A functional gene LrRNaseH for regulating the transformation of black wolfberry thorns into side branches and its application

Abstract

The present invention discloses a functional gene, LrRNaseH, for regulating the softening of thorns transformed into side branches or thorn tips of black wolfberry and its application. Specifically, the functional gene LrRNaseH of black wolfberry has a nucleotide sequence as shown in SEQ ID No: 3, or encodes an amino acid sequence as shown in SEQ ID No: 4, which can regulate the softening of thorns transformed into side branches or thorn tips of black wolfberry.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology. Specifically, the present invention relates to the use of a ruthenic wolfberry LrRNaseH gene and its encoded protein in inhibiting the occurrence of thorns on ruthenic wolfberry branches and promoting the growth of stems and leaves. Background Art

[0002] Lycium ruthenicum (Lycium ruthenicum Murr) is a perennial shrub of the Solanaceae family, growing approximately 20 to 50 cm tall. It is primarily distributed in desert regions of Ningxia, Qinghai, Gansu, and Xinjiang, where it grows in patches and thickets. It is drought-tolerant and infertile, making it a dominant and dominant species in the desert communities of the Ejina region of the Heihe River Basin, playing a vital role in soil and water conservation, windbreak, and sand fixation. The fruit of Lycium ruthenicum Murr contains a rich supply of active ingredients, including amino acids, anthocyanins, polysaccharides, and polyphenols. Proanthocyanidins are the highest content in the fruit, resulting in high medicinal value and a common remedy in Uyghur and Tibetan medicine. As the ecological and economic value of Lycium ruthenicum has gradually gained recognition, research into its genetic function has intensified. Current research on Lycium ruthenicum focuses on its nutrient content, medicinal components, pigment and polysaccharide extraction techniques, and pharmacological analysis. Studies have shown that its fruit components possess anti-radiation, intestinal microbiome regulation, antioxidant, anti-cancer, anti-fatigue, immune-enhancing, and anti-aging properties. Furthermore, recent studies have found that Lycium ruthenicum polyphenols can delay the onset and progression of oxidative stress-related neurodegenerative diseases, and that Lycium ruthenicum polysaccharides exert neuroprotective effects against oxygen-glucose deprivation / reoxygenation-induced damage in primary rat cortical neurons. Lycium ruthenicum is also known as the "king of natural proanthocyanidins." Lycium ruthenicum pigment is non-toxic and food-safe. Its strong coloring power, excellent stability, and simple processing make it widely applicable in the pharmaceutical, food, and textile industries. In summary, Lycium ruthenicum is an important ecological and economical shrub with broad prospects for development and application.

[0003] The thorns of black wolfberry are branch thorns. Black wolfberry itself has high economic value. However, due to the dense thorns on the branches of wild or cultivated black wolfberry in arid soil conditions and the extremely thin peel of the fruit, the formation of these thorns during manual picking significantly reduces picking efficiency and increases picking costs, ultimately leading to an extremely high market price for black wolfberry. Cultivating thornless black wolfberry varieties is feasible, and thornless varieties should be easier to cultivate as economic forest trees. Therefore, cultivating high-quality thornless black wolfberries is of great significance.

[0004] Based on this, developing a method for inhibiting the occurrence of black wolfberry branch thorns while promoting the growth of stems and leaves is a technical problem that needs to be solved urgently. Summary of the Invention

[0005] In view of this, the present invention provides a functional gene LrRNaseH for regulating the formation of thorns of black wolfberry, characterized in that the nucleotide sequence of the open reading frame of the functional gene is shown in SEQ ID No: 3.

[0006] In one embodiment, the amino acid sequence encoded by the open reading frame of the functional gene is shown in SEQ ID NO:4.

[0007] It should be understood that, taking into account codon degeneracy, modifications to the base sequence of the cDNA reading frame without altering the amino acid sequence also fall within the scope of the present invention. Substitutions, deletions, additions of one or more amino acids, or terminal modifications to the amino acid sequence of the LrRNaseH protein from L. ruthenica also fall within the scope of the present invention without affecting the structure and activity of the LrRNaseH protein.

[0008] On the other hand, the present invention also provides an application of the functional gene LrRNaseH in the genetic breeding of Lycium ruthenicum.

[0009] On the other hand, the present invention also provides a gene silencing system of Lycium barbarum based on the functional gene LrRNaseH, characterized in that it includes a recombinant vector; wherein the recombinant vector contains a target gene fragment, and the target gene fragment is a specific fragment of the above-mentioned functional gene LrRNaseH.

[0010] On the other hand, the present invention also provides a method for constructing a gene silencing system of Lycium ruthenicum based on the functional gene LrRNaseH, characterized in that it comprises the following steps:

[0011] S1. Extract RNA from leaves and tender stems of Lycium ruthenicum and reverse transcribe it into Lycium ruthenicum cDNA;

[0012] S2. Using a set of specific primer pairs, PCR amplification of the cDNA fragment of the black wolfberry fruit was performed, and the specific fragment of the functional gene LrRNaseH was purified;

[0013] S3, using two sets of specific primer pairs to perform PCR amplification on the functional gene LrRNaseH specific fragment in step S2, and recombinantly ligating the amplified products with the vector to obtain a recombinant vector containing the target gene;

[0014] S4, transforming the recombinant vector in step S3 into Agrobacterium competent cells to obtain a bacterial solution containing the recombinant vector;

[0015] S5. Co-culture the bacterial solution containing the recombinant vector in step S4 with the leaf tips of Lycium ruthenicum to screen and obtain transgenic plants in which the functional gene LrRNaseH is silenced.

[0016] In one embodiment, the specific primer pair in step S2 is primer 1 and primer 2, the nucleotide sequence of primer 1 is shown in SEQ ID NO: 1, and the nucleotide sequence of primer 2 is shown in SEQ ID NO: 2.

[0017] In one embodiment, the two sets of specific primer pairs in step S3 are primer 3 and primer 4, and primer 5 and primer 6, the nucleotide sequence of primer 3 is shown in SEQ ID NO: 5, the nucleotide sequence of primer 4 is shown in SEQ ID NO: 6, the nucleotide sequence of primer 5 is shown in SEQ ID NO: 7, and the nucleotide sequence of primer 6 is shown in SEQ ID NO: 8.

[0018] In one embodiment, the reaction procedure of PCR amplification in step S2 is: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 58.3°C for 30 seconds, extension at 72°C for 1 minute, and 34 cycles are set; the reaction procedure of PCR amplification in step S3 is: pre-denaturation at 98°C for 1 minute; denaturation at 98°C for 10 seconds, annealing at 53.5°C for 30 seconds, extension at 72°C for 10 seconds, and 34 cycles are set.

[0019] On the other hand, the present invention also provides a method for softening the thorns of black wolfberry by transforming them into side branches or thorn tips, comprising reducing the expression level of the above-mentioned functional gene LrRNaseH in the target black wolfberry, thereby promoting the transformation of the black wolfberry thorns into side branches or the forking of the thorn tips to weaken the thorn tips.

[0020] In one embodiment, the functional gene LrRNaseH in the target Lycium ruthenicum is silenced by RNAi to achieve a reduction in the expression level of the functional gene LrRNaseH.

[0021] The present invention provides a LrRNaseH-based gene silencing system and construction method for Lycium ruthenicum, which can be used to verify the function of the LrRNaseH gene in Lycium ruthenicum. This system has application value in the study of genes related to thorn formation in Lycium ruthenicum. It is also of great significance for studying the transformation of thorns into lateral branches and the softening of thorn tips. It not only improves the branching ability of Lycium ruthenicum, but also helps solve the picking problem caused by sharp thorns during production, laying the foundation for the next step of functional genomics and gene function research in Lycium ruthenicum. This method can also be used to cultivate new varieties of Lycium ruthenicum. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without making any creative efforts.

[0023] Figure 1 This is the electropherogram of PCR verification of pMD18-T ligation products.

[0024] Figure 2 This is the electrophoresis diagram of the forward and reverse fragments of pRNAi-LrRNaseH PCR.

[0025] Figure 3 is the relative expression level of LrRNaseH in Lycium ruthenicum mutants and WT plants.

[0026] Figure 4 This is the "thorn" of the black-fruited wolfberry mutant plant 45 days after transplantation.

[0027] Figure 5 These are the thorns of the WT plant of Lycium ruthenicum at 45 days after transplantation.

[0028] Figure 6 This is the "thorn" of the black-fruited wolfberry mutant plant 52 days after transplantation.

[0029] Figure 7 These are the thorns of the WT plant of Lycium ruthenicum at 52 days after transplantation.

[0030] Detailed Description of the Invention

[0031] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] the term

[0033] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0034] As used herein, the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0035] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0036] It should be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably and should generally be understood to be open-ended and non-restrictive, e.g., not excluding other unrecited elements or steps. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail. Regarding the "comprising", "including", "having", "containing" and the like used herein, they are all open terms, i.e., meaning including but not limited to.

[0038] The reagents, methods, and equipment used in the present invention are conventional in the art. Unless otherwise specified, all reagents used in this example are commercially available. The quantitative tests in the present invention were performed in triplicate, and the results were averaged.

[0039] The reagents used in the following examples include TIANGEN plant tissue RNA rapid extraction kit, Vazyme Hi Script III RT Super Mix for qPCR (gDNAwiper), Takara pMD TM 18-T Vector Cloning Kit, Kangwei Century Company 2×ES Taq Master Mix (Dye), 2×Taq Master Mix (Dye), Super Pfx DNA Polymerase (Dye), Plus recombinase, Kangwei Century Agarose Gel Rapid DNA Recovery Kit, Kangwei Century Plant Genomic DNA Extraction Kit, Novozymes ChamQ Universal qPCR SYBR Green Master Mix, Lamblitolide Kpn I, EcoRI, XbaI, and SmaI endonucleases. Ampicillin (Amp), rifampicin (Rif), ceftriaxone sodium (Cef), acetosyringone (AS), and kanamycin (Kan).

[0040] The RNAi expression vector pRNAi-E in the following examples is recorded in the document “Song Mengru, Chen Keqin, Guo Yunna, et al. Construction of a new RNAi vector for plant gene silencing [J]. 2017(6).”

[0041] The culture medium and its components used in the following examples are shown in the following table:

[0042]

[0043]

[0044] Example 1 Obtaining the CDS sequence of the LrRNaseH gene of Lycium ruthenicum

[0045] Prior art CN114303766A indicates that the exogenous auxin IAA significantly inhibits thorn formation, while treatment with the auxin inhibitor PCIB significantly increases the rate of thorn formation in thornless branches. Therefore, four samples were selected for RNA-Seq transcriptome analysis: naturally thorny terminal buds (ThoCK), naturally thornless terminal buds (TleCK), terminal buds transformed from thorny to thornless after IAA treatment (TleIAA), and terminal buds transformed from thornless to thorny after PCIB treatment (ThoPCI).

[0046] The results showed that 457 differentially expressed genes were shared across the three comparison groups (ThoCK vs. TleCK, ThoCK vs. TleIAA, and TleCK vs. ThoPCI). Among these 457 genes, the gene implicated in this study shared the highest homology with the putative ribonuclease H gene from Arabidopsis thaliana, as determined by protein family analysis (PFAM). Therefore, the gene in Lycium ruthenicum was tentatively designated LrRNaseH. This gene was significantly upregulated in the terminal buds of thornless branches under exogenous IAA treatment.

[0047] RNA was extracted from leaves and tender stems of Lycium ruthenicum clones and reverse transcribed into cDNA. Cloning primers containing the CDS sequence for LrRNaseH were designed based on the transcript sequence. The primer sequences are as follows:

[0048] T-LrRNaseH-F CAGCTAAGCGATGGTCA SEQ ID NO: 1 T-LrRNaseH-R TGTAGTAGGAAGCGTGCC SEQ ID NO: 2

[0049] Using Kangwei Century's 2× ES Taq MasterMix (Dye) as the premixed enzyme and dye, a PCR premix was prepared using cDNA from a sample of Lycium ruthenicum strain 11 as the template and T-LrRNaseH-F and T-LrRNaseH-R as primers: 12.5 μl of 2× ES Taq MasterMix (Dye), 1 μl of each upstream and downstream primer, 1 μl of cDNA, and 9.5 μl of ddH2O. The PCR reaction program was as follows: 94°C denaturation for 2 min; 34 cycles of denaturation at 94°C for 30 s, annealing at 58.3°C for 30 s, and extension at 72°C for 1 min; 5 min of extension at 72°C; and storage at 16°C.

[0050] The PCR amplification products were separated by electrophoresis in 1.5% agarose gel. After separation, the agarose gel DNA rapid recovery kit provided by Kangwei Century Company was used to extract the PCR amplified LrRNaseH transcript fragment according to the instructions. TM 18-T Vector Cloning Kit was used for TA cloning. The T vector and the obtained gene CDS were connected at 16°C overnight according to the instructions. The PCR verification results of the connection product were as follows. Figure 1 , LrRNaseH gene CDS fragment sequencing results:

[0051] ATGGAGACCATTGCAAAATTTCATGAAACTGGAGAGTTCGAGAAAAGTCTTAATGCATCTTTCATCACTATTGTGCCGAAGAAGGAAAGGGTGATGAGCATCAAAGATTACAGGCCTATTAGTCTGATGGGTAGCATCTTTAAGATTATCTCGAAGGTGCTTTCTAATAGACTAAAAAAGGTCTTGGATGAGACAGTATCCTCCTCTCAAAATGCTTTTGTGGAAGGAAGACAGATCCTCGATGCAGCTTTAGTGGCGAATGAAGTGGTTGACTCTAGAAAGAAGAAAGGAGAGCCTGGAATTTTATGTAAGTTGGATCTGGAGAAAGCTTATGATCATGTGAATTGGGACTTTCTAGACTTTACAATGCAAAAGATGGGGTTTGGGGACAAGTGGAGGAAATGGATGAATGCTTGCATCTCTACTGCCACGTTTTCTGTTTTGGTGAATGGGAGTCCTTGCGGTTTCTTTGGGAGCTCGAGGGGGTTAAGACAGGGGGATCCTTTATCCCCTATGCTTTTCATTCTTGTTATAGAAGCATTGAGCAAAATGATTGATAGGGCAGTCACTGGAGGCTTTTTGAAGGGTTTTGATGTTTCATTCAGAGGACACAGTAGTGTGAGGTTTCACATCTCTTATTTGGCGGATGATACCTTGGTATTTTGTGATGCTGAGGTCTCCCCGGTAGATTACTTGAGACAGATACTTATTTGGTTTCAGGTTGTGTCAGGCCTCACGATCAATCTTAGGAAATGTGAAATCACTCCGGTAGGAGAAGTGGTTAATATGGATGAGATTGCACAAGTGTTGAATTGGATGAGTCACTGGAGGCTTTTTGAAGGGTTTTGA(SEQ ID NO:3)

[0052] The translated amino acid sequence is as follows:

[0053] METIAKFHETGEFEKSLNASFITIVPKKERVMSIKDYRPISLMGSIFKIISKVLSNRLKKVLDETVSSSQNAFVEGRQILDAALVANEVVDSRKKKGEPGILCKLDLEKAYDHVNWDFLDFTMQKMGFGDKWRKWMNACISTA TFSVLVNGSPCGFFGSSRGLRQGDPLSPMLFILVIEALSKMIDRAVTGGFLKGFDVSFRGHSSVRFHISYLADDTLVFCDAEVSPVDYLRQILIWFQVVSGLTINLRKCEITPVGEVVNMDEIAQVLNWMSHWRLFEGF(SEQ ID NO:4)

[0054] Example 2 Construction of recombinant RNAi expression vector

[0055] Select two sets of restriction enzyme cleavage sites on the RNAi expression vector pRNAi-E: Kpn I and EcoR I (antisense strand); Xba I and Sma I (sense strand). Design forward and reverse primer homology arms based on the upstream and downstream sequences of the restriction enzyme cleavage sites, and design upstream and downstream amplification primers based on the selected siRNA target sequence. The siRNA target sequence homologous recombination primers are as follows:

[0056]

[0057] The LrRNaseH gene TA cloning vector was diluted to 100 ng / μl as a template, and 12.5 μl of 2× Super Pfx Master Mix (Dye) was added, along with 1.25 μl of each upstream and downstream primers in a total volume of 25 μl. The upstream and downstream primers were the two sets of RNAi-1-LrRNaseH-F and RNAi-1-LrRNaseH-R, and RNAi-2-LrRNaseH-F and RNAi-2-LrRNaseH-R, respectively, as listed in the table above. The PCR reaction procedure was as follows: 98°C denaturation for 1 min; 34 cycles of denaturation at 98°C for 10 s, annealing at 53.5°C for 30 s, and extension at 72°C for 10 s; extension at 72°C for 5 min; and storage at 16°C.

