sequence shown in SEQ ID NO.3 is shown in SEQ ID NO.16.

[0046] MESSSSSSSSKRAKAAPGNIAHCLVDGCNADLSECREYHRRHKVCEVHSKTAKVTIARGRDQRFCQQCSRFHSLVEFDDGKRSCRKRLDGHNRRRRRKPQPDSMAKNSGLLFGQQGTKLLSFSSQQIFPSAVVSSAWAGVVKTDSDMVLYNRSHMNGMDSQN SFPDSSGHSYTGGSQFQFMQGSDHSLTESSSICQPLFEHPTSAAGISSSGQKIFSSGLNDIVDSDRALSLLSSAPAVTREIGLSHMVQQPASIPRSQSQGLQYDGLSHFPFAQDFNSKPQDSHVSNSSSPLHFHDMLQNAQDGSSTSGGSQQTLAFMWD.

[0047] This invention also provides an RNAi silencing vector for the potato StTRG gene described in the above-mentioned technical solution, wherein the RNAi silencing vector contains an interfering fragment sequence of StTRG as shown in SEQ ID NO.4. Specifically, SEQ ID NO.4 of this invention is:

[0048] The RNAi silencing vector is 296 bp in length. As one embodiment, the entry vector for constructing the RNAi silencing vector includes pENTR; the backbone vector for the RNAi silencing vector includes pHELLSGATE 12. As one embodiment, the RNAi silencing vector of the present invention can also be constructed using other equivalent entry and backbone vectors known in the art. As one embodiment, the pENTR of the present invention... TMThe / D-TOPO vector insertion sites are attL1 and attL2, both containing GTGG adapters, allowing the target fragment to be inserted into the vector via homologous recombination. As one embodiment, the pHELLSGATE 12 vector insertion sites of this invention are attL1, attL2 and attR1, attR2, which are associated with the pENTR vector carrying the target fragment. TM The / D-TOP vector replaces the target fragment into the pHELLSGATE 12 vector through homologous recombination, thus constructing the silencing vector.

[0049] As one implementation method, the nucleotide sequence of the upstream primer of the primer pair for constructing the RNAi silencing vector of the present invention is shown in SEQ ID NO. 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 6. Specifically, SEQ ID NO. 5 of the present invention is: CACCCTCGTTCCCTGATTCTTCGG; and SEQ ID NO. 6 of the present invention is: GTGACTGGGAACGTGGGATA.

[0050] As one implementation method, the construction of the initiation vector described in this invention can be performed with reference to the instructions of a commercial initiation vector construction kit. As another implementation method, the commercial initiation vector construction kit described in this invention can be pENTR Directional. Cloning Kits. As one embodiment, the reaction system of the initiator vector of this invention comprises the following components: salt solution (1 μL), interfering fragment PCR product (0.5 μL), pENTR vector (1 μL), and ddH2O (2.5 μL). As one embodiment, the initiator vector of this invention is constructed and then processed using pENTR... TM The vector universal primers M13For and M13Rev were used for PCR verification; the nucleotide sequence of M13For is shown in SEQ ID NO.7; the nucleotide sequence of M13Rev is shown in SEQ ID NO.8.

[0051] In one embodiment, the LR reaction for constructing the RNAi silencing vector of the present invention can be performed according to the instructions of a commercial LR reaction kit. In one embodiment, the commercial LR reaction kit of the present invention can be LRClonase™ II Enzyme Mix. In one embodiment, the LR reaction system for constructing the RNAi silencing vector of the present invention comprises the following components: TE Buffer (5 μL), pHellsGate 12 (1 μL), pENTR plasmid containing the target fragment (2 μL), and LR Clonase™ II Enzyme (2 μL). In one embodiment, after the components of the LR reaction system of the present invention are mixed, the mixture is incubated at 25°C for 1 h, and then 1 μL of proteinase K is added to terminate the reaction. In one embodiment, the present invention uses vector primers I3 and I5 in combination with target gene primers StTRG-RNAi-For (SEQ ID NO.5) and StTRG-RNAi-Rev (SEQ ID NO.6) to perform positive identification of the RNAi silencing vector; the nucleotide sequence of the vector primer I3 is shown in SEQ ID NO.9; the nucleotide sequence of the vector primer I5 is shown in SEQ ID NO.10.

