MayUCCA2, a gene that promotes adventitious root development, and its applications.

Abstract

This invention relates to a gene, MaYUCCA2, that promotes adventitious root development and its applications, belonging to the field of plant growth and development genetic engineering. The nucleotide sequence of the MaYUCCA2 gene is shown in Sequence 3; its amino acid sequence is shown in Sequence 4. This invention, by transferring MaYUCCA2 into *Populus alba*, produces transgenic poplar trees named MaYUCCA2-OE. Compared with non-transgenic poplar trees (WT), the results show that MaYUCCA2-OE has a significantly higher endogenous auxin content, and during asexual propagation tissue culture, adventitious roots are more easily formed and their number is significantly increased. Furthermore, the callus tissue of MaYUCCA2-OE transgenic poplar is more significantly enlarged and deeper red in color compared to WT, and it is more likely to induce the production of adventitious buds.

Description

Technical Field

[0001] This invention relates to a gene, MaYUCCA2, that promotes adventitious root development and its application, belonging to the field of plant growth and development genetic engineering. Background Technology

[0002] Adventitious roots are roots that originate from non-periosteal tissues such as plant stems or leaves. The occurrence of adventitious roots is not only a component of plant organ differentiation but also relates to asexual reproduction and the regeneration of complete plants. The occurrence of adventitious roots in most woody plants is difficult, thus hindering the application of asexual reproduction techniques, especially restricting the industrialized propagation of many fruit trees, forest trees, and woody flowering plants, as well as the breeding of superior varieties.

[0003] Mulberry trees can be used not only for silkworm breeding but also for processing mulberry leaves into tea and for landscaping. However, factors such as the difficulty of asexual propagation, the slow and difficult development of adventitious roots, and the difficulty in establishing a mulberry tissue culture seedling regeneration system have hindered the breeding, promotion, and demonstration application of superior mulberry varieties.

[0004] Poplar, as an important model species of woody plants, possesses a stable genetic transformation system and plays a crucial role in studying gene function in woody plants. Flavin monooxygenase (YUCCA) is a key enzyme in the auxin biosynthesis pathway. It catalyzes the conversion of indole-3-pyruvic acid (IPyA) into the auxin indole-3-acetic acid (IAA), thereby directly influencing the synthesis and accumulation of auxin in plants.

[0005] Research on mulberry genes that promote adventitious root development and their application in promoting adventitious root development in poplar trees is of great significance for solving the difficulties in asexual propagation of woody plants. Summary of the Invention

[0006] In view of the current situation that most superior woody plant germplasms are difficult to propagate and root, and the propagation is slow, this invention provides a gene MaYUCCA2 that promotes adventitious root development and its application.

[0007] The present invention adopts the following technical solution:

[0008] A gene that promotes the development of adventitious roots, MaYUCCA2, has the nucleotide sequence shown in Sequence 3 and the amino acid sequence shown in Sequence 4.

[0009] This invention also provides a method for cloning the gene MaYUCCA2, which promotes adventitious root development, comprising the following steps:

[0010] Step 1: Using the superior mulberry variety Lu Sang No. 1 (Morus alba L., provided by the Sericulture Research Institute of Shandong Academy of Agricultural Sciences) as experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China); cDNA of Lu Sang No. 1 was obtained by reverse transcription of RNA using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA, USA).

[0011] Step 2: Obtain the cDNA sequence of the target gene MayUCCA2 from the website (https: / / morus.biodb.org / ), and design primers using Oligo7 or Primer5 software; the MayUCCA2-F forward primer is shown in Sequence 1; the MayUCCA2-R reverse primer is shown in Sequence 2.

[0012] Step 3: Amplification of the target gene cDNA sequence;

[0013] Using the cDNA of Lusang No. 1 as a template, and amplification was performed using TaKaRa PrimeSTAR HS (premix) R040A high-fidelity enzyme in a 25μL reaction system and PCR reaction program, the full-length 1338bp cDNA sequence of the gene was finally obtained and named the MayUCCA2 gene.

[0014] Preferably, in step 2, the primer length is 15-25 bp, and the Tm value of the designed primer is suitable at 65℃, and a GATEWAY adapter is added to the primer.

