CN116804199A - China rose RhSEP3 gene and application thereof in regulating and controlling plant flower organ development - Google Patents
China rose RhSEP3 gene and application thereof in regulating and controlling plant flower organ development Download PDFInfo
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Abstract
The invention discloses a China rose RhSEP3 gene and application thereof in regulating and controlling plant flower organ development; the nucleotide sequence of the China rose 'kala' RhSEP3 gene is shown as SEQ ID No. 1; the sequence of the corresponding protein is shown as SEQ ID NO: 2. The RhSEP3 gene is over-expressed in tobacco, so that the number of tobacco sepals and petals can be increased, and the color of the petals can be changed. The invention lays a foundation for research on the molecular mechanism of China rose malformation, provides excellent genes for cultivating new China rose varieties through a gene editing technology in the future, and has practical significance for cultivating China rose varieties.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a China rose RhSEP3 gene and application thereof in regulating and controlling plant flower organ development.
Background
High temperature is one of main abiotic stress factors affecting plant growth and development and propagation, and is used as an important environmental incentive affecting the growth and development of China rose, which can cause plant dysplasia of cut flower China rose card rollers, cause physiological diseases (Dan Liyun and 2018) such as leaf-changing disease, stem bending phenomenon and the like, and cause flower deformity, thereby seriously affecting ornamental properties and economic value of cut flower China rose. The phenomenon of changing leaves of China rose refers to that the flower organ is partially or completely replaced by a leaf-like structure, and an abnormal state of changing leaves of China rose is formed (Li Jingbo, etc., 2010). The phenomenon of rose stem bending is different from neck bending caused by water stress, and is that flower organs cannot be opened normally due to the bending of the flower stem to one side in the growth and development process of the rose (Dan Liyun, etc., 2020). The sepal of the first round organ of the flower in the rose stem bending plant has the phenomena of leaf variation, area enlargement and falling, and the sepal of the second round petal occurs. Research has shown that uneven distribution of hormones during the process of rose plant growth from vegetative to reproductive growth is the main cause of sepal leaf changes, and at the same time causes sepal changes in petals (Cook O F,1926; bond T, 1945). In addition, MADS-box gene family is used as an important regulatory factor for flower organ development, and the malformation phenomenon is probably related to abnormal expression of transcription factors of the family, and the molecular mechanism of the malformation is still to be further explored.
A large set of redundant MADS-box genes, designated SEPALLATA (SEP 1), SEP2, SEP3 and SEP4, were revealed by reverse genetics analysis in Arabidopsis thaliana. In the sep1, sep2, sep3 triple mutants, all the rotagenic organs of the flowers develop into sepals and into new inflorescences from the central region of the flower meristem, so that the development of the flowers becomes uncertain (Pelaz et al, 2000); in the four-fold mutants of sep1, sep2, sep3, sep4, vegetative leaves, but not sepals, were produced in all round of indeterminate "flowers" (Ditta G et al, 2004). In arabidopsis, the SEP gene is an important factor in determining the flowering state of plants. SEP3 is a class E functional gene, a member of the SEP MADS-box subfamily (Zahn et al 2005). Although the function of the SEP gene is highly redundant, SEP3 appears to be more important in flower and ovule development than SEPs 1, 2, 4 (Castillejo C et al,2005;Favaro R et al,2003). Studies of SEP subfamily phylogenetic development have shown (Zahn et al, 2005) that gene duplication of angiosperms resulted in two SEP lineages, one containing SEP3 and the other undergoing at least two more duplicated events, resulting in SEP1, SEP2 and SEP4, approximately 3 hundred million years ago. Since SEP3 evolved much longer than other SEP genes, it is possible to obtain a function to distinguish it from other SEP genes during this time.
In summary, the improvement of plant flower type by gene editing technology is one of the effective methods.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a China rose RhSEP3 gene and application thereof in regulating and controlling plant flower organ development; firstly, cloning a nucleotide coding sequence of a RhSEP3 gene; constructing an expression vector, combining a complete translation region of the RhSEP3 gene with a cauliflower mosaic virus promoter, and transferring the combined expression vector into a plant body (host tobacco) to obtain a RhSEP3 overexpression plant; phenotype observation is carried out on the transgenic tobacco so as to evaluate the application prospect of the gene in regulating the development of plant flowers.
The number of sepals and petals in the flower organ of the transgenic tobacco is increased, the color of the petals becomes light, and the flowering period is obviously advanced. The invention has important reference significance for changing plant flower types and cultivating new rose varieties by over-expressing the genes through a gene editing technology in the future.
In order to achieve the aim, the invention designs a China rose RhSEP3 gene, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
The invention also provides a protein RhSEP3 coded by the gene, and the amino acid sequence of the protein RhSEP3 is shown as SEQ ID No. 2.
The invention also provides a primer pair for obtaining the RhSEP3 gene, which is:
RhSEP3 forward primer: 5'-GAGAGAAGAGAAATGGGGAGGGG-3' the number of the individual pieces of the plastic,
RhSEP3 reverse primer: 5'-GGCTTCACCACACAGAGTTGCTT-3'.
The method for obtaining the RhSEP3 gene comprises the following steps:
PCR amplification and purification are carried out by taking China rose 'kala' flower bud cDNA as a template and a primer pair of the RhSEP3 gene to obtain the RhSEP3 gene; wherein, the liquid crystal display device comprises a liquid crystal display device,
PCR system:
PCR conditions:
the invention also provides a recombinant expression vector which contains the plant expression vector of the RhSEP3 gene, wherein the plant expression vector is pCAMBIA2300s.
The construction method of the recombinant expression vector comprises the following steps:
the target fragment is connected with a cloning vector pClone007 versatile Simple Vector Kit, then cloning vectors pClone007-RhSEP3 and pCAMBIA2300s containing target genes are subjected to double enzyme digestion by SmaI and XbaI to be subjected to enzyme digestion, and the obtained target fragment is connected with an enzyme-digested pCAMBIA2300s vector to obtain a recombinant expression vector pCAMBIA2300s-RhSEP3.
The invention also provides a host cell containing the recombinant expression vector, and the host cell is agrobacterium GV3101.
Use of one of the following for regulating plant floral organ development, comprising:
(1) The RhSEP3 gene;
(2) The recombinant expression vector described above;
(3) The host cell described above.
Preferably, the plant is tobacco or rose.
Use of one of the following for growing new plant varieties of new flower types, comprising:
(1) The RhSEP3 gene;
(2) The recombinant expression vector described above;
(3) The host cell described above.
The invention has the beneficial effects that:
the gene of the invention is named as RhSEP3. The transgenic tobacco is obtained by constructing an over-expression vector pCAMBIA2300s-RhSEP3 and introducing the over-expression vector into tobacco. Compared with control tobacco, the transgenic grass has obviously advanced flowering time, increased sepals and petals and changed petal color. The RhSEP3 gene provides a molecular biological foundation for regulating the flower development of plants by means of plant genetic engineering. The application of the gene is helpful for changing the flower shape of plants, shortening the flowering time, improving the plant quality and cultivating new varieties with good characters.
To sum up: the invention can regulate and control the development of plant flower organs through a gene editing technology, change the flower type of plants and improve the yield and ornamental value of the plants.
Drawings
FIG. 1 is a schematic diagram of the structure of the original plant over-expression vector pCAMBIA2300s (vector size 11630 bp);
FIG. 2 is a diagram showing the expression of RhSEP3 gene in China rose 'kala' at different developmental stages of flower buds in curved stem plants;
FIG. 3 is a diagram showing the expression of RhSEP3 genes in different organs of flowers in China rose 'kala' plants;
FIG. 4 is a graph of a flower type analysis of wild type tobacco and RhSEP3 transgenic tobacco of the present invention;
FIG. 5 is a construction diagram of the expression vector pCAMBIA2300s-RhSEP3 (insert size 735 bp).
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
Example 1: isolation of RhSEP3 Gene
RhSEP3 gene is separated from China rose 'kala', and the sequence of the RhSEP3 gene is shown as SEQ ID No. 1:
ATGGGGAGGGGGAGAGTTGAGCTGAAGAGGATTGAGAACAAGATCAACAGGCAGGTGACCTTTGCAAAGAGAAGGAACGGACTATTGAAGAAAGCCTACGAGCTTTCCGTTCTTTGCGATGCCGAGGTTGCTCTCATCATCTTCTCCAATAGAGGAAAGCTGTACGAGTTTTGCAGTAGTTCAAGCATGCTCAAAACACTTGAGAGGTACCAGAAGTGCAACTATGGAGCACCGGAGACAAATGTATCTACAAGGGAGGCCTTGGAATTGAGCAGCCAGCAGGAGTATTTGAAGCTCAAGGCACGTTATGAAGCCCTACAAAGAAACCAAAGGAATCTTCTTGGAGAAGATCTTGGCCCTTTAAGTAGCAAAGAGCTTGAATCACTTGAAAGGCAGCTGGACATGTCATTGAAGCAGATCAGATCAACACGGACCCAATGCATGCTGGATCAGCTCACAGATCTTCAGCGAAAGGAACACATGCTTAATGAAGCAAACAGGACACTGAAACAAAGGTTGTTTGAGGGATACAATGTACATCAACTCCAACTGAATGCAAATGCTGAGGATGTGGGATATGGCCGGCAACAAGCTCATCATCAACCTCAGGGCGAGGGCTTCTTCCAACCCTTAGAGTGCGAGCCCACGTