Gene RhWOX11 for promoting rhododendron callus cell division and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene RhWOX11 for promoting rhododendron callus cell division and application of the gene RhWOX11, and the nucleotide sequence of the gene RhWOX11 is as shown in SEQ ID NO.1. The amino acid sequence of the expression protein of the gene RhWOX11 is as shown in SEQ ID NO. 2. The invention also comprises application of the gene RhWOX11 for promoting the growth of the rhododendron calluses in accelerating the growth and development of the calluses, the key gene RhWOX11 for promoting the division of the rhododendron calluses is obtained through a genetic engineering means, and the key gene RhWOX11 is introduced into tobacco plant cells, so that transgenic calluses with higher division speed can be cultivated, and the application of the gene RhWOX11 for promoting the growth of the rhododendron calluses in accelerating the growth and development of the calluses is realized. Therefore, the key gene RhWOX11 can be applied to plant callus division, the success rate of callus division of plants difficult to regenerate is increased, and a technical means support is provided for large-scale breeding of seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a gene RhWOX11 for promoting the division of rhododendron callus cells and an application thereof. Background Art

[0002] Azalea belongs to the genus Rhododendron in the family Ericaceae. It is a world-famous flower with rich germplasm resources and high ornamental value. It has a market share of about 65% in the New Year flower market. Some varieties can be eaten, used as medicine, and used to extract essential oils, etc., and have important medicinal and edible value. Alpine azalea horticultural cultivars are recognized as high-end potted flowers because of their huge inflorescences, bright colors, and beautiful flowers. They are also important flowers for improving the quality of urban landscapes. Due to the low flowering and fruiting rate of azaleas and the low survival rate of cuttings, large-scale breeding of seedlings is limited. Plant callus is widely used because of its good genetic stability, fast reproduction speed and high reproduction efficiency. Focusing on the cultivation of callus provides good materials for studying the molecular mechanism of plant embryogenesis. Embryonic callus is also an important medium for the establishment of Agrobacterium-mediated genetic transformation system. It is a good receptor for exogenous genes and is of great significance for revealing cell differentiation, morphogenesis, and zygotic embryogenesis.

[0003] Plant growth regulators can induce callus cell division in plants with difficulty in regeneration and promote somatic embryogenesis, but embryonic callus is relatively difficult to generate in vitro, with a high proportion of deformed embryos and difficulty in synchronization. This problem has become an important factor restricting the rapid breeding of excellent plants using embryonic callus generation technology, and also makes genetic transformation of species with specific genotypes difficult, thus hindering functional genomic research and genetic improvement of traits. Genome editing technology continues to advance, but the current lack of embryonic callus generation capacity is still an important obstacle to future molecular design breeding.

[0004] The WOX transcription factor family is a plant-specific family of transcription factors that plays a regulatory role in the development of plant stem cells. WOX family genes have the ability to regulate somatic embryo division, differentiation, dynamic balance and plant organ development.

[0005] The technical problem to be solved by the present invention is to provide the RhWOX11 gene and its expression protein and application. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a cuckoo RhWOX11 gene and its expression protein and application.

[0007] The specific technical solution adopted by the present invention is:

[0008] A gene RhWOX11 for promoting the division of rhododendron callus cells. The nucleic acid sequence of the gene RhWOX11 is shown as SEQ ID NO.1.

[0009] The details are as follows:

[0010] ATGGAAGATCATGGCGGCCAAGAAGATGCTGGTGGTAGTAGAGAGAGAAACAACGAGGGAGTTAGATCAAGGTGGACTCCCAAACCAGAGCAAATCCTCATACTGGAATCAATCTTCAACAGTGGGATGGTAAATCCCCGAAAGATGAAACGGTGAGGATCAGAAAACTACTGGAGAAATTCGGTTCAGTCGGG GACGCAAATGTCTTTTACTGGTTCCAGAACCGGAGGTCCCGGTCCCGCCGCCGGCAGCGCCAGCAGATTCATGAGGCGGCGGCCACCACCACCAGCCATGCCACGGCCACCCAACAACAACAGCAGCATGGTTATTATCAGACCACCAACGAAAGCGGTGGCGGTGGTAGTGGTGGTGGTGGTGCAATTCAT CAGTATGAGATGAGGTGGTTCTTCTTCTTCCTCGGGCGGCGGTGGTGGCGGTGGTGATTTTGTGAATGAGCATCTCTGCTCTTTTTCGGGTGAAATTGGTTTTTCCAGGGTACTTCAGTGAGCAGATTTCCTCCTCCCCTTCAGTTTCGGCCCCGTTAGATACTTCTTCAACTTCTTTGCCATACCAATCTCCAGGA TTTATAACGGTGTTCATAAATGGGGTGTCAACAGAAGTAGGGAGGGGCCCGATAGACATGAAAGGAACGTTTGGTGAAGATTTGGTGTTAGTCCACTCTTCTGGAATGCCAGTTGTCCCTTTCAACGACTTCGGCTTTTCCCTTCACACTTTGCACCATGGAGAGAGCTATTTCCTGGTTCCAAGGCCTGGTTAA

