Rosa chinensis RhARF6 gene and application thereof in regulation and control of plant flower development

Abstract

The invention discloses a rosa chinensis RhARF6 gene and application thereof in regulation and control of plant flower development. The nucleotide sequence of the Chinese rose 'Corola' RhARF6 gene is as shown in SEQ ID No. 1; the sequence of the corresponding protein is as shown in SEQ ID NO: 2. The RhARF6 gene is excessively expressed in tobacco, so that the number of tobacco petals can be increased, and the color of the petals can be changed. The invention lays a foundation for the molecular mechanism research of abnormal flower development and flower color regulation and control of the Chinese rose, provides an excellent gene for the cultivation of new flower varieties of the Chinese rose through a gene editing technology in future, and has practical significance for the cultivation of the Chinese rose varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to a RhARF6 gene of rose and its application in regulating plant flower development. Background Art

[0002] In the annual production of cut roses, the phenomenon of bent pedicels in roses (BPP) often occurs, and high temperature is one of the reasons for the bent pedicel phenomenon. In the flower organs of rose plants with bent pedicels, there are phenomena of leaf transformation, ectopy and sepal petalization, the petals are reduced and the flower heads are smaller (Shi Liyun et al., 2020), which do not meet the requirements of the commodity standard for upright flower heads and complete flower organs. The flower heads of rose plants with bent pedicels are easily broken during transportation, are not resistant to storage, and the maintenance cannot reach the normal opening degree. Research shows that the formation reason of the bent pedicel phenomenon may be the asymmetric distribution of IAA, and auxin affects the morphogenesis of flower organs and pedicels in roses. The endogenous IAA (3-indoleacetic acid) in the flower buds of the cut rose "Beast" (Rosa Hydrada L. "Beast") maintains a high level during the flower development process in BPP, and is only at a relatively high level in the early stage of flower bud differentiation in the flower buds of normal plants. At the same time, there is no significant difference in the content of gibberellin in normal buds and BPP buds (Seo JH et al., 2013).

[0003] Current research has clearly shown that there are differences in the content of auxin in the malformed flower organs and the asymmetric growth of the pedicel part where the bent pedicel appears, but the specific molecular mechanism regulating the polar transport of IAA needs to be further studied. In addition, as an important regulatory factor of auxin, the ARF gene family may be related to the abnormal expression of transcription factors in this family for this malformation phenomenon, and its molecular mechanism also needs to be further explored. ARFs play a crucial role in regulating the growth, development, senescence and abscission of roots, leaves, flowers, fruits and seeds (Fan Lu, 2019), the response to biotic and abiotic stresses, and the crosstalk of plant hormone signals. With the continuous generation of whole-plant genome sequences, the ARF gene families of many plant species have been systematically analyzed.

[0004] Auxin affects all aspects of plant growth and development, including floral organ development (Cheng Y et al., 2007). Auxin response factor 1 and auxin response factor 2 regulate senescence and floral organ abscission in Arabidopsis thaliana (Yamada K et al., 2009). The arf2 mutant of Arabidopsis thaliana shows delayed flowering, dense and long inflorescences, abnormal flower morphology, and early floral sterility (Okushima Y et al., 2005). The Arabidopsis thaliana member AtARF3 determines the number of floral organ primordia and affects the development of primordia. The loss of its function leads to an increase in the number of perianth organs such as sepals and petals, a decrease in the number of stamens and anther formation, and a disordered pistil structure (Sessions R A et al., 1995). MP / ARF5 can induce the expression of several direct targets, and its activity is concentrated in the initiation of floral primordia (Przemeck G K H et al., 1996). The arf6 and arf8 double mutants show short petals, short stamen filaments, and non-dividing anthers (no pollen release). It has also been reported that AtARF6 and AtARF8 play redundant roles in flower maturation; the arf6 and arf8 single mutant plants show delayed stamen development and reduced fertility, while the arf6 and arf8 double mutant plants show a complete blockage of flower maturation (Nagpal P et al., 2005). In addition, AtARF8 can interact with BIGPETALp (BPEp), thereby affecting cell expansion and restricting the growth of Arabidopsis thaliana petals (Varaud E et al., 2011). AtARF17 plays an important role in pollen wall formation and development (Yang J et al., 2013). In tomatoes, high expression levels of SlARF1, 9, 11, 15, and 16 were observed during flowering, indicating that these genes may be related to flower development (Kumar R et al., 2011).

