Application of RcHDA19 gene in regulation and control of drought resistance and salt resistance of Chinese rose

Abstract

The invention discloses an application of an RcHDA19 gene in regulation and control of drought resistance and salt tolerance of Chinese rose, belongs to the technical field of agricultural biological genetic engineering, and discloses the application of the RcHDA19 gene in regulation and control of drought resistance and salt tolerance of Chinese rose, and the nucleotide sequence of the RcHDA19 gene is as shown in SEQ ID NO.1. According to the application, a new choice is provided for promoting the drought resistance and the salt resistance of the rosa chinensis, the RcHDA19 gene is integrated into the rosa chinensis through a silent vector, the tolerance of a rosa chinensis plant to drought and salt stress is improved, and the important application value is achieved for rosa chinensis breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-genetic engineering technology, specifically involving RcHDA19 Application of genes in regulating drought resistance and salt tolerance in roses. Background Technology

[0002] Rose ( Rosa Roses (Rosa spp.) belong to the genus Rosa in the family Rosaceae. They are among the most widely cultivated and economically valuable ornamental plants globally. With their rich flower colors, diverse flower shapes and plant forms, long flowering period, and multi-season flowering characteristics, they are known as the "Queen of Flowers" and are widely used in landscaping, cut flower production, and potted ornamental plants. As a major country in rose cultivation and application, my country has over 100 varieties of roses, with its annual cultivation area and market size ranking among the world's top. The development of this industry plays a vital supporting role in the ornamental horticulture economy. However, in the process of large-scale production and outdoor application of roses, abiotic stresses, especially drought and salt stress, have become key bottlenecks restricting their growth, development, and quality improvement. Drought environments lead to decreased root water absorption capacity and imbalanced leaf transpiration, resulting in problems such as plant wilting, leaf yellowing, delayed flowering, smaller and duller flowers, and in severe cases, even plant death, causing significant loss of ornamental value and a decline in economic benefits. Statistics show that in arid and semi-arid regions of northern my country, seasonal drought reduces the survival rate of outdoor cultivated roses by 30%-50% compared to suitable water conditions. Therefore, cultivating rose varieties with high resistance to drought and salt stress, or developing technologies to enhance the drought and salt tolerance of roses, is a core need that the current rose industry needs to address.

[0003] Epigenetic modifications play a crucial regulatory role in the molecular mechanisms of plant responses to drought and salt stress. Among these, histone acetylation, as an important epigenetic regulatory mechanism, influences chromatin structure and gene transcription efficiency by dynamically regulating the acetylation levels of histones and non-histone proteins, thereby participating in the fine regulation of plant growth and development, stress response, and metabolic processes. Histone deacetylases (HDA) are the core regulators of histone acetylation modification. They can inhibit the expression of downstream target genes by removing acetyl groups from histones. Members of this family exhibit high functional diversity in plant stress responses—for example, the AtHDA19 gene in Arabidopsis has been shown to participate in the regulation of low-temperature stress response. However, research on the function of this family of genes in roses is still in its early stages. Existing studies have shown that there are 23 genes encoding histone deacetylases in the rose genome, forming a multi-member HDA family. However, research on the specific functions, regulatory mechanisms, and application value of this family of genes in the drought and salt stress responses of roses is extremely limited. Summary of the Invention

[0004] This invention aims to provide RcHDA19 The application of genes in regulating drought and salt tolerance in roses provides a new option for promoting these traits. This application involves... RcHDA19 Genes are integrated into roses through silencing vectors, improving the plants' tolerance to drought and salt stress, which has significant application value for rose breeding.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: RcHDA19 The application of genes in regulating drought resistance and salt tolerance in roses, the aforementioned RcHDA19 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] Preferably, the RcHDA19 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0007] Preferably, by lowering RcHDA19 Gene expression level or repression RcHDA19 Gene-encoded protein activity enhances the drought resistance and salt tolerance of roses.

[0008] The present invention also provides a silencing carrier for enhancing the drought resistance and salt tolerance of roses, the silencing carrier comprising a component for silencing the... RcHDA19 Interference fragments in genes.

[0009] Preferably, the interfering fragment comprises a nucleotide sequence as shown in SEQ ID NO:3.

[0010] The present invention also provides a strain that enhances the drought resistance and salt tolerance of roses, the strain comprising the silencing vector described above.

[0011] The present invention also provides a method for utilizing the above-mentioned RcHDA19 A method for regulating the drought resistance and salt tolerance of roses using genes, characterized by comprising the following steps: Will RcHDA19 A specific fragment of the gene is introduced into a silencing vector, which is then transferred into Agrobacterium tumefaciens to infect rose plants, resulting in transgenic plants.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses RcHDA19 The application of genes in regulating drought resistance and salt tolerance in roses, through cloning RcHDA19 Genes will RcHDA19A specific fragment of the gene is introduced into a silencing vector, which is then transferred into Agrobacterium tumefaciens to infect rose plants, resulting in transgenic plants. These plants can effectively improve the water use efficiency, photosynthetic capacity, and stress resistance of roses, as well as enhance their drought resistance and salt tolerance, thus effectively promoting their adaptability to the external environment.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 for RcHDA19 Bioinformatics analysis of genes, among which, Figure 1 A in the text is RcHDA19 Phylogenetic tree of histone deacetylase genes in Arabidopsis thaliana Figure 1 B in the text is RcHDA19 Gene sequence exons, Figure 1 In this context, C represents the FPK index of the rose deacetylase gene in different rose tissues. Figure 1 D in RcHDA19 Subcellular localization in tobacco; Figure 2 for RcHDA19 A schematic diagram of the protein sequence structure, wherein, Figure 2 A in the text is RcHDA19 Protein structure model, Figure 2 B in the diagram represents the protein sequence structure. Figure 3 for RcHDA19 Schematic diagram of silent carrier construction. Figure 3 A in the text is RcHDA19 Recombinant vector plasmid map, Figure 3 B in the text is RcHDA19 Schematic diagram of gene silencing fragments; Figure 4 This is a gel electrophoresis image of nucleic acid, where, Figure 4 In the image, A represents the gel electrophoresis result of double-enzyme digestion of the pTRV2 vector nucleic acid. Figure 4 B in the text is RcHDA19 DNA gel electrophoresis images of homologous arm clones of gene silencing fragments. Figure 4 C in the text is RcHDA19 Gene silencing fragment recombinant plasmid was transformed into E. coli, and the bacterial culture was analyzed by PCR and nucleic acid gel electrophoresis. Figure 4 D in RcHDA19 Gene silencing fragment recombinant plasmid was transformed into Agrobacterium (GV3101), and the bacterial culture was analyzed by PCR nucleic acid gel electrophoresis. Figure 5 for RcHDA19 The response of transgenic plants to drought and salt stress, among which, Figure 5In the diagram, A represents four transgenic Agrobacterium bacterial cultures (transient overexpression control group pSuper, transient overexpression group pSuper-). RcHDA19, Transient silencing of TRV in the control group and transient silencing of TRV- RcHDA19 The growth phenotypes of transgenic seedlings obtained by infecting roses under drought stress (left) and salt stress (right). Figure 5 In the figure, B represents the DAB and NBT staining of leaves from four transgenic rose plants subjected to drought and salt stress. Figure 6 for RcHDA19 Physiological responses of transgenic plants under drought stress, among which... Figure 6 In the figure, A represents one of the four types of transgenic rose plants. RcHDA19 The relative expression levels of genes. Figure 6 In the figure, B represents the relative water content of four transgenic rose plants at different time points after being subjected to drought stress. Figure 6 In the figure, C represents the ion permeability of four transgenic rose plants at different time points after being subjected to drought stress. Figure 6 In the figure, D represents the ion permeability of four transgenic rose plants at different time points after being subjected to salt stress. Figure 6 In the figure, E represents the Fv / Fm ratio of the four transgenic rose plants after salt stress. Figure 6 F in the figure represents the photosynthetic YII values ​​of four transgenic rose plants after being subjected to salt stress. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0017] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0018] Example 1 Rose RcHDA19 Bioinformatics analysis of genes, such as Figure 1 As shown.

[0019] RcHDA19 Phylogenetic tree of histone deacetylase genes in Arabidopsis thaliana ( Figure 1 A) Comparison in the middle. RcHDA19 and Arabidopsis AtHDA19 Highly homologous; the gene sequence encoding the protein region consists of seven exons and is 1476 bp in length.

[0020] Depend on Figure 1 It can be seen that, RcHDA19 The conserved domain of deacetylase is located from amino acid 15 to amino acid 324. RcHDA19 It is located in the cell nucleus.

[0021] RcHDA19 A schematic diagram of the protein sequence structure is shown below. Figure 2 As shown, the amino acid composition of the Helix region is (25-29, 41-52, 78-86, 92-101, 242-260, 286-298, 313-328, 364-379 aa) and the amino acid composition of the Sheet region is (19-22, 60-62, 139-142, 178-182, 201-208, 231-236, 264-268, 302-306 aa).