[0011] The amino acid sequence of the expressed protein of gene RhWOX11 is shown in SEQ ID NO.2.

[0012] The details are as follows:

[0013] MEDHGGQEDAGGSRERNNEGVRSRWTPKPEQILILESIFNSGMVNPPKDETVRIRKLLEKFGSVGDANVFYWFQNRRSRSRRRRQRQQIHEAAATTTSHATATQQQQQHGYYQTTNESGGGGSGGGGAIH QYEMSGSSSSSSGGGGGGGDFVNEHLCSFSGEIGFPGYFSEQISSSPSVSAPLDTSSTSLPYQSPGFITVFINGVSTEVGRGPIDMKGTFGEDLVLVHSSGMPVVPFNDFGFSLHTLHHGESYFLVPRPG*

[0014] An application of a gene RhWOX11 for promoting the division of rhododendron callus in accelerating the growth and development of callus, the application specifically comprising:

[0015] 1) Constructing the vector of RhWOX11 gene, a key gene for callus division in rhododendron;

[0016] 2) Transforming the constructed vector of the key gene RhWOX11 for callus division of rhododendron into plant cells;

[0017] 3) Cultivate and screen transgenic callus tissue that grows faster than untreated materials.

[0018] The vector is a plant expression vector.

[0019] The plant cells in step 2) are plant leaf cells, and the vector transformation method is Agrobacterium-mediated method.

[0020] The plant is tobacco.

[0021] The beneficial effects of the present invention are:

[0022] The present invention obtains the key gene RhWOX11 for promoting the division of rhododendron callus by genetic engineering means, and introduces the key gene RhWOX11 into plant cells such as tobacco, so as to cultivate transgenic callus with a faster division speed. Therefore, the key gene RhWOX11 can be used for the division of plant callus, thereby improving the success rate of callus division of plants with regeneration difficulties, and providing technical support for large-scale breeding of seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the 1% agarose gel electrophoresis image of the full-length PCR product of RhWOX11 gene;

[0024] Figure 2This is the 1% agarose gel electrophoresis image of the pBI121WOX Agrobacterium single colony PCR;

[0025] Figure 3 This is the constructed RhWOX11 overexpression vector map;

[0026] Figure 4 This is the PCR test result of the positive transgenic tobacco plants overexpressing RhWOX11;

[0027] Figure 5 is the comparison phenotype of WT and RhWOX11 in transgenic tobacco leaves overexpressing RhWOX11; DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments:

[0029] The invention discloses a gene RhWOX11 for promoting the division of rhododendron callus, and the nucleic acid sequence of the gene is shown as SEQ ID NO.1.

[0030] The details are as follows:

[0031] ATGGAAGATCATGGCGGCCAAGAAGATGCTGGTGGTAGTAGAGAGAGAAACAACGAGGGAGTTAGATCAAGGTGGACTCCCAAACCAGAGCAAATCCTCATACTGGAATCAATCTTCAACAGTGGGATGGTAAATCCCCCGAAAGATGAAACGGTGAGGATCAGAAAACTACTGGAGAAATTCGGTTCAGTCGGGGACGCAAATGTCTTTTACTGGTTCCAGAACCGGAGGTCCCGGTCCCGCCGCCGGCAGCGCCAGCAGATTCATGAGGCGGCGGCCACCACCACCAGCCATGCCACGGCCACCCAACAACAACAACAGCAGCATGGTTATTATCAGACCACCAACGAAAGCGGTGGCGGTGGTAGTGGTGGTGGTGGTGCAATTCATCAGTATGAGATGAGTGGTTCTTCTTCTTCCTCGGGCGGCGGTGGTGGCGGTGGTGATTTTGTGAATGAGCATCTCTGCTCTTTTTCGGGTGAAATTGGTTTTCCAGGGTACTTCAGTGAGCAGATTTCCTCCTCCCCTTCAGTTTCGGCCCCGTTAGATACTTCTTCAACTTCTTTGCCATACCAATCTCCAGGATTTATAACGGTGTTCATAAATGGGGTGTCAACAGAAGTAGGGAGGGGCCCGATAGACATGAAAGGAACGTTTGGTGAAGATTTGGTGTTAGTCCACTCTTCTGGAATGCCAGTTGTCCCTTTCAACGACTTCGGCTTTTCCCTTCACACTTTGCACCATGGAGAGAGCTATTTCCTGGTTCCAAGGCCTGGTTAA

[0032] The expressed protein of gene RhWOX11 has an amino acid sequence as shown in SEQ ID NO.2.

[0033] Specifically as follows:

[0034] MEDHGGQEDAGGSRERNNEGVRSRWTPKPEQILILESIFNSGMVNPPKDETVRIRKLLEKFGSVGDANVFYWFQNRRSRSRRRRQRQQIHEAAATTTSHATATQQQQQHGYYQTTNESGGGGSGGGGAIH QYEMSGSSSSSSGGGGGGGDFVNEHLCSFSGEIGFPGYFSEQISSSPSVSAPLDTSSTSLPYQSPGFITVFINGVSTEVGRGPIDMKGTFGEDLVLVHSSGMPVVPFNDFGFSLHTLHHGESYFLVPRPG*

[0035] A use of a gene RhWOX11 for promoting callus division in azalea in accelerating the division and development of callus, the application specifically comprising:

[0036] 1) constructing a vector of the key gene RhWOX11 for callus division in rhododendron; Figure 3 As shown,

[0037] 2) Transforming the constructed vector of the key gene RhWOX11 for callus division of rhododendron into plant cells;

[0038] 3) Cultivate and screen transgenic callus tissue that grows and divides faster than untreated materials.

[0039] The vector is a plant expression vector.

[0040] The plant cells in step 2) are plant leaf cells, and the vector transformation method is Agrobacterium-mediated method.

[0041] The plant is tobacco.

[0042] The present invention relates to the extraction of total RNA from azalea callus, and the specific steps are: using azalea leaf-induced callus as experimental material, extracting RNA, reversing cDNA, designing corresponding primers for PCR, recovering target bands after agarose gel electrophoresis, connecting with pMD19-T as a vector, transferring into Agrobacterium, sequencing and analyzing.

[0043] Total RNA extraction: The callus tissue induced by rhododendron leaves was used as the experimental material, and RNA was extracted according to the operating procedures of the finished kit. The results of 1% agarose gel electrophoresis of total RNA from callus tissue induced by rhododendron leaves are as follows: Figure 1 shown.

[0044] cDNA acquisition: Invitrogen cDNA kit was used to obtain RNA as a template and reverse transcribed to obtain cDNA.

[0045] Homologous cloning of the target gene: Use oligo to design degenerate primers, perform gradient PCR to obtain the target product, perform conversion sequencing, and finally perform comparative analysis.

[0046] The amplification primer sequences are:

[0047] ·RD024593-f:ATGGAAGATCATGGCGGCCAAG

[0048] ·RD024593-r:TTAACCAGGCCTTGGAACCAGG

[0049] PCR reaction conditions: 94℃, 4min, 94℃, 30s, 54℃, 30s, 72℃, 45s, 36Cycles, 72℃, 10min; 4℃, hold. Take the PCR product, and run the result of 1% agarose gel electrophoresis to recover the target band.

[0050] The present invention Figure 1 — Figure 4 Obtaining the RhWOX11 gene and verifying its function in tobacco;

[0051] like Figure 1 , is a 1% agarose gel electrophoresis diagram of the full-length PCR product of the RhWOX11 gene; in the figure, a, b: the full-length RhWOX11 gene obtained by PCR using cDNA as a template; M: 2000 bp Marker;

[0052] DNA molecular weight standard M: 2000bp Marker, used to estimate the size of DNA fragments in the sample.

[0053] Lanes a and b show the full length of the RhWOX11 gene obtained by PCR using cDNA as a template. It can be seen that there are obvious DNA bands in lanes a and b.

[0054] Figure 2 This is a 1% agarose gel electrophoresis diagram of pBI121WOX Agrobacterium single colony PCR; in the figure, af: Agrobacterium transformed with RhWOX11 gene; g: ddH2O; M: 2000bp Marker;

[0055] Among them, the primers for Agrobacterium transformation colony detection are:

[0056] RD024593-f:ATGGAAGATCATGGCGGCCAAG

[0057] NOS-r: accggcaacaggattcaatc

[0058] After the RhWOX11 gene was introduced into the colonies of Agrobacterium by genetic engineering, single colony PCR and electrophoresis were performed, such as Figure 2 As shown, it can be confirmed that Agrobacterium successfully carries the RhWOX11 gene.

[0059] Figure 4 In the process of detecting the DNA of the transgenic positive seedlings, it is used to detect whether the RhWOX11 gene has been successfully transferred into the tobacco plants.

[0060] 1-20: DNA of tobacco plants transfected with RhWOX11 gene;

[0061] H2O: ddH2O, used for negative control;

[0062] P: plasmid DNA, used for positive control;

[0063] M: used to determine the size of DNA fragments;

[0064] Figure 4 DNA detection of positive seedlings expressing transgenic

[0065] M = D2000, P = plasmid control

[0066] Detection primers:

[0067] RD024593-f:ATGGAAGATCATGGCGGCCAAG

[0068] NOS-r: accggcaacaggattcaatc

[0069] Among them, Marker (M): is used to determine the size of DNA fragments. Figure 4 In the middle, M: 2000bp Marker, which can be used to determine the size of DNA fragments in other samples.

[0070] Positive control (P): Plasmid DNA shows the expected bands, indicating the effectiveness of the primers and detection method.

[0071] Negative control (H2O): deionized water showed no bands, indicating no contamination.

[0072] Tobacco plants transformed with RhWOX11 gene (1-20): Bands appeared in samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 13, 15, 16, 17, 18, 19, and 20, proving that the RhWOX11 gene was successfully transformed into tobacco plants. No bands appeared in samples 11, 12, and 14, indicating that the RhWOX11 gene was not successfully transformed into tobacco plants.

[0073] like Figure 5, indicating that the growth and phenotype differences between wild-type (WT) and transgenic (RhWOX11) tobacco leaves under specific conditions were compared. It can be seen that overexpression of RhWOX11 increased the efficiency of tobacco-induced callus division, among which Figure 5 The left side is the record of 18 days of culture, and the right side is the record of 23 days of culture. The left side of the blue line in the figure is the transgenic (RhWOX11) tobacco leaf, and the right side is the wild type (WT). It is not difficult to see that the callus division incidence rate of tobacco leaves treated with transgenic (RhWOX11) is significantly higher than that of the untreated wild type (WT) tobacco leaves on the right, and the transgenic (RhWOX11) treated tobacco leaves have a higher synchronization rate, and the callus growth and division are highly uniform. It can be used for rapid breeding of excellent plants and provide technical support for large-scale breeding of seedlings.

Claims

1. A gene RhWOX11 that promotes the division of rhododendron callus cells, characterized in that: The nucleic acid sequence of the gene RhWOX11 is shown in SEQ ID NO.

1.

2. The gene RhWOX11 according to claim 1, characterized in that: The amino acid sequence of the expressed protein of gene RhWOX11 is shown in SEQ ID NO.

2.

3. An application of the gene RhWOX11 for promoting the division of rhododendron callus cells according to claim 1 in accelerating the growth and development of callus tissue, characterized in that: The application is specifically: 1) Constructing the vector of RhWOX11 gene, a key gene for callus cell division in rhododendron; 2) Transforming the constructed vector of the key gene RhWOX11 for callus cell division of rhododendron into plant cells; 3) Cultivate and screen transgenic callus cells that grow faster than untreated materials.

4. The use according to claim 3, characterized in that: The vector is a plant expression vector.

5. The use according to claim 3, characterized in that: The plant cells in step 2) are plant leaf cells, and the vector transformation method is Agrobacterium-mediated method.

6. The use according to claim 5, characterized in that: The plant is tobacco.

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