[0005] In summary, using gene editing technology to improve plant flower types has become one of the effective methods. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Rosa hybrida RhARF6 gene and its application in regulating plant flower development. First, the nucleotide coding sequence of the RhARF6 gene was cloned, and an expression vector was constructed. Then, the complete translation region of the RhARF6 gene was combined with the cauliflower mosaic virus promoter and transferred into a plant body (host tobacco) to obtain RhARF6 overexpressing plants. Phenotypic observations were made on the transgenic tobacco to evaluate the application prospects of this gene in regulating plant flower development.

[0007] In the floral organs of transgenic tobacco, the number of petals increases and the petal color becomes lighter. The present invention has important reference significance for overexpressing this gene through gene editing technology in the future to change plant flower types and cultivate new varieties of roses.

[0008] To achieve the above object, the technical solution designed by the present invention is as follows:

[0009] The present invention provides a RhARF6 gene of rose, and its nucleotide sequence is shown as SEQ ID No.1.

[0010] The present invention also provides a protein RhARF6 encoded by the above gene, and its amino acid sequence is shown as SEQ ID No.2.

[0011] The present invention also provides a primer pair for the above RhARF6 gene, and the primer pair is:

[0012] Forward primer of RhARF6: 5’-GCTTGCTCGTATGTTTGGCCTTG-3’, Reverse primer of RhARF6: 5’-GGAGAGCCTCTGCATTGGGACA-3’.

[0013] The method for obtaining the above RhARF6 gene is as follows:

[0014] Using the cDNA of rose ‘Carola’ flower buds as a template and the above primer pair of RhARF6 gene, perform PCR amplification, and purify to obtain the RhARF6 gene; wherein,

[0015] PCR system:

[0016]

[0017] PCR conditions:

[0018]

[0019] The present invention also provides a recombinant expression vector, and the recombinant expression vector contains a plant expression vector of the above RhARF6 gene, wherein the plant expression vector is pCAMBIA2300s.

[0020] The construction method of the above recombinant expression vector includes the following steps:

[0021] Connect the target fragment into the cloning vector pClone007 versatile Simple Vector Kit, then perform double digestion of the cloning vector pClone007-RhARF6 containing the target gene and pCAMBIA2300s with KpnI and XbaⅠ, and then connect the obtained target fragment to the digested pCAMBIA2300s vector to obtain the recombinant expression vector pCAMBIA2300s-RhARF6.

[0022] The present invention also provides a host cell containing the above-mentioned recombinant expression vector, and the host cell is Agrobacterium tumefaciens GV3101.

[0023] Use of any one of the following in regulating plant flower development, which includes:

[0024] (1) The above-mentioned RhARF6 gene;

[0025] (2) The above-mentioned recombinant expression vector;

[0026] (3) The above-mentioned host cell.

[0027] Use of any one of the following in regulating the flower shape and / or flower color of plants, which includes:

[0028] (1) The above-mentioned RhARF6 gene;

[0029] (2) The above-mentioned recombinant expression vector;

[0030] (3) The above-mentioned host cell.

[0031] Further, the plant is tobacco or rose.

[0032] Use of any one of the following in cultivating new plant varieties with different flower shapes and / or different flower colors, which includes:

[0033] (1) The above-mentioned RhARF6 gene;

[0034] (2) The above-mentioned recombinant expression vector;

[0035] (3) The above-mentioned host cell.

[0036] Still further, the plant is tobacco or rose.

[0037] Advantages of the present invention:

[0038] The gene of the present invention is named RhARF6. By constructing an overexpression vector pCAMBIA2300s-RhARF6 and introducing it into tobacco, transgenic tobacco is obtained. Compared with the control tobacco, the transgenic tobacco has an increased number of petals and a change in petal color. The RhARF6 gene provides a molecular biological basis for us to regulate plant flower development by means of plant genetic engineering. The application of this gene will help to change the flower shape of plants, improve the quality of plants, and cultivate new varieties with excellent traits.

[0039] In summary: The present invention can regulate the development of plant floral organs through gene editing technology, change the flower shape of plants, and improve the yield and ornamental value of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1It is a schematic diagram of the structure of the original plant overexpression vector pCAMBIA2300s (the vector size is 11630bp);

[0041] Figure 2 It is a diagram showing the expression of the RhARF6 gene,

[0042] In the figure, A is a diagram showing the expression of the RhARF6 gene at different developmental stages of flower buds in normal and bent-stem plants of Rosa hybrida 'Carola',

[0043] B is a diagram showing the expression of the RhARF6 gene in different organs of flowers in Rosa hybrida 'Carola' plants,

[0044] C is a legend for different developmental stages of flower buds in normal and bent-stem plants of Rosa hybrida 'Carola',

[0045] D is a legend for different organs of flowers in Rosa hybrida 'Carola' plants.

[0046] Note: Normal: normal plant, Bend: bent-stem plant, NS: normal sepal, NP: normal petal, BS: falling sepal, BP: transformed sepal, BU: dorsal side of bent stem, BD: lateral side of bent stem, N: normal flower stalk.

[0047] Figure 3 It is a construction diagram of the expression vector pCAMBIA2300s-RhARF6 (the inserted fragment size is 2679bp).

[0048] Figure 4 It is a diagram for analyzing the flower types of wild-type tobacco and RhARF6 transgenic tobacco.

[0049] In the figure, A is the corolla (front view), B is the stamen, C is the sepal, D is the pistil, and E is the corolla (side view). Specific implementation manners

[0050] The following further describes the present invention in detail with reference to specific embodiments for those skilled in the art to understand.

[0051] Example 1 Isolation of the RhARF6 gene

[0052] The RhARF6 gene was isolated from Rosa hybrida 'Carola', and its sequence is shown as SEQ ID No.1:

[0053]

[0054] Protein RhARF6, whose amino acid sequence is shown in SEQ ID No. 2:

[0055] MRLSSAGFSPQPQEGEKRVLNSELWHACAGPLVCLPAVGSRVVYFPQGHSEQVAASTNKEVDAHIPNYPSLPPQLICQLHNVTMHADVETDEVYAQMTLQPLNPQEQKDGYLPAGLGSPNKQPTNYFCKTLTASDTSTHGGFSVPRRAAEKVFPPLDFSQQPPAQELIARDLHDNEWKFRHIFRGQPKRHLLTTGWSVFVSAKRLVAGDSVLFIWNEKNQLLLGIRRANRPQTVMPSSVLSSDSMHLGLLAAAAHAASTNSRFTIFYNPRASPSEFVIPLAKYIKAVYHTCISVGMRFRMLFETEESSVRRYMGTITGISDLDAARWPNSHWRSVKVGWDESTAGERQPRVSLWEIEPLTTFPMYPSPFPLRLKRPWPPGLPSFTGMREDDLSMNTPLMWLRGDTGDRGIQSLNYHGIGVTPWMQPRLDVSMIGLQTDMYQAMAAAALQEMRAVDPSKSLHTSLLQFQQTQNLPSRSAALMQPQMVQESQPQPAFLQGVQENHRQSHSQTPTQSHLQHQLQHQNSFSNQQQQLLDHQQIPSAVSSMNHFASASQSQSPSLQVITSLCQQQSFSDSNGNPATSTILSPLQSLMGSFSQDESSNLLNMPRTNSLISSSGWPSKRAAIEPLLSSGVSQCVLPQVEQLGPPHTTISQNPISLPPFPGRECSIDQEGSTDPQTHLLFGINIESSPLLMQNGMSNLRGVGSDSDSTTIHFPSNYMSPTGTDFSLNPAVTPSSCIDESGFLQSSENVGHENPPNGNFVKVYKSGSFGRSLDITKFSSYHELRSELARMFGLEGELEDPVRSGWQLVFVDRENDVLLLGDDPWPEFVNSVWCIKILSPQEVQQMGKRGLELLKSVPMQRLSGNSCDEYGGSRQDSRNLSSGITSVGSLEY

[0056] Using the cDNA of Rosa hybrida 'Carola' flower buds as a template to amplify the required fragment, the specific steps are as follows:

[0057] Design a primer pair for the RhARF6 gene according to the RhARF6 sequence shown in SEQ ID No.1:

[0058] RhARF6 forward primer: 5’-GCTTGCTCGTATGTTTGGCCTTG-3’, RhARF6 reverse primer: 5’-GGAGAGCCTCTGCATTGGGACA-3’.

[0059] Using the cDNA obtained by reverse transcription of the RNA extracted from Rosa hybrida 'Carola' flower buds as a template and the above-mentioned primer pair of the RhARF6 gene for PCR,

[0060] PCR system:

[0061] T3 Super PCR Mix 22 μl Forward primer 1 μl Reverse primer 1 μl Template 1 μl Total 25 μl

[0062] PCR conditions:

[0063] Pre-denaturation at 94°C 3 min 94℃ 30 sec 60℃ 30 sec 72℃ 1 min Extension at 72°C 5 min

[0064] Purify the amplification product to obtain the RhARF6 gene;

[0065] Ligate the amplified RhARF6 gene into the pClone007 vector (purchased from Tsingke Biotechnology Co., Ltd.), screen positive clones and sequence them to obtain the required full-length gene; this clone is named pClone007-RhARF6 plasmid.

[0066] Example 2 Detection of RhARF6 gene expression during the development of deformed flowers of Rosa hybrida 'Carola'

[0067] The total RNA extraction method for Rosa hybrida uses the CTAB method to extract the RNA of samples at different developmental stages of flower buds in normal and bent-stem plants. Use the Evo M-MLV PIus cDNA synthesis kit for reverse transcription, and the specific steps can be found in the operation manual (CodeNo.AG11615). Use the qRT-PCR method to detect the expression of the RhARF6 gene. Use the TAKARA fluorescence quantitative PCR kit (RR430A, TaKaRa) for qRT-PCR reaction, and the instrument model used is the iQ5 RT-PCR detectionsystem (Bio-Rad, USA). Each sample is subjected to three technical replicates, and the 2 -△△CT method is used to calculate the relative expression level, and the SPSS software is used to statistically analyze the original data.

[0068] The specific primer sequences for the qRT-PCR reaction are:

[0069] qP-RhARF6-F: 5’-AGGAGTTGGCAGCGATAGTGATTC-3’;

[0070] qP-RhARF6-R: 5’-TGGAAGGTGTAACTGCTGGATTGAG-3’;

[0071] The RcGAPDH gene was selected as the internal reference gene, and the primer sequences were:

[0072] qGAPDH-F: 5’-CTCAGACTCCTCCTTGATAGC-3’;

[0073] qGAPDH-R: 5’-TTCTGCCTGCTCTCAATGG-3’.

[0074] Real-time fluorescence quantitative detection found that the expression level of RhARF6 was the highest in NP (normal petals), and the expression level in BS (falling sepals) was significantly higher than that in NS (normal sepals), while the expression level in BP (transformed sepals) was significantly lower than that in NP (normal petals). The expression level in BU (dorsal side of the bent pedicel) was higher than that in BD (lateral side of the bent pedicel), and both were higher than that in N (normal pedicel). RhARF6 was relatively high in the late stage of flower bud development (S4 stage) of normal plants and the highest in the early stage of flower bud development (S2 stage) of bent-pedicel plants ( Figure 2 ).

[0075] Example 3 Construction and transformation of the RhARF6 gene overexpression vector

[0076] To clarify the function of this gene, the cloned gene fragment was overexpressed in tobacco, and its function was verified by observing the phenotypes of transgenic tobacco. The specific steps are as follows:

[0077] First, the positive clone pClone007-RhARF6 plasmid obtained in Example 1 was double-digested with KpnI and XbaⅠ to recover the target fragment; at the same time, the overexpression vector pCAMBIA2300s was digested with the same method ( Figure 1 ). After digestion, a ligation reaction was performed using the digested fragment containing the RhARF6 gene and the digested pCAMBIA2300s ( Figure 1 ) vector, and then transformed into Escherichia coli DH5α (the Escherichia coli strain was purchased from Tsingke Biotechnology Co., Ltd.). Positive clones were screened by PCR amplification to obtain the transformation vector, which was named pCAMBIA2300s-RhARF6 ( Figure 3 ).

[0078] By means of the Agrobacterium-mediated tobacco genetic transformation method, pCAMBIA2300s-RhARF6 was introduced into wild-type tobacco. Through infection, co-culture, and screening, transformed seedlings with resistance were obtained.

[0079] Then, through conventional steps such as rooting, acclimatizing the seedlings, and transplanting, transgenic plants were obtained.

[0080] The main steps and application reagents of the above genetic transformation are as follows:

[0081] Abbreviations of reagents and solutions: The abbreviations of plant hormones used in the culture medium are as follows: 6-BA (6-BenzylaminoPurine, 6-benzylaminopurine); NAA (Naphthalene acetic acid, naphthaleneacetic acid); Kan (Kanamycin, kanamycin); Cef (Cefotaxime, cefotaxime).