[0022] RcHDA19 The gene is the nucleotide sequence shown in SEQ ID NO:1.

[0023] SEQ ID NO:1:

[0024] RcHDA19 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0025] SEQ ID NO:2:MDTGGNSLASGPDGVKRKVSYFYDPEVGNYY YGQGHPMKPHRIRMTHALLAHYGLLQHMQVLKPYPARDRDLCRFHADDYVAFLRSITPETQQDQLRQLKRFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHGICDISINWAGG LHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYYSLNVPLDDGIDDESYHYLFKPLIGKVMEIFRP GAVVLQCGADSLSGDRLGCFNLSIKGHAECVRYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGAEIEDKMPQHEYYEYFGPDYTLHVAPSNMENKNSHQLLEEIRSKLLEN LSRLQHAPSVQFHERPPENELPEEDEDQDDRDERWDADSDMELDDRPIAARRVKKEIVEPEVKDPKGAVENSRGYDAGLDEITTSAKALDMGSGSMEEPSVKVEQESLNKPADQM.

[0026] for RcHDA19 Construction of recombinant vectors, recombinant vector plasmids (pTRV2- RcHDA19 ) Atlas as Figure 3 As shown in A in the diagram. Figure 3 B in the text is RcHDA19 A schematic diagram of the gene silencing fragment, showing the silencing region as 934bp-1255bp. The silencing vector contains the gene silencing fragment. RcHDA19 The interference fragment of the gene, containing the nucleotide sequence shown in SEQ ID NO:3, significantly reduces RcHDA19 Expression level.

[0027] SEQ ID NO:3: ACCATTCGCAATGTTGCTCGTTGCTGGTGCTA CGAGACTGGTGTTGCACTTGGAGCAGAAATTGAGGACAAAATGCCACAGCATGAGTATTATGAGTATTTTGGTCCAGACTATACTCTTCATGTTGCCCCTAGTAATATGGAAAACAAGAATTCTCATCAGTTACTGGAAGAAATA CGATCGAAACTTCTTGAAAATCTCTCCAGGCTTCAGCATGCACCTAGTGTCCAATTTCATGAAAGGCCTCCTGAAAATGAGCTTCCAGAGGAAGATGAGGATCAGGATGACAGAGACGAGAGATGGGATGCCGATTCTGATATGG.

[0028] The system obtained by double digestion of vector pTRV2 is shown in Table 1: Table 1 Reaction System

[0029] In a PCR instrument, after enzyme digestion at 37°C for 2 hours and heat inactivation at 80°C, nucleic acid agarose gel electrophoresis was performed. The gel was then cut and recovered. Figure 4 As shown.

[0030] RcHDA19 The gene silencing fragment addition homologous arm cloning system is shown in Table 2 below: Table 2 Cloning System

[0031] The primer homologous arm F sequence is shown in SEQ ID NO:4, and the primer homologous arm R sequence is shown in SEQ ID NO:5.

[0032] SEQ ID NO:4: cctccatggggatccggtaccACCATTCGCAATGTTGCTCG.

[0033] SEQ ID NO:5: gagacgcgtgagctcggtaccCCATATCAGAATCGGCATCCC.

[0034] After the routine clonal amplification reaction in the PCR instrument is completed, nucleic acid agarose gel electrophoresis is performed. The gel is then cut and recovered, as follows: Figure 4 As shown. Linearized pTRV2 vector and vector with added homologous arms. RcHDA19 Homologous recombination of gene-silenced fragments was performed, as shown in Table 3: Table 3 Reaction System

[0035] The cells were heat-shocked at 50℃ for 5 min, incubated on ice for 2 min, and then the recombinant plasmid was transformed into E. coli according to the instructions of the Coollab DH5α E. coli competent cells. Subsequent identification of positive E. coli clones using the recombinant plasmid was performed. The bacterial culture PCR system is shown in Table 4. Table 4 Bacterial PCR System

[0036] The TRV-F sequence is shown in SEQ ID NO:6, and the TRV-R sequence is shown in SEQ ID NO:7.

[0037] SEQ ID NO:6: TGGGAGATGATACGCTGTT.

[0038] SEQ ID NO:7: CCTAAAACTTCAGACACG.

[0039] After the bacterial culture PCR was completed, nucleic acid agarose gel electrophoresis was performed. Positive clones were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After confirming the sequencing accuracy, recombinant plasmid transformation was performed according to the instructions for Agrobacterium tumefaciens (CV3101) competent cells. The Agrobacterium tumefaciens PCR reaction system was the same as that for Escherichia coli PCR, and the nucleic acid agarose gel electrophoresis results are as follows. Figure 4 As shown.

[0040] The infection solution system for transforming rose plants is shown in Table 5 below: Table 5. Infection solution system for infected and transformed rose plants

[0041] pTRV1, pTRV2 and pTRV- RcHDA19 Agrobacterium bacterial suspension was incubated overnight at 28°C, and the bacterial pellet was collected by centrifugation. The bacterial cells were resuspended in the infection solution. After uniformly adjusting the absorbance to 0.8 at 600 nm, pTRV1 and pTRV2, pTRV2- RcHDA19 Equal volumes were mixed and left to stand in the dark for 3 hours. The bacterial solution was then pumped into the plants using a vacuum pump and incubated in the dark for three days. Drought and salt stress experiments were then conducted, and relevant indicators reflecting pTRV2- were measured. RcHDA19To improve the drought and salt stress tolerance of rose plants, the specific experimental protocol was as follows: Transgenic plants were placed in a 1 / 4 Hoagland solution of 10% PEG6000 for drought stress. Salt stress was achieved by adjusting the NaCl molar concentration in the 1 / 4 Hoagland solution to 200 mM. When different genotypes of rose seedlings showed phenotypic differences in stress tolerance under the two stresses, relevant phenotypic records and index measurements were performed. DAB, NBT, and Trpan staining were performed on transgenic rose leaves. After staining for 8-12 hours, the leaves were heated at 90°C with a low concentration of 50% alcohol. After destroying the cell structure, the staining was reduced to 75% alcohol and then destained by heating. Finally, chlorophyll was destained using a high concentration of 95% alcohol. The photosynthetic coefficient of the leaves was measured using a portable photosynthesis meter. The results are as follows: Figure 5 and Figure 6 As shown.

[0042] Depend on Figure 5 It can be seen that the four transgenic rose plants exhibited different drought and salt tolerance under drought and salt stress, respectively, and overexpression... RcHDA19 It reduced the rose's tolerance to drought and salt stress, while silence RcHDA19 The rose varieties exhibited stronger drought and salt tolerance. DAB and NBT staining also reflected this. RcHDA19 Plants overexpressing this gene showed more severe damage from drought and salt stress, and accumulated more reactive oxygen species. Roses... RcHDA19 Silent strains experienced less stress damage and accumulated less reactive oxygen species (ROS), indicating a stronger ROS scavenging ability, suggesting that TRV- RcHDA19 Rose varieties have stronger drought and salt tolerance.

[0043] Depend on Figure 6 It can be seen that among the four types of transgenic rose plants RcHDA19 The relative expression levels showed significant differences, indicating that different [elements / types] were successfully obtained. RcHDA19 Transgenic rose lines. After two days of drought stress, TRV- RcHDA19 The relative water content decreased the least, at 65%. Two days after rehydration, the relative water content of the rose plants increased significantly, TRV- RcHDA19 The relative moisture content can be restored to normal levels (70%), and as Figure 6 The C in the TRV- RcHDA19 With lower ion permeability, they experience less damage from drought stress and exhibit stronger drought resistance. After one day of salt stress treatment, such as... Figure 6 The D in the TRV- RcHDA19 It has lower ion permeability and is less susceptible to damage from salt stress. Furthermore... Figure 6 E in 6 and F in 6 demonstrate TRV- RcHDA19 The photosynthetic correlation coefficient Fv / Fm ratio and YⅡ value were significantly higher than those of the control, further revealing that TRV- RcHDA19 It has stronger tolerance to salt stress.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. RcHDA19 The application of genes in regulating drought resistance and salt tolerance in roses is characterized by, The RcHDA19 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The RcHDA19 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, By lowering RcHDA19 Gene expression level or repression RcHDA19 Gene-encoded protein activity enhances the drought resistance and salt tolerance of roses.

4. A silent carrier for enhancing the drought resistance and salt tolerance of roses, characterized in that, The silencing carrier comprises a component for silencing the claim 1. RcHDA19 Interference fragments in genes.

5. The silencing carrier according to claim 4, characterized in that, The interfering fragment contains a nucleotide sequence as shown in SEQ ID NO:

3.

6. A strain that enhances the drought resistance and salt tolerance of roses, characterized in that, The strain includes the silencing vector as described in claim 4.

7. A method utilizing the method described in claim 1 RcHDA19 A method for regulating the drought resistance and salt tolerance of roses using genes, characterized in that, Includes the following steps: Will RcHDA19 A specific fragment of the gene is introduced into a silencing vector, which is then transferred into Agrobacterium tumefaciens to infect rose plants, resulting in transgenic plants.

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