[0058] The pRNAi-E original plasmid and the gel-recovered LrRNaseH gene antisense fragment were digested with restriction endonucleases Kpn I and EcoRI. Simultaneous digestion was performed using a PCR instrument at 37°C for 1.5-3 hours. Ligation was performed using the Nearshore Protein Plus OneStep PCR Cloning Kit, using a vector:fragment ratio of 1:2 and a total vector volume of 200 ng. Ligation was performed at 50°C for 15 minutes.

[0059] After the reaction is completed, the recombinant plasmid is transformed into E. coli and the bacterial solution is used as a template to perform PCR experiments. After confirming that the antisense chain is successfully connected, the sense chain is connected in the same way to complete the RNAi vector construction. The PCR verification results of the sense chain and antisense chain connection products are shown in Figure 2 .

[0060] Example 3 Construction of a mutant that inhibits the LrRNaseH gene

[0061] The asexual tissue culture seedlings of the black-fruited wolfberry plantlet propagated from the terminal bud for 45 days were selected. The tips of the leaves (1-1.5 cm) were cut as explants in a clean bench and inoculated on MS solid culture medium without plant growth regulators and antibiotics. The plants were pre-cultured for 5 days in a tissue culture room with a photoperiod of 12 h light / 12 h dark and a temperature of 25±2°C.

[0062] The constructed RNAi vector was transformed into GV3101 competent Agrobacterium cells and plated onto solid culture plates containing 100 mg / mL rifampicin and 100 mg / mL YEP. The plates were then incubated upside down at 28°C for 48 hours. After incubation, single colonies with smooth, rounded edges were selected and inoculated into 1 mL of YEP liquid culture medium containing 100 mg / mL rifampicin and 100 mg / mL Kan. The culture was shaken at 28°C and 220 rpm for 12 to 15 hours until the culture became turbid. Finally, the culture was transferred to 50 mL of YEP liquid culture medium containing the appropriate antibiotic and continued to be shaken for 12 to 15 hours under the same conditions to obtain the culture medium for infection.

[0063] The leaf tips of the black wolfberry clones, pre-cultured 5 days in advance, were immersed in the prepared infection solution under a sterile environment and manually shaken for 10 minutes. The tips were then removed and the bacterial solution was blotted dry with sterile filter paper. The leaf tips were then inoculated onto MS solid medium supplemented with AS. The co-culture was carried out in a tissue culture room under the same conditions as above for 48 hours in the dark. The explants that had sprouted were cultured individually until they formed complete plants.

[0064] Example 4 Verification of transgenic plants

[0065] RNA was extracted from leaves of the Lycium ruthenicum mutant tissue culture seedlings cultured in Example 3 and leaves of an untreated control group of Lycium ruthenicum tissue culture seedlings of the same age and reverse transcribed into cDNA.

[0066] Specific quantitative primers were designed based on the CDS sequence of LrRNaseH, and real-time fluorescence quantitative PCR experiments were performed using GAPDH as the internal reference primer. The quantitative primers used were designed as follows:

[0067] DL-LrRNaseH-F: 5′-CAGTATCCCTCCTCTCAAAATGCTT-3′ (SEQ ID NO: 9)

[0068] DL-LrRNaseH-R: 5′-GCTTTCTCCAGATCCAACTTACAT-3′ (SEQ ID NO: 10)

[0069] The internal reference primers used are as follows:

[0070] GAPDH-F: 5′-TTCAATCGTCCGTCTTCG-3′ (SEQ ID NO: 11)

[0071] GAPDH-R: 5′-TACCAACCCTTGTCTTCC-3′ (SEQ ID NO: 12)

[0072] qPCR was performed using ChamQ Universal qPCR SYBR Green Master Mix, with 40 cycles of pre-denaturation at 95°C for 30s and annealing at 60°C for 30s. -△△Ct The relative expression level of LrRNaseH in the leaves of RNAi-resistant seedlings was obtained by analysis and calculation. Figure 3 As shown in Figure 3, the expression level of LrRNaseH was significantly downregulated.