[0015] Another objective of this invention is to provide a plant expression vector carrying the mulberry MayUCCA2 gene. This is achieved through the following technical solution:

[0016] Step 1: Construction of the introductory vector: Using the mulberry coding genome sequence as a template, the coding sequence of the MayUCCA2 gene was obtained by PCR amplification using specific PCR primers (MaYUCCA2-F and MayUCCA2-R), and then inserted into the multiple cloning site of the introductory vector PDNOR207 to obtain the constructed introductory vector. The sequence of the introductory vector PDNOR207 is shown in Sequence 5.

[0017] According to the reaction system, fresh PCR product, PDNOR207 entry vector and BP enzyme were added respectively. Then, the mixture was placed in a 25℃ metal bath and reacted for 4-5 hours to obtain the constructed entry vector. The mixture after the above reaction was transformed into E. coli and plated on LB solid selection medium (LB solid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 8g / L, gentamicin 50mg / L). Positive clones were picked from the selection medium for PCR detection and sequencing verification to confirm that the MayUCCA2 entry vector was successfully constructed.

[0018] Step 2: Expression vector construction: According to the reaction system, the constructed entry vector, PMDC32 expression vector and LR enzyme were added respectively, and then placed in a 25℃ metal bath for 4-5 hours. After the LR reaction, the mixture was transformed into E. coli and plated on LB solid selection medium (LB solid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 8g / L, kanamycin 50mg / L). Positive clones were picked from the selection medium for PCR detection and sequencing verification to confirm that the MayUCCA2 expression vector was successfully constructed and named MayUCCA2-OE.

[0019] The MayUCCA2 gene in the constructed MayUCCA2-OE vector has a strong 35S promoter assembled at its 5' end, enabling efficient expression of the MayUCCA2 protein in poplar. Simultaneously, the expression vector PMDC32 contains hygromycin phosphotransferase (HPT), serving as a selection marker for transgenic poplar, allowing for selection using hygromycin. It also contains LB and RB sequences, facilitating the integration of the 35S::MayUCCA2 expression framework and the selection marker gene HPT into the poplar chromosome, thus obtaining MayUCCA2-OE transgenic poplar.

[0020] Preferably, in step 2, the sequence of the plant expression vector PMDC32 is shown in sequence 6.

[0021] Another object of the present invention is to provide the application of the aforementioned gene Mayuca2, which promotes adventitious root development, in enhancing the adventitious root development of poplar trees. This is achieved through the following scheme:

[0022] The genetic transformation of MaYUCCA2 transgenic poplar trees was carried out through the following steps: The constructed MaYUCCA2-OE expression vector was transferred into Agrobacterium tumefaciena GV3101 by electroporation. Then, using Agrobacterium-mediated transformation, the 35S::MaYUCCA2 expression framework and the selection marker gene HPT were integrated into the chromosome of Populus alba x Populus glandulosa (84K poplar) to create MaYUCCA2 overexpression transgenic poplar trees (MaYUCCA2-OE).

[0023] Preferably, the specific transformation steps are as follows: One-month-old 84K poplar tissue culture seedlings are cultured at a temperature of 23-25℃ and a light intensity of 16 / 8h (day / night). Agrobacterium containing the MayUCCA2-OE expression vector is cultured in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, kanamycin 50mg / L, gentamicin 50mg / L, rifampin 20mg / L) until OD. 600When the pH value is 0.6–0.8, the leaf discs of 84K poplar are infected, and the infected leaves are transferred to a co-culture medium (2.2 g / L Murashige-Skoog (MS) + 0.5 g / L 4-Morpholineethanesulfonic acid (MES) + 20 g / L sucrose + 4 g / L plant gel + 100 μmol / L Acetosyringone (AS)) and co-cultured in the dark for 3–4 days. The co-cultured leaves are then transferred to an adventitious shoot induction medium (2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-Benzyl Aminopurine (6-BA) + 0.05 mg / L Naphthaleneacetic acid) containing 3 mg / L hygromycin B (Hyg) and 200 mg / L tim. Resistant adventitious shoots were induced and screened on a medium containing 3 mg / L Hydral-3-butyric acid (NAA) + 20 g / L sucrose + 4 g / L plant gel. After 20 days of induction culture, the resistant adventitious shoots were transferred to a rooting medium containing 3 mg / L Hydral-3-butyric acid (IBA) and 200 mg / L Tim (2.2 g / L MS + 0.5 g / L MES + 0.05 mg / L Indole-3-butyric acid (IBA) + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder) until adventitious roots were induced. DNA was extracted from the leaves of the rooted plants that had developed hygromycin resistance, and PCR verification was performed using the MayUCCA2 cloning primers. Finally, transgenic 84K poplar containing the MayUCCA2-OE vector sequence was obtained.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention creates a transgenic poplar tree, MaYUCCA2-OE, that overexpresses the key Arabidopsis thaliana auxin synthesis gene AtYUCCA2, a homolog of mulberry, MaYUCCA2. This resulted in transgenic poplar lines (OE1, OE2, and OE3) with significantly increased endogenous auxin content. Phenotypic analysis during asexual propagation revealed that adventitious root development in the MaYUCCA2-OE transgenic poplar was significantly earlier than in the non-transgenic WT poplar, and the number of adventitious roots in the later stages was significantly greater than in WT. Simultaneously, in callus induction experiments, the callus induced by the leaves of the MaYUCCA2-OE transgenic poplar was significantly greater than that WT. In conclusion, the MaYUCCA2-OE transgenic poplar promotes adventitious root development and callus formation in woody plants by increasing auxin synthesis and accumulation. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the MayUCCA2 overexpression vector constructed in this invention;