[0082] 1) Culture medium formula for tobacco genetic transformation

[0083] Table 1 lists the components and dosages of the culture medium for tobacco genetic transformation

[0084]

[0085] Adjust the pH of the culture medium to 6.0 before sterilization.

[0086] 2) Agrobacterium infection of tobacco tissue and acquisition of resistant seedlings:

[0087] a. Cut the fully expanded and tender upper leaves of wild-type tobacco sterile seedlings into small pieces of 1 cm x 1 cm and place them in a sterile beaker.

[0088] b. Pour the prepared bacterial solution into the beaker and gently shake the beaker. Soak the leaves in the bacterial solution for 8 min.

[0089] c. Take out the leaves in step b, transfer them to sterilized filter paper to dry; then place them on the co-culture medium as described in Table 1 and incubate them in the dark for three days at a culture temperature of 28°C.

[0090] d. After three days, transfer the leaves to the budding selection medium as described in Table 1 and culture them alternately with light and dark (light intensity: 1000 - 1500 lx, light time: 16 h / d, dark time: 8 h / d) for screening and differentiation of resistant buds at a culture temperature of 28°C.

[0091] e. After the formation of resistant buds, cut them off and transfer them to the strong seedling selection medium as described in Table 1.

[0092] Alternate cultivation with light and dark culture (light intensity: 1000 - 1500 lx, light duration: 16 h / d,

[0093] dark duration: 8 h / d) was used for screening resistant seedlings, and the cultivation temperature was 28°C;

[0094] f. Transfer the screened resistant seedlings to the rooting selection medium described in Table 1 to make them root.

[0095] Alternate cultivation with light and dark culture (light intensity: 1000 - 1500 lx, light duration: 16 h / d,

[0096] dark duration: 8 h / d) was carried out, and the cultivation temperature was 28°C.

[0097] Example 4: Observation on the flower phenotype of transgenic tobacco RhARF6

[0098] Carry out flower phenotype observation on the control and transgenic tobacco plants that have grown to flowering as Figure 4 can be seen: At the same time, in the overexpressing plants, the petals of RhARF6 transgenic tobacco differentiated into 6 pieces, with 1 more sepal and petal each compared to the control tobacco. It can be seen that RhARF6 is involved in the formation of sepal and petal primordia. At the same time, the longitudinal splitting of the petals of RhARF6 transgenic tobacco deepened compared to the control, and the color became lighter compared to the control tobacco. The flower color of the control tobacco was pink, and the petal color of RhARF6 transgenic tobacco changed to light pink. It can be seen that RhARF6 may be involved in the synthesis or accumulation of anthocyanins.

[0099] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A rose RhARF6 gene, whose nucleotide sequence is shown in SEQ ID No.

1.

2. A protein RhARF6 encoded by the gene according to claim 1, whose amino acid sequence is shown in SEQ ID No.

2.

3. A primer pair for obtaining the RhARF6 gene according to claim 1, characterized in that: The primer pairs are: RhARF6 forward primer: 5'-GCTTGCTCGTATGTTTGGCCTTG-3', RhARF6 reverse primer: 5′-GGAGAGCCTCTGCATTGGGACA-3′.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the plant expression vector of the RhARF6 gene according to claim 1, wherein the plant expression vector is pCAMBIA2300s.

5. A host cell containing the recombinant expression vector according to claim 4, characterized in that: The host cell is Agrobacterium GV3101.

6. Use of any one of the following in regulating plant flower development, characterized in that: It includes: (1) The RhARF6 gene according to claim 1; (2) The recombinant expression vector according to claim 4; (3) The host cell according to claim 5.

7. Use of any of the following for regulating the flower shape and / or flower color of a plant, characterized in that: It includes: (1) The RhARF6 gene according to claim 1; (2) The recombinant expression vector according to claim 4; (3) The host cell according to claim 5.

8. The use according to claim 6 or 7, characterized in that: The plant is tobacco or rose.

9. Use of any of the following in breeding new plant varieties with different flower shapes and / or different flower colors, characterized in that: It includes: (1) The RhARF6 gene according to claim 1; (2) The recombinant expression vector according to claim 4; (3) The host cell according to claim 5.

10. The use according to claim 9, characterized in that: The plant is tobacco or rose.

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