[0052] This invention also provides the application of the potato StTRG gene or the RNAi silencing vector described in the above-mentioned technical solutions in improving potato yield and / or cultivating high-yield potatoes.

[0053] In one embodiment, the increased potato yield described in this invention is achieved by increasing the number of potato tubers. In another embodiment, the RNAi silencing vector described in this invention can reduce the expression level of the StTRG gene and significantly increase the number of potato tubers.

[0054] This invention also provides the application of the potato StTRG gene or the RNAi silencing vector described in the above-mentioned technical solutions in promoting early tuber formation and / or cultivating early-maturing potatoes.

[0055] In one embodiment, the method of promoting early tuber formation in potatoes according to the present invention includes promoting early flowering in potatoes. In another embodiment, the RNAi silencing vector of the present invention can reduce the expression level of the StTRG gene and cause potato plants to flower earlier. In yet another embodiment, the RNAi silencing vector of the present invention can also promote early tuber formation in potatoes.

[0056] The present invention also provides a method for increasing potato yield and / or promoting early tuber formation in potatoes, the method comprising the following steps:

[0057] The expression level of the StTRG gene is reduced using the RNAi silencing vector described in the above technical solution.

[0058] This invention utilizes the RNAi silencing vector described in the above-mentioned technical solution to reduce the expression level of the StTRG gene, thereby increasing potato yield and / or promoting early tuber formation. As one embodiment, the RNAi silencing vector of this invention can achieve genetic transformation of potatoes via Agrobacterium-mediated transformation. As one embodiment, the preparation of the Agrobacterium infection solution for the Agrobacterium-mediated transformation of this invention includes the following steps: Agrobacterium carrying the StTRG-RNAi vector with the silenced fragment is cultured in LB liquid medium. When the bacterial concentration reaches an OD600 of 0.8–1.0, the bacterial solution is centrifuged for 10 min, the supernatant is discarded, and MS culture medium is added to adjust the bacterial concentration to 0.4–0.6. As one embodiment, the Agrobacterium-mediated genetic transformation process of the present invention is as follows: Under aseptic conditions, four-week-old Atlantic test-tube seedlings are cut into 0.5 cm long segments, immersed in the Agrobacterium infection solution for 15 min, removed, and excess liquid is blotted with filter paper. The segments are then placed in ZIG medium for dark incubation. Two days later, they are placed in ZIG medium containing kanamycin and termetidine resistance for light-protected culture to obtain the transformed stem segments. As one embodiment, the light-protected culture conditions of the present invention are: 16 h light / 8 h darkness, 21 °C, and 50%–60% humidity.

[0059] As one implementation method, after the transformed stem segments grow into differentiated seedlings of about 1 cm, the present invention identifies transgenic potatoes, including the following steps: the potato differentiated seedlings are transferred to MS medium and propagated after they have rooted; after secondary rooting, leaves are taken, genomic DNA is extracted using the plant CTAB method, and PCR positive transgenic plants are identified using primers M15F and StTRG-RNAi-Rev combination.

[0060] The present invention also provides a method for cultivating high-yield and / or early-maturing potatoes, the method comprising the following steps:

[0061] Potatoes with reduced StTRG gene expression levels as described in the above technical solution were used as parents for breeding.

[0062] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a potato StTRG gene, a StTRG-RNAi silencing vector, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] StTRG gene discovery and silencing vector construction

[0065] 1. RNA extraction and reverse transcription

[0066] Using laboratory-preserved Atlantic potato tissue culture seedlings as material, tender leaves from the top of the seedlings after four weeks of growth were taken and ground into powder with liquid nitrogen. Total RNA was extracted from the leaves using Trizol reagent (Tiangen Pharmaceuticals), following the reagent instructions. The reverse transcription process consisted of two steps: (1) and (2): System (1) reaction conditions: 42℃×2min, then placed at 4℃; System (2) reaction conditions: 37℃×15min, 85℃×5s. The reverse transcription system is as follows:

[0067] Table 1 Reverse transcription reaction system (1)

[0068]

[0069]

[0070] Table 2 Reverse transcription reaction system (2)

[0071] reagents Volume / μL Reaction solution from Step 1 10 5×PrimeScriptBuffer(FRealTime) 4 PrimeScriptRTEnzymeMix1 1 RTPrimerMix 1 RNase-freeH2O 4 Total 20

[0072] 2. Analysis of StTRG gene sequence and expression pattern

[0073] Gene sequence comparison analysis: The CDS sequence of the potato Atlantic StTRG gene was obtained as shown in SEQ ID NO:1. The sequence ID published in the Potato Genome Database PGSC (http: / / spuddb.uga.edu / index.shtml) is Soltu.DM.05G012120.1. The two sequences are completely identical.

[0074] Expression pattern analysis: Tissue-specific expression analysis was performed using transcriptome data from diploid potato RH (RH89-039-16) and DM (DM1-3) published by PGSC. The expression levels of the StTRG gene in potato DM and RH were obtained as follows: Figure 1 As shown, the StTRG gene was found to be specifically highly expressed in stolons, suggesting that it may be involved in stolon development and tuber initiation.

[0075] 3. Primer design

[0076] The StTRG sequence was input into the siRNA primer design websites Invitrogen Block-iT RNAiDesigner and siDirect. A fragment (296 bp in size) containing multiple siRNA regions and with conserved sequences among alleles was selected as the target sequence. The specific sequence is shown in SEQ ID NO.4. Primers for positive identification of StTRG silencing vector and detection of gene expression levels were also designed. The specific sequences are detailed in Table 3.

[0077] Table 3 Primer sequences

[0078]

[0079] 4. Construction of StTRG-RNAi silencing vector

[0080] Using the above cDNA as a template, the interference fragment sequence of StTRG (296 bp) was cloned. The upstream and downstream primers were StTRG-RNAi-For and StTRG-RNAi-Rev, respectively. The reaction system is shown in Tables 4 and 5. The entry vector and silencing vector were pENTR and pHELLSGATE 12, respectively.

[0081] The initial carrier construction refers to pENTR Directional According to the CloningKits vector instructions, mix the components in Table 3 and let stand for 5 minutes. Take 2 μL and transform competent E. coli DH5α, heat shock at 42℃ for 45 seconds, add 500 μL of LB medium for activation, then plate and incubate overnight at 37℃. Pick single colonies using pENTR. TM PCR verification was performed using universal primers M13For and M13Rev. Single clones with fragment sizes consistent with expectations were selected, and plasmids were extracted and sent for sequencing after amplification.

[0082] Table 4. Construction System of Introductory Carrier

[0083] Element Volume / μL Saltsolution 1 PCR products 0.5 pENTR vector 1 <![CDATA[ddH2O]]> 2.5

[0084] pENTR plasmids containing the non-mutated target sequence were selected for silencing vector construction. Silent vector construction was performed according to LRClonase. TM II. Enzyme Mix Kit Instructions: Mix the components in Table 4 thoroughly, incubate at 25°C for 1 hour, then add 1 μL of proteinase K to terminate the reaction. The transformation and detection methods for *E. coli* are the same as above. Amplify positive colonies and extract plasmids. Gently mix 2 μL of plasmid with 100 μL of *Agrobacterium* competent cells GV3101-P19, transfer to a 2 mm electrode cup, and transform using electroporation at 2500 V and 2.6 ms. After transformation, add 500 μL of LB medium and incubate at 28°C with a shaker at 180 rpm for 2 hours. Spread the bacterial culture on LB medium containing Kan and Rif resistance and incubate at 28°C for 2 days. Use vector primers I3 and I5, combined with target gene primers StTRG-RNAi-For and StTRG-RNAi-Rev, respectively. PCR screening of positive single clones is performed for sequencing to confirm the insertion of the target sequence.

[0085] Table 5 LR reaction system

[0086] Element Volume / μL TEBuffer 5 pHellsGate12 1 pENTR plasmid with target fragment 2 LRClonaseTMIIEnzyme 2 <![CDATA[H20]]> to10

[0087] Example 2

[0088] Agrobacterium-mediated potato genetic transformation

[0089] 1. Preparation of Agrobacterium infection solution

[0090] Agrobacterium carrying the StTRG-RNAi vector with the silencing fragment was cultured in LB liquid medium. When the bacterial concentration reached OD600 of 0.8-1.0, the bacterial culture was centrifuged for 10 min, the supernatant was discarded, and MS culture medium was added to adjust the bacterial concentration to 0.4-0.6.

[0091] 2. Agrobacterium genetic transformation

[0092] Under aseptic conditions, the stem segments of four-week-old Atlantic test-tube seedlings were cut into 0.5cm long segments, soaked in the above bacterial solution for 15 minutes, removed and dried with filter paper, and placed in ZIG medium for dark incubation. Two days later, they were placed in ZIG medium containing kanamycin and termethin resistance and cultured in the dark under the following conditions: 16h light / 8h dark, 21℃, and 50%–60% humidity.

[0093] 3. Identification of transgenic plants

[0094] After the transformed stem segments have grown to about 1 cm in length, they are transferred to MS medium and propagated after rooting. Leaves are harvested after secondary rooting, and genomic DNA is extracted using the plant CTAB method. PCR-positive transgenic plants are then identified using primers M15F and StTRG-RNAi-Rev.

[0095] 4. Identification of silencing efficiency in transgenic plants

[0096] RNA was extracted from the leaves of the above-mentioned positive transgenic plants and reverse transcribed into cDNA (method as in Example 1). Quantitative PCR was used to detect the expression level of the StTRG gene. Primers were q-StTRG-For and q-StTRG-Rev (sequence information detailed in Table 3). Reaction conditions were: 95℃ for 30s, 1 cycle; 95℃ for 5s, 60℃ for 34s, 40 cycles; 95℃ for 15s, 60℃ for 1min, 95℃ for 15s. The reaction system is shown in Table 6. 2 -ΔΔCT The calculation method was used to analyze the changes in StTRG expression levels in control and transgenic plants. Three transgenic lines with a StTRG gene expression level decrease of more than 50% were selected: RNAi-9, RNAi-11, and RNAi-13. The StTRG gene expression levels of these three StTRG silencing lines relative to the WT control group were measured as follows: Figure 9 As shown, the silencing efficiencies of the three StTRG silencing lines were 62%, 71%, and 80%, respectively.

[0097] Table 6 RT-qPCR reaction system

[0098] Element Volume / μL TBGreenExTaqⅡ(2×) 10.0 PrimerFor(10μM) 0.8 PrimerRev (10μM) 0.8 cDNA 1.0 ROXReferenceDyeⅡ(50×) 0.4 <![CDATA[ddH2O]]> 7.0

[0099] 5. Phenotypic evaluation of transgenic plants

[0100] Three StTRG silent lines (RNAi-9, RNAi-11, and RNAi-13) were selected for propagation and phenotypic evaluation. They were grown in an artificial climate chamber under long-day conditions: 16 hours of light at 24°C and 8 hours of darkness at 20°C. After 3 weeks of long-day growth, the transgenic test-tube seedlings were transplanted into pots. Tuber formation and flowering phenotypes were investigated at 4 weeks, 7 weeks, and at harvest (15 weeks). Tuber initiation time was investigated, primarily based on whether the stolons swelled or formed tubers, and the percentage of each plant type was statistically analyzed. Flowering time was determined by the opening of the first flower on each plant, and the flowering percentage of each line was statistically analyzed.

[0101] After 4 weeks of growth, the tuber formation initiation status of the control plants and three StTRG silent lines (4 weeks) was obtained as follows: Figure 2 As shown (red arrows represent enlarged stolons or tubers), the statistical results of the percentage of tuber formation in StTRG silent lines are as follows: Figure 3 As shown in the figure, only a few runners in the control plants swelled and initiated tuber formation, while most of the three StTRG-silenced lines initiated tuber formation. The percentage of plants with stolon swelling and tuber formation was calculated; in the control, less than 20% of plants initiated tuber formation by runners, while in the three StTRG-silenced lines, over 80% of plants initiated tuber formation by runners. These results indicate that reducing StTRG expression can promote early tuber formation.