[0027] Figure 2 This invention relates to the identification and phenotypic analysis of the Mayu CCA2-OE transgenic poplar. A is a semi-quantitative electrophoresis diagram of the Mayu CCA2-OE transgenic poplar; B is a phenotypic diagram of WT and Mayu CCA2-OE transgenic poplar after multiple asexual propagations; C is an analysis diagram of auxin content at the shoot tips of WT and Mayu CCA2-OE transgenic poplar; D is a phenotypic diagram of WT and Mayu CCA2-OE transgenic poplar after one month of soil cultivation. The t-test p-value is indicated by asterisks: one asterisk indicates p < 0.05, and two asterisks indicate p < 0.01.

[0028] Figure 3 This invention provides a statistical analysis of the number of roots formed by WT and Mayu CCA2-OE transgenic poplar trees. A is a comparison chart of adventitious root formation in the rooting medium on days 11, 21, and 60 of the WT and Mayu CCA2-OE transgenic poplar trees. B is a bar chart showing the number of adventitious roots formed by WT and Mayu CCA2-OE transgenic poplar trees after 30 days of growth in the rooting medium. The t-test p-value is indicated by asterisks: one asterisk indicates p < 0.05, and two asterisks indicate p < 0.01.

[0029] Figure 4 Comparative figures of callus induction in leaves of WT and MayUCCA2-OE transgenic poplar (OE1, OE2 and OE3); where A represents day 0 and B represents day 60. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. It should be understood that the following text is only used to describe one or more specific embodiments of the present application and does not strictly limit the scope of protection specifically claimed in the present application. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0031] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0032] Example

[0033] 1. Cloning of the MaYUCCA2 gene

[0034] In view of the current situation that most superior woody plant germplasms are difficult to propagate and root, and the propagation is slow, this invention provides a gene that promotes the development of adventitious roots and a transgenic poplar with a significant increase in adventitious roots.

[0035] First, the main rate-limiting enzyme gene YUCCA in auxin synthesis was analyzed, and the homolog of the Arabidopsis thaliana AtYUCCA2 gene, MaYUCCA2, was selected for cloning; the specific steps are as follows:

[0036] (1) Using the mulberry variety Lu Sang No. 1 (Morus alba L., provided by the Sericulture Research Institute of Shandong Academy of Agricultural Sciences) as experimental material, RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China); cDNA was synthesized by reverse transcription of RNA using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA, USA);

[0037] (2) Obtain the cDNA sequence of the target gene MayUCCA2 through the mulberry genome website (https: / / morus.biodb.org / ). Use Oligo7 or Primer5 software to design primers with a length of 15-25 bp. At the same time, the Tm value of the designed primers is suitable at 65℃ (Gateway adapter is added to the primers).

[0038] The forward primer of MayUCCA2-F is shown in sequence 1; the reverse primer of MayUCCA2-R is shown in sequence 2.

[0039] (3) Using the cDNA of Lusang No. 1 as a template, and using TaKaRa Prime STAR HS (premix) R040A high-fidelity enzyme for amplification, the 25μL reaction system is shown in Table 1 below;

[0040] Table 1

[0041]

[0042] The PCR reaction procedure is shown in Table 2 below;

[0043] Table 2

[0044]

[0045] The final cDNA sequence of the gene, 1338 bp in length, was obtained and named the MayUCCA2 gene; its nucleotide sequence is shown in Sequence 3; the amino acid sequence of the MayUCCA2 gene is shown in Sequence 4.

[0046] 2. Construction of plant expression vector for the MaYUCCA2 gene

[0047] The method for constructing the plant expression vector of the MaYUCCA2 gene is as follows:

[0048] An overexpression vector for the MayUCCA2 gene was constructed using cloning technology. Specific PCR primers (MayUCCA2-F and MayUCCA2-R primers) were used to construct the coding sequence fragment of the MayUCCA2 gene obtained from the PCR amplification product into an entry vector, which was PDNOR207, and its sequence is shown in Sequence 5.

[0049] Construction of the introductory vector: According to the reaction system shown in Table 3 below, add fresh PCR product, PDNOR207 introductory vector and BP enzyme respectively, and then place in a 25℃ metal bath for 4-5 h.

[0050] Table 3

[0051]

[0052] The mixture after the above reaction was transformed into Escherichia coli and plated on selection medium (LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 8 g / L, gentamicin 50 mg / L). Positive clones were picked from the selection medium for PCR detection and sequencing verification, and the plant expression vector PMDC32 was constructed by LR reaction in the Gateway system. Its sequence is shown in sequence 6.

[0053] Expression vector construction: According to the reaction system shown in Table 4 below, the constructed cloning vector, PMDC32 expression vector and LR enzyme were added respectively, and then placed in a metal bath at 25℃ for 4-5 h.

[0054] Table 4

[0055]

[0056] After the LR reaction, the MayUCCA2 gene was constructed into the expression vector PMDC32. The MayUCCA2 overexpression vector was successfully constructed and named MayUCCA2-OE by transforming E. coli and selecting single clones for sequencing verification.

[0057] The Mayuca2 gene in the MaYUCCA2-OE vector has a strong expression promoter 35S assembled at its 5' end, enabling efficient expression of the Mayuca2 protein in poplar. Simultaneously, the PMDC32 expression vector contains hygromycin phosphotransferase (HPT), serving as a selection marker for transgenic poplar, allowing for selection using hygromycin. It also contains LB and RB sequences, facilitating the integration of the assembled 35S::MaYUCCA2 expression framework and the selection marker gene HPT into the chromosome of 84K poplar, thus obtaining Mayuca2 overexpressing transgenic poplar.

[0058] 3. Genetic transformation of MaYUCCA2 transgenic poplar

[0059] The genetic transformation steps of MaYUCCA2 transgenic poplar are as follows:

[0060] The constructed MayUCCA2-OE expression vector was transformed into Agrobacterium GV3101 by electroporation; then, the MayUCCA2-OE expression vector was integrated into the 84K poplar chromosome by Agrobacterium-mediated transformation, thereby obtaining MayUCCA2-OE transgenic poplar.

[0061] The specific transformation steps are as follows: One-month-old 84K poplar tissue culture seedlings used for genetic transformation were cultured at a temperature of 23-25℃ and a light intensity of 16 / 8h (day / night); GV3101 Agrobacterium cells containing the MayUCCA2-OE expression vector were cultured in LB liquid medium (tryptic peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, kanamycin 50mg / L, gentamicin 50mg / L, rifampin 20mg / L) until OD. 600When the concentration of α = 0.6–0.8, the leaf discs of 84K were infected, and the infected leaves were transferred to a co-culture medium (2.2 g / L Murashige-Skoog (MS) + 0.5 g / L 4-Morpholineethanesulfonic acid (MES) + 20 g / L sucrose + 4 g / L plant gel + 100 μmol / L Acetosyringone (AS)) and co-cultured in the dark for 3–4 days. The co-cultured leaves were then transferred to an adventitious shoot induction medium containing 3 mg / L hygromycin B (Hyg) and 200 mg / L Timentin (Tim) (2.2 g / L MS + 0.5 g / L MES + 0.5 mg / L 6-Benzyl Aminopurine (6-BA) + 0.05 mg / L Naphthaleneacetic acid). Resistant adventitious shoots were induced and screened on a medium containing 3 mg / L Hydral-3-butyric acid (NAA) + 20 g / L sucrose + 4 g / L plant gel. After 20 days of induction culture, the resistant adventitious shoots were transferred to a rooting medium containing 3 mg / L Hydral-3-butyric acid (IBA) and 200 mg / L Tim (2.2 g / L MS + 0.5 g / L MES + 0.05 mg / L Indole-3-butyric acid (IBA) + 0.02 mg / L NAA + 20 g / L sucrose + 8 g / L agar powder) until adventitious roots were induced. DNA was extracted from the leaves of the rooted plants that had developed hygromycin resistance, and PCR verification was performed using the MayUCCA2 cloning primers. Finally, transgenic 84K poplar containing the MayUCCA2-OE vector sequence was obtained.