[0073] Example 5 Observation of RNAi plant phenotype

[0074] The terminal buds and stem segments of RNAi plants with significantly downregulated expression were cut with sterile scissors and tweezers and inserted into sterile 1 / 2 MS medium supplemented with 300 mg / L Cef and 5 mg / L Kan. After new terminal buds emerged, they were propagated again. Clonal tissue culture seedlings were propagated using the same method and medium as a control. After 45 days of propagation, the RNAi and clonal tissue culture seedlings were removed from the culture room and acclimated under natural light for 7 days before transplanting.

[0075] In the LrRNaseH gene RNAi plants of black wolfberry 45 days after transplantation, the phenotypic change of thorns gradually transforming into side branches was observed. In contrast, the thorns of the untransformed wild-type (WT) black wolfberry asexual line plants were more densely packed, such as Figure 4-5 As shown in Figure 5. 52 days after transplantation, the phenotypic changes of the LrRNaseH gene RNAi plants were more significant. In contrast, the spines of the WT plants remained in their original state, as shown in Figure 5. Figure 6-7 shown.

[0076] Finally, it should be noted that the above-mentioned more specific embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above-mentioned embodiments. It should be pointed out that for those skilled in the relevant fields, several variations and improvements can be made without departing from the conceptual framework of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims, and those modifications or equivalent replacements of the technical solution of the present invention, without departing from the purpose and scope of the technical solution, shall be included in the scope of the claims of the present invention.

Claims

1. A functional gene LrRNaseH for regulating the formation of thorns of Lycium ruthenicum, characterized in that: The amino acid sequence encoded by the open reading frame of the functional gene is shown in SEQ ID NO:

4.

2. The functional gene LrRNaseH according to claim 1, characterized in that The nucleotide sequence of the open reading frame of the functional gene is shown in SEQ ID No:

3.

3. Use of the functional gene LrRNaseH as claimed in claim 1 or 2 in the genetic breeding of Lycium barbarum.

4. A method for constructing a gene silencing system of Lycium ruthenicum based on the functional gene LrRNaseH, characterized in that: The steps include: S1. Extract RNA from leaves and tender stems of Lycium ruthenicum and reverse transcribe into cDNA; S2. Using a set of specific primer pairs, PCR amplification of cDNA fragments from leaves and tender stems of Lycium ruthenicum was performed, and the specific fragment of the functional gene LrRNaseH was purified; S3, using two sets of specific primer pairs to perform PCR amplification on the functional gene LrRNaseH specific fragment in step S2, and recombinantly ligating the amplified products with the pRNAi-E vector to obtain a recombinant vector containing the target gene; S4, transforming the recombinant vector in step S3 into Agrobacterium competent cells to obtain a bacterial solution containing the recombinant vector; S5. Co-culturing the bacterial solution containing the recombinant vector in step S4 with the leaf tips of Lycium ruthenicum to screen for transgenic plants in which the functional gene LrRNaseH is silenced; Wherein, the specific primer pair in step S2 is primer 1 and primer 2, the nucleotide sequence of primer 1 is shown in SEQ ID NO: 1, and the nucleotide sequence of primer 2 is shown in SEQ ID NO: 2; Among them, the two sets of specific primer pairs in step S3 are primer 3 and primer 4, and primer 5 and primer 6, the nucleotide sequence of primer 3 is shown in SEQ ID NO: 5, the nucleotide sequence of primer 4 is shown in SEQ ID NO: 6, the nucleotide sequence of primer 5 is shown in SEQ ID NO: 7, and the nucleotide sequence of primer 6 is shown in SEQ ID NO:

8.

5. The construction method according to claim 4, characterized in that The reaction procedure of PCR amplification in step S2 is: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 58.3°C for 30 seconds, extension at 72°C for 1 minute, and 34 cycles; the reaction procedure of PCR amplification in step S3 is: pre-denaturation at 98°C for 1 minute, denaturation at 98°C for 10 seconds, annealing at 53.5°C for 30 seconds, extension at 72°C for 10 seconds, and 34 cycles.

6. A method for softening the thorns of black wolfberry by converting them into side branches or thorn tips, characterized in that: The method comprises reducing the expression level of the functional gene LrRNaseH as claimed in claim 2 in the target black wolfberry, thereby promoting the transformation of black wolfberry thorns into side branches or the forking of the thorn tops to weaken the thorn tips.

7. The method according to claim 6, characterized in that The functional gene LrRNaseH in the target black wolfberry is silenced by RNAi, so as to reduce the expression level of the functional gene LrRNaseH.

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