[0102] After 7 weeks of growth, the number of tubers per plant was obtained from the control plant and three StTRG silent lines (7 weeks). Figure 4 As shown, the statistical results of tuber formation in the StTRG silent lines are as follows: Figure 5 As shown.

[0103] The control plants had an average of 2.8 tubers per plant, while the three StTRG silent lines (RNAi-9, RNAi-11, and RNAi-13) had averages of 8.5, 5.6, and 7.6 tubers per plant, respectively. The tuber count was more than two times that of the control, and the differences were highly significant in a t-test. After 15 weeks of potato growth, the number of tubers per plant was counted.

[0104] The tuber formation phenotype and statistical results of the StTRG silent lines are as follows: Figure 6 and Figure 7As shown in the figure, the average number of tubers per plant in the control plants was 5.8, while the average number of tubers per plant in the three StTRG silencing lines (RNAi-9, RNAi-11, and RNAi-13) was 12.0, 8.3, and 9.5, respectively. The number of tubers in the StTRG silencing lines was significantly increased compared to the control. These results indicate that reducing StTRG expression can significantly increase the number of tubers.

[0105] In addition, the percentage of flowering plants was recorded and analyzed between 47 and 59 days after transplanting. At 47 days, 3.3% of the plants in the control group were flowering, while the percentages of flowering plants in the three StTRG silent lines RNAi-9, RNAi-11, and RNAi-13 were 15.8%, 9.5%, and 21.1%, respectively. At 55 days, 100% of the plants in RNAi-11 and RNAi-13 were flowering, 89.5% of the plants in RNAi-9 were flowering, while only 73.3% of the plants in the control group were flowering (e.g., ...). Figure 8 (As shown in the figure). Based on the calculation of flowering time with 75% of plants flowering, the StTRG-silenced lines flowered 2-4 days earlier than the control, indicating that reducing StTRG expression can promote earlier flowering.

[0106] As can be seen from the above, the StTRG-RNAi silencing vector described in this invention can significantly reduce the expression level of the StTRG gene and promote early tuber formation and increase the number of tubers in potatoes, mainly manifested in early flowering, early tuber formation, and increased tuber number.

[0107] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A potato StTRG gene, characterized in that, The nucleotide sequence of CDS includes at least one of the following: ①The nucleotide sequence shown in SEQ ID NO.1; ②The nucleotide sequence shown in SEQ ID NO.2; ③The nucleotide sequence shown in SEQ ID NO.3; ④ A nucleotide sequence that has at least 98% identity with any of the sequences in ① to ③.

2. The RNAi silencing vector for the potato StTRG gene as described in claim 1, characterized in that, The RNAi silencing vector contains an interfering fragment sequence of StTRG as shown in SEQ ID NO.

4.

3. The RNAi silencing vector according to claim 2, characterized in that, The introductory vector for constructing the RNAi silencing vector includes pENTR; the backbone vector for constructing the RNAi silencing vector includes pHELLSGATE 12.

4. The RNAi silencing vector according to claim 2, characterized in that, The nucleotide sequence of the upstream primer for constructing the RNAi silencing vector is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

6.

5. The application of the potato StTRG gene of claim 1 or the RNAi silencing vector of any one of claims 2 to 4 in increasing potato yield and / or cultivating high-yield potatoes.

6. The application according to claim 5, characterized in that, The increase in potato yield is achieved by increasing the number of tubers formed in the potatoes.

7. The use of the potato StTRG gene of claim 1 or the RNAi silencing vector of any one of claims 2 to 4 in promoting early tuber formation in potatoes and / or cultivating early-maturing potatoes.

8. The application according to claim 7, characterized in that, Promoting early potato tuber formation includes promoting early flowering of potatoes.

9. A method for increasing potato yield and / or promoting early tuber formation in potatoes, characterized in that, The method includes the following steps: The expression level of the StTRG gene is reduced using the RNAi silencing vector according to any one of claims 2 to 4.

10. A method for cultivating high-yielding and / or early-maturing potatoes, characterized in that, The method includes the following steps: Potatoes with reduced StTRG gene expression levels as described in claim 9 were used as parents for breeding.

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