CN115261397A - Chrysanthemum DEAD-box RNA helicase gene CmRH56 and application thereof in plant breeding - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a chrysanthemum DEAD-box RNA helicase gene CmRH56 and its application in plant breeding. Specifically, the present invention provides the CmRH56 gene, the open reading frame of the gene has the nucleotide sequence shown in SEQ ID NO.1. The present invention also provides a protein, which is highly identical to the sequence shown in SEQ ID NO. 2 and has the same biological function. The present invention also provides the application of the gene or the protein in the regulation of plant rhizome number and/or the regulation of plant drought tolerance. The present invention also provides a method for plant breeding, which comprises the step of altering the overexpression of the gene described in the first aspect of the present invention or the protein described in the second aspect of the present invention in plants by means of genetic engineering technology. The invention can realize the convenient and effective regulation of plant rhizome and drought tolerance.

Description

Chrysanthemum DEAD-box RNA helicase gene CmRH56 and its role in plant breeding application

technical field

The invention relates to the technical field of genetic engineering, in particular to a chrysanthemum DEAD-box RNA helicase gene CmRH56 and its application in plant breeding.

Background technique

Rhizomes are composed of leaves, stems or internodes and nodes and grow horizontally below the soil surface. Rhizomes contain meristems that give rise to daughter plants. As a reproductive strategy and storage organ, rhizomes support plant growth under abiotic stress and enable plants to perennialize and survive stress damage. Many plants have evolved altered organs that exhibit specific activities in response to specific environmental conditions, and rhizome growth is known to be induced upon major changes in the environment. This growth could slow significantly, or even be suppressed altogether, until more favorable conditions are restored. However, the specific mechanisms that regulate rhizome growth under harsh conditions remain unclear.

Chrysanthemum (Chrysanthemum morifolium (Ramat.) Kitamura) is a perennial herb with a long history of cultivation and culture. It is an important flower for ornamental, edible, tea and medicinal purposes. It is widely used in cut flowers, potted flowers and landscapes. landscaping in progress. Long-term natural selection has led plants to evolve a series of survival strategies to face adversities such as drought. The occurrence of chrysanthemum rhizomes is a key factor that reflects the ability of plants to adapt to adverse environments, asexual reproduction, and plant regeneration. The regulation of chrysanthemum rhizomes The mechanism has not been determined. Therefore, there is an urgent need to cultivate new chrysanthemum varieties with strong drought tolerance by regulating the rhizome of chrysanthemums.

Contents of the invention

The inventors cloned a DEAD-box RNA helicase gene CmRH56, the full length is 1,675bp, encoding 428 amino acids, the amino acid sequence has 9 conserved motifs of the DExD/H RNA helicase family, according to the motif II Specific amino acid sequence (D-E-C-D), assigning it to the DEAD-box RNA helicase subfamily. The gene is localized in the nucleus, down-regulated by drought stress and ABA induction; it is expressed in all organs of chrysanthemum, but the highest expression level is in the root at the seedling stage, and the highest expression level is in the rhizome at the seedling stage, indicating that the expression of CmRH56 gene varies with changes during plant growth and development.

The inventors have found through research that in chrysanthemums, overexpression of CmRH56 can increase the number of chrysanthemum rhizomes and advance the germination time, and silencing CmRH56 (CmRH56-RNAi) can reduce the number of chrysanthemum rhizomes and delay the germination time. Drought stress treatment of WT and CmRH56 transgenic chrysanthemums found that the drought stress tolerance of CmRH56 overexpression lines was weakened, and the drought stress tolerance of CmRH56-RNAi lines was enhanced. Drought stress can reduce the number of rhizomes of each line of chrysanthemum to varying degrees. The CmRH56-RNAi line enhanced the tolerance of the parent to drought stress due to its own small number of rhizomes. With the intensification of drought stress, the survival rate of CmRH56-RNAi lines was significantly higher than that of WT plants. RNA-seq results showed that GA2ox6 gene expression was significantly upregulated in CmRH56-RNAi lines with enhanced drought stress tolerance. Exogenous GA treatment and CmGA2ox6 silencing resulted in increased rhizome number. These results indicated that CmRH56 suppressed rhizome growth under drought stress by inhibiting GA biosynthesis. All in all, the present invention finds a new way to cultivate new chrysanthemum varieties with stronger drought tolerance, that is, to improve the drought tolerance of chrysanthemums by inhibiting the germination and quantity of rhizomes of chrysanthemums. The DEAD-box RNA helicase gene CmRH56 is an effective regulator for regulating the number of rhizomes of chrysanthemum, and silencing CmRH56 gene can breed new varieties with strong drought tolerance.

Based on the above findings, the present invention provides the CmRH56 gene in the first aspect, the open reading frame of the gene has the nucleotide sequence shown in SEQ ID NO.1.

Preferably, the gene can encode a protein having the amino acid sequence shown in SEQ ID NO.2.

In a second aspect, the present invention provides a protein, which has 80%, preferably 90%, more preferably 95%, further preferably 99% identity with respect to the sequence shown in SEQ ID NO.2 and has the same biological functional protein.

Preferably, the amino acid sequence of the protein is shown in SEQ ID NO.2.

Preferably, the biological function is to regulate the growth of plant rhizomes and/or regulate the drought tolerance of plants; more preferably, the regulation of the biological function is realized through the GA pathway.

The third aspect of the present invention provides the application of the gene according to the first aspect of the present invention or the protein according to the second aspect of the present invention in the regulation of plant rhizome number and/or the regulation of plant drought tolerance.

Preferably, the plant is Compositae; more preferably, the plant is Chrysanthemum.

Preferably, said regulation is achieved through the GA pathway.

Preferably, the application is realized by changing the expression of the gene or the protein in the plant, thereby affecting the function of CmGA2ox6 in the plant, thereby realizing the regulation.

Genetic engineering techniques are well known to those skilled in the art, and methods or techniques for changing the expression of genes in plants are also known. Those skilled in the art can fully select the gene that can change known genes according to the needs according to the content disclosed in this application. expressed in plants. For example, gene expression in plants can be blocked or reduced by gene silencing such as various known RNA interference (RNAi) techniques. Promoting the expression of the gene or the protein in the plant can be achieved by introducing the gene into the plant and overexpressing the gene in the plant through various known transgenic techniques.

Thus, the present invention provides a plant breeding method in a fourth aspect, characterized in that the method includes changing the gene described in the first aspect of the present invention or the protein described in the second aspect of the present invention by means of genetic engineering techniques overexpressed in plants.

Compared with the prior art, the present invention has at least the following technical effects: by changing the expression of CmRH56, the number of rhizomes of plants can be regulated. For example, by overexpressing CmRH56 in plants, the number of plant rhizomes can be increased. Conversely, the expression of CmRH56 in plants can be inhibited to reduce the number of plant rhizomes and improve the drought tolerance of plants.

Description of drawings

Figure 1 shows the change of rhizome number of chrysanthemum under drought stress. Among them, (a) Rhizome phenotypes under different degrees of drought stress. The plants were subjected to different degrees of drought stress by keeping the irrigation amount at 50%, 20%, and 10% relative water content (RWC) for 30 days. (b) The number of rhizomes under different degrees of drought stress. Results are mean values of ten replicates with standard deviations. Letters indicate significant differences (P<0.05) according to the Tukey-Kramer test. Scale bar, 5 cm.

Figure 2 shows the expression and subcellular localization of the CmRH56 GFP fusion protein. Among them, (a) in situ hybridization showing the localization of CMRH56 mRNA in rhizome and axillary meristem. The sense CmRH56 probe was hybridized against the rhizome meristem. Scale bar represents 200 μm. (b) Quantitative real-time fluorescence (qRT)-PCR was used to detect the expression of CmRH56 in the rhizomes of chrysanthemums grown under different degrees of drought stress. Plants were grown under different degrees of drought stress, with irrigation controlled at 20% or 10% of RWC. Ubiquitin was used as a control gene for qRT-PCR, and statistical significance between different drought treatments and controls was determined by Dunnett's test (**P<0.01). (c) Subcellular localization of CmRH56-GFP fusion protein in Arabidopsis protoplasts. Images are shown as bright field (left), dark field (middle), and merged field (right).

Figure 3 shows the drought tolerance of CmRH56 OX and CmRH56 RNAi chrysanthemum plants. Among them, (a) Phenotypes of CmRH56 OX and CmRH56 RNAi plants under dehydration conditions. OX-1 and OX-2 correspond to two independent CmRH56 OX lines. RNAi-1 and RNAi-2 correspond to two independent CmRH56 RNAi lines. OX, RNAi, and wild-type (WT) plants were grown in a single pot and maintained with a 35-day water supply before a 30-day recovery period and watered regularly. Arrows indicate resurrected axillary buds, and red arrows indicate resurrected rhizome buds. The scale bar represents 5 cm. (b) Expression of CmRH56 in CmRH56 OX, CmRH56 RNAi and WT plants. Results are mean values of five replicates with standard deviations shown. (c) Rhizome and axillary bud survival of CmRH56 OX, CmRH56 RNAi, and WT plants grown under drought stress. Three independent experiments were performed using 10 plants per row in each experiment. Letters indicate significant differences (P<0.05) according to the Tukey-Kramer test.

Figure 4 shows the rhizome growth of CmRH56 overexpression (OX) and RNAi chrysanthemum plants under normal growth and drought stress conditions. Among them, (a) Phenotypes of CmRH56 OX and CmRH56 RNAi chrysanthemum plants grown under normal and drought stress conditions. Plants were grown under normal and drought stress conditions with watering maintained at 20% relative water content (RWC) for 30 days. (b) Phenotypes of rhizome emergence 14 days after transplantation of CmRH56 OX and CmRH56 RNAi chrysanthemums. Arrows indicate rhizomes. The scale bar represents 5 cm. (c) Rhizome number of CmRH56 OX and CmRH56 RNAi chrysanthemums under normal and drought stress conditions. Results are mean values of 12 replicates with standard deviations shown. According to the Tukey-Kramer test, letters indicate significant differences (P<0.05), uppercase letters and lowercase letters indicate significant differences between transgenic plants and wild type under normal conditions (control) and drought stress (20% RWC), respectively. (d) Days when rhizomes were first observed in CmRH56 OX and CmRH56 RNAi chrysanthemum plants grown under normal conditions. Results are mean values of 12 replicates with standard deviations shown. Letters indicate significant differences (P<0.05) according to the Tukey-Kramer test.

Figure 5 shows that CmRH56 RNAi chrysanthemums are defective in mRNA export. Among them, using the leaves of WT and CmRH56 RNAi chrysanthemums, in situ hybridization was performed using an Alexa-488-labeled oligonucleotide (dT) probe. Scale bar represents 60 μm.

Figure 6 shows the number of rhizomes of CmRH56 RNAi chrysanthemums treated with GA and PAC. Among them, (a) Rhizome phenotypes of CmRH56 RNAi and wild-type (WT) chrysanthemums treated with GA or PAC. (b) The number of rhizomes of CmRH56 RNAi and WT chrysanthemum plants grown under GA and PAC treatments. Statistical significance between different drought treatments and controls was determined using Dunnett's test (**P<0.01). The scale bar represents 5 cm.

Figure 7 shows that CmRH56 affects rhizome growth by regulating GA biosynthesis. Among them, (a) the expression of CmGA2ox6 in CmRH56 RNAi and WT plants was determined by qRT PCR. (b) Localization of CmGA2ox6 mRNA in rhizome and axillary meristems. The rhizome meristem was used as a control, and hybridization was performed using the sense CmGA2ox6 probe. Scale bar represents 200 μm. (c) The expression pattern of CmGA2ox6 in rhizomes grown under different degrees of drought stress was determined by qRT-PCR. (d) Rhizome phenotypes of CmGA2ox6RNAi and WT chrysanthemums under drought stress. Under drought stress conditions, chrysanthemums were grown by controlling the amount of irrigation at 20% relative water content (RWC).

Figure 8 shows the phenotypic characteristics of CmRH56 transgenic chrysanthemum plants.

Figure 9 shows the expression of GA pathway genes in CmRH56 RNAi and WT plants detected by qRT-PCR. Ubiquitin was used as a control gene for qRT-PCR. Chrysanthemum GA pathway genes were obtained from the Chrysanthemum Transcriptome Database (http://www.icugi.org/chrysanthemum/). Statistical significance between WT plants and different transgenic lines was determined using Dunnett's test (**P<0.01).

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be described more clearly and completely below in conjunction with the embodiments. However, it should be understood that the described embodiments are some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example

Materials and methods

Plant material and handling

In this study, chrysanthemum species (Chrysanthemum morifolium cv. Fall Color) were used as materials. Chrysanthemum plants are propagated by tissue culture. 40-day-old plants were transplanted into 14 cm diameter pots containing peat and vermiculite (1:1, v/v; 144 g/pot). The growth conditions were 23±1°C, 100 μmol m ⁻² s ⁻¹ , and fluorescent lamps (SINOL, SN-T5, 16W) were used for illumination under a 16-hour light/8-hour dark photoperiod. Another 144g of soil was dried at 65°C for 72 hours (Wd). Relative water content (RWC) was calculated as follows: RWC (%)=(Wt-Wd)/Wt×100%.

For different degrees of drought stress treatment, the plants grown for 2 weeks after transplanting were watered to saturation, and then water control treatment was carried out to gradually reduce the RWC of the soil to 50%, 20% and 10%, which took 17 days and 25 days respectively. days and 30 days. To maintain RWC at 50%, 20% and 10%, daily rehydration was performed by injecting water into the soil around the plant roots.

To determine survival under drought stress, plants grown 2 weeks after transplanting were first fully watered and then left for an additional 35 days before being watered again. After 30 days of rehydration, the survival rate of the plants and the regeneration rate of rhizomes were measured.

For GA3 and PAC (paclobutrazol) treatments, plants were irrigated with 100 μM GA3 or 100 μM PAC solution. The GA3 or PAC solution was applied every 10 days, and the number of rhizomes was observed after 50 days of treatment.

Subcellular localization of CmRH56

Use primer PCR to amplify the CmRH56 open reading frame (ORF, see SEQ ID NO.1 for gene sequence, see SEQ ID NO.2 for protein sequence) without stop codon, introduce BamHI and SalI restriction sites into the amplified fragment, It was then cloned into the pEZS NL vector. See Table 1 for primers. Using PEG-mediated transformation, the resulting construct was transformed into Arabidopsis protoplasts, where after culturing the transformed cells at 22°C for 16-20 h, the Olympus FV1000 confocal laser scanning microscope (Olympus) (488 nm excitation, 525 nm launch) to capture the image.

Real-time quantitative PCR analysis

Total RNA was extracted from chrysanthemum rhizomes using TRIzol reagent (Invitrogen), and cDNA was synthesized by TianScript IIRT kit (Tiangen Biotech). qRT PCR was performed in standard mode using the ABI StepOne Real-Time PCR System (Tiangen Biotech). Each gene was analyzed by 3 biological and 3 technical replicates. Chrysanthemum ubiquitin gene (GenBank No. EU862325) was used as an internal control. Primers used for qRT-PCR are listed in Table 1.

Chrysanthemum transformation

To construct the 35S:CmRH56 overexpression vector, a cDNA fragment containing the CmRH56 ORF was amplified and cloned into pBIG vector.

In order to construct the CmRH56 RNAi and CmGA2ox6 RNAi vectors, the 261bp CmRH56 and 238bp CmGA2ox6 sense and antisense fragments were amplified and inserted into the pHANNIBAL vector on both sides of the Pdk intron. The ihpRNA construct was then cloned into the pART27 binary vector.

The resulting plasmid was transformed into chrysanthemum flowers using the Agrobacterium-mediated leaf disc transformation method (Wei, Q. et al. Control of chrysanthemum flowering through integration with an aging pathway. NatCommun 8, 829 (2017).). The PCR primers used for vector construction are listed in Table 1.

in situ hybridization

Chrysanthemum rhizomes and axillary bud tops were fixed with 3.7% formaldehyde-acetic acid, sectioned and hybridized (see also Zhang, X. et al. Transcription repressor HANABA TARANU controls flower development by integrating the actions of multiple hormones, floral organ specification genes, and GATA3 family genes in Arabidopsis. Plant Cell 25, 83-101 (2013)). CmRH56 probe (236bp) and CmGA2ox6 probe (299bp) are linearized fragments designed according to the unique region of the corresponding coding sequence. Primers are listed in Table 1.

Detection of Poly(A)RNA by in situ hybridization

The leaves of 40-day-old WT and CmRH56-RNAi chrysanthemums were immersed in 1 ml of fixation solution (120 mM NaCl, 7 mM Na ₂ HPO ₄ , 3 mM NaH ₂ PO ₄ , 2.7 mM KCl, 0.1% Tween 20, EGTA80, 5% formaldehyde, 10% DMSO and 50% heptane). Samples were incubated 2 times in 1 ml methanol, 3 times in 1 ml ethanol, and then immersed in 1 ml ethanol/xylene (1:1) for 30 min at room temperature. Add formaldehyde-free methanol/fixation solution (1:1) twice for 5 min each. Samples were incubated in 1 ml PerfectHyb Plus (Sigma) for 1 hour at 50°C. Samples were incubated overnight at 50°C in 1 μl of 10 μM Alexa Fluor-488 labeled 48-mer oligo d(T) (Invitrogen). Images were taken with an Olympus FV1000 confocal laser scanning microscope (Olympus) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Table 1. List of primers used in the examples

Example 1: Rhizome growth is significantly reduced under drought stress

In order to study the effect of drought stress on the rhizome growth of Chrysanthemum chrysanthemum, the inventors first studied the field changes under normal irrigation and drought stress conditions. The inventors observed that plants grown under drought stress had significantly fewer rhizomes than plants grown under normal irrigated conditions (Fig. 1a, b). Then, the present inventors studied the changes of rhizomes under different degrees of drought stress in a controlled environment growth chamber. Water was maintained at 50% relative water content (RWC, mild drought), 20% RWC (moderate drought) and 10% RWC (severe drought) for 30 days. Compared to control plants, growth (plant height) was reduced and the number of rhizomes was significantly reduced under all different drought conditions and decreased significantly with decreasing content (Fig. 1c,d).

Example 2: Decreased expression in rhizomes of CmRH56 under drought stress

In the study of the present inventors on the expression of drought-response genes in rhizomes, a gene encoding DEAD-box RNA helicase CmRH65 was found, and the expression of the gene was significantly changed in rhizomes. qRT-PCR revealed that the expression of CmRH56 was significantly downregulated in rhizomes exposed to different degrees of drought stress (Fig. 2b). Furthermore, in situ hybridization revealed that CmRH56 was highly expressed at the apex of rhizomes, but its expression was not detected at the apex of axillary buds (Fig. 2a).

The present inventors examined the subcellular localization of CmRH56 by a fusion protein comprising CmRH56 fused to green fluorescent protein (CmRH56 GFP), which was transiently expressed in onion epidermal cells. The CmRH56-GFP fusion protein was localized to the nucleus, whereas GFP protein was only detected in the cytoplasm and nucleus, suggesting that CmRH56 is a nuclear protein (Fig. 2c).

Example 3: DEAD-box RNA helicase CmRH56 is involved in the growth of rhizomes under drought stress

To elucidate the role of CmRH56 in drought tolerance, the inventors established transgenic chrysanthemum lines in which CmRH56 was suppressed by RNA interference (RNAi) or ectopically overexpressed. The results showed that when grown under non-stress conditions, the aerial parts of CmRH56 RNAi or overexpressed (OX) plants exhibited normal phenotypes based on comparison with wild-type (WT) plants (Fig. 3a,b; Fig. 8). However, under drought stress, CmRH56RNAi plants had a higher survival rate, whereas CmRH56 OX plants had a lower survival rate than wild-type plants (Fig. 3a, c). The inventors observed that most of the surviving RNAi plants were revived from the axillary buds of the main plant. In contrast, CmRH56 OX plants revived through rhizome survival (Fig. 3a,c).

To further study the effect of ectopic CmRH56 expression on drought stress tolerance associated with rhizomes, the present inventors also detected rhizomes of CmRH56 OX and -RNAi chrysanthemums. Under normal and drought stress conditions, the rhizome number of CmRH56 RNAi plants was significantly lower than that of wild-type plants, but significantly higher than that of CmRH56 OX plants (Fig. 4a,c). Under drought conditions, the number of rhizomes of CmRH56 RNAi plants was significantly less than that of wild-type plants, but higher than that of wild-type CmRH56 OX plants (Fig. 4a, c). Furthermore, the inventors observed that rhizome development was earlier in CmRH56 OX plants, whereas CmRH56 silencing delayed rhizome development (Fig. 4b,d). The rhizomes of CmRH56 OX lines appeared about 3 days earlier than those of WT lines, while those of CmRH56 RNAi lines appeared about 7 days later than those of WT lines (Fig. 4d).

The inventors also tested the function of CmRH56 in chrysanthemum mRNA export. The results showed that the total mRNA export of CmRH56-RNAi chrysanthemum was blocked compared with WT plants (Fig. 5), suggesting that CmRH56 is involved in mRNA export.

Example 4: GA biosynthesis is involved in CmRH56 regulation of rhizome response to drought

In view of the role of GA in rhizome growth, the present inventors investigated whether CmRH56 might be involved in rhizome growth by affecting the GA pathway. In WT plants, we observed that GA treatment resulted in an increase in the number of rhizomes, whereas treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) resulted in a decrease in the number of rhizomes, suggesting a role for GA in controlling rhizome growth in chrysanthemums (Fig. 6). . Application of GA to CmRH56-RNAi plants rescued the reduced rhizome number phenotype (Fig. 6), suggesting that GA treatment blocked the inhibitory effect of CmRH56 silencing.

The present inventors then analyzed the expression of GA pathway genes in CmRH56-RNAi and WT plants by qRT-PCR ( FIG. 9 ). It was found that the expression of putative members of the GA biosynthesis/catabolism gene family GA2ox was significantly upregulated in CmRH56-RNAi plants compared with WT plants (Fig. 7a). The expression of CmGA2ox6 was significantly increased in the rhizomes of WT plants under drought stress (Fig. 7c), and in situ hybridization showed that CmGA2ox6 was highly expressed at the tops of rhizomes, but not detected in the tops of axillary buds (Fig. 7b).

The present inventors also suppressed CmGA2ox6 in chrysanthemums using RNAi (Fig. 7e), and observed significantly more rhizomes in CmGA2ox6-RNAi plants than in WT plants (Fig. 7d,f).

Both rhizomes and aboveground buds arise from the axillary buds of the mother plant, although their developmental patterns differ. In chrysanthemums, the formation of axillary meristems can be identified at early developmental stages of leaf primordia. Therefore, changes in rhizome morphology in response to the environment are related to rhizome growth and development. However, the molecular mechanism by which rhizomes grow under unfavorable growth conditions is unclear, and the present inventors demonstrated that DEAD-box RNA helicase CmRH56 is a regulator of chrysanthemum rhizome growth.

Rhizomes have a major advantage in allowing plants to survive harsh environments. In harsh environments, rhizomes can survive underground, storing and distributing nutrients to support plant growth, and when conditions are suitable for growth, they can develop into above-ground shoots. In addition, the rhizome meristem produces nodes and adventitious roots to extend the root system of the parent plant. Many molecular mechanisms involved in the response of rhizome initiation, growth and development to drought stress are unknown.

The inventors' results showed that under drought stress, the expression of CmRH56 was reduced (Fig. 2b), suggesting that it is involved in rhizome growth in response to drought stress. Furthermore, the survival strategies of CmRH56 RNAi and CmRH56 OX chrysanthemum plants were altered under severe drought stress (Fig. 3a, c). CmRH56 RNAi plants were revived from the axillary buds of the main plant, whereas CmRH56OX plants were revived through rhizome survival (Fig. 3a, c). In the present study, the inventors regulated the expression of GA2ox6 by CmRH56 in a tissue-specific manner, providing evidence for the involvement of the GA pathway in rhizome growth under drought stress.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

SEQUENCE LISTING

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<213> Artificial sequence

<400> 4

accccgggtt atgatggcat gtatgtcgaa g 31

<210> 5

<211> 33

<212>DNA

<213> Artificial sequence

<400> 5

catctcgagg agaagtctgt gttcaagttg atc 33

<210> 6

<211> 34

<212>DNA

<213> Artificial sequence

<400> 6

gagaattcgc taaacattat aatactaaac cacc 34

<210> 7

<211> 32

<212>DNA

<213> Artificial sequence

<400> 7

attctagaga gaagtctgtg ttcaagttga tc 32

<210> 8

<211> 35

<212>DNA

<213> Artificial sequence

<400> 8

cgaaagcttg ctaaacatta taatactaaa ccacc 35

<210> 9

<211> 34

<212>DNA

<213> Artificial sequence

<400> 9

ccgctcgagt ataaatgttc catatattgg aact 34

<210> 10

<211> 33

<212>DNA

<213> Artificial sequence

<400> 10

ggggtaccaa gttgataatt gtctaaagca aag 33

<210> 11

<211> 34

<212>DNA

<213> Artificial sequence

<400> 11

cccaagctta agttgataat tgtctaaagc aaag 34

<210> 12

<211> 34

<212>DNA

<213> Artificial sequence

<400> 12

tgctctagat ataaatgttc catatattgg aact 34

<210> 13

<211> 20

<212>DNA

<213> Artificial sequence

<400> 13

agctgagcag actcccgatg 20

<210> 14

<211> 22

<212>DNA

<213> Artificial sequence

<400> 14

aggcgaatca tcagtaccaa gt 22

<210> 15

<211> 25

<212>DNA

<213> Artificial sequence

<400> 15

gtagcatctg cttcagactc tgatg 25

<210> 16

<211> 23

<212>DNA

<213> Artificial sequence

<400> 16

tgcaaataag atacacgctc cat 23

<210> 17

<211> 20

<212>DNA

<213> Artificial sequence

<400> 17

gcccgtttgg atatgggtgt 20

<210> 18

<211> 20

<212>DNA

<213> Artificial sequence

<400> 18

cgagccaagg gcaatagagt 20

<210> 19

<211> 35

<212>DNA

<213> Artificial sequence

<400> 19

ctggatccca tgatggcatg tatgtcgaag tatca 35

<210> 20

<211> 35

<212>DNA

<213> Artificial sequence

<400> 20

cttgtcgacc aaccaagatc agggacagtg aagtg 35

<210> 21

<211> 50

<212>DNA

<213> Artificial sequence

<400> 21

gatttaggtg acactataga atgctggaga agtctgtgtt caagttgatc 50

<210> 22

<211> 51

<212>DNA

<213> Artificial sequence

<400> 22

tgtaatacga ctcactatag ggccaactag tcttcaagat actaatacca g 51

<210> 23

<211> 45

<212>DNA

<213> Artificial sequence

<400> 23

gatttaggtg acactataga atgcttggct atctccgatc ccaca 45

<210> 24

<211> 42

<212>DNA

<213> Artificial sequence

<400> 24

tgtaatacga ctcactatag ggagacacag tctgcgcatc tt 42

Claims (10)

1. CmRH56 gene, characterized in that the open reading frame of said gene has the nucleotide sequence shown in SEQ ID NO.1. 2. The gene according to claim 1, characterized in that the gene can encode a protein having the amino acid sequence shown in SEQ ID NO.2. 3. A protein, characterized in that the protein has 80%, preferably 90%, more preferably 95, and further preferably 99% identity with respect to the sequence shown in SEQ ID NO.2 and has the same biological function protein. 4. The protein according to claim 3, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2. 5. The protein according to claim 3 or 4, wherein the biological function is regulation of the number of plant rhizomes and/or drought tolerance regulation; more preferably, the regulation of the biological function is achieved by GA way to achieve. 6. The application of the gene according to claim 1 or 2 or the protein according to any one of claims 3 to 5 in the regulation of plant rhizome number and/or drought tolerance regulation. 7. The application according to claim 6, characterized in that, the plant is Compositae; preferably, the plant is chrysanthemum. 8. The application according to claim 6 or 7, characterized in that the regulation is realized through the GA pathway. 9. The application according to any one of claims 6 to 8, characterized in that, the application is realized by changing the expression of the gene or the protein in plants, thereby affecting the expression of CmGA2ox6 in plants function, thereby realizing the regulation. 10. A method for plant breeding, characterized in that the method comprises changing the gene described in claim 1 or 2 or the protein described in any one of claims 3 to 5 in plants by means of genetic engineering techniques Express.

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