[0062] 4. Phenotypic analysis of MaYUCCA2-OE transgenic poplar

[0063] This invention cloned the mulberry MayUCCA2 gene and constructed it into the PMDC32 expression vector. Figure 1 Subsequently, using Agrobacterium-mediated genetic transformation, transgenic poplar trees overexpressing MayUCCA2 (MayUCCA2-OE) were obtained. Finally, through semi-quantitative experiments, the obtained MayUCCA2-OE (OE1, OE2, and OE3) positive lines were identified, with the expression levels of the MayUCCA2 gene in the three lines showing: OE2 > OE3 > OE1. Figure 2 (A); A comparative analysis of WT and Mayu CCA2-OE transgenic poplars grown in tissue culture for one month revealed that the number of adventitious roots in Mayu CCA2-OE transgenic poplars was significantly increased ( Figure 2(B); Subsequently, analysis of the auxin content in the shoot tips of poplars of different genotypes revealed that the auxin content in the Mayu CCA2-OE transgenic poplar was significantly higher than that in the WT genotype, and the auxin content in the shoot tips of different transgenic poplars was positively correlated with the expression level of Mayu CCA2. Figure 2 (C) Finally, WT and Mayuca2-OE (OE1, OE2, and OE3) plants that had grown for one month were transplanted to a soil culture environment and grown for another month. Phenotypic observation revealed that the plant height of the line OE1, which had the lowest Mayuca2 overexpression level, was significantly higher than that of WT, while the plant height of the lines OE2 and OE3, which had significantly increased Mayuca2 expression, was significantly shorter than that of WT. Figure 2 (D) Based on this, it is inferred that overexpression of the Mayuca2 gene promotes adventitious root development while regulating plant height growth, and its positive or negative regulation of plant height growth depends on the expression level of Mayuca2.

[0064] To further analyze the function of MaYUCCA2 in the development of adventitious roots in woody plants, this invention analyzed the adventitious root development of WT and MaYUCCA2-OE transgenic poplar tissue culture seedlings. It was found that adventitious root development in the MaYUCCA2-OE transgenic poplar line occurred significantly earlier than in the WT line, and the number of adventitious roots was significantly greater in the MaYUCCA2-OE line. Figure 3 Meanwhile, this invention used leaves from one-month-old WT and MayUCCA2-OE transgenic poplar tissue culture seedlings as materials to conduct callus induction experiments, and found that the leaves of the MayUCCA2 overexpression line produced significantly more callus than those of the WT line. Figure 4 This indicates that the MayUCCA2-OE transgenic poplar promotes callus formation by increasing auxin synthesis.

[0065] This invention utilizes the Mayuca2 gene, which regulates the synthesis of auxin, a growth regulator in woody plants. Using the coding genome sequence of *Lusang No. 1* as a template, the Mayuca2 gene was cloned. Subsequently, the Mayuca2 coding sequence was constructed into the plant overexpression vector PMDC32 via the Gateway system. Transgenic poplars overexpressing Mayuca2 (Mayuca2-OE, OE1, OE2, and OE3) were obtained through stable genetic transformation in poplar. Finally, by comparing and analyzing the auxin content, adventitious root formation, and callus formation in Mayuca2-OE and WT poplars, this invention found that the endogenous auxin content, adventitious root formation, and callus formation were all positively correlated with the Mayuca2 gene expression level. Ultimately, this invention clarifies that Mayuca2 promotes adventitious root development and callus formation by increasing the accumulation of auxin in the plant, and simultaneously obtains new Mayuca2-OE transgenic poplar germplasm with a significant increase in adventitious roots. The application of this invention will alleviate the problems of difficult rooting and slow propagation of woody plants that are difficult to root by cuttings during asexual propagation.

Claims

1. Key genes for auxin synthesis in mulberry trees MaYUCCA2 Application in promoting adventitious root development and callus formation in poplar; genes promoting adventitious root and callus formation. MaYUCCA2 The nucleotide sequence is shown in Sequence 3 of the sequence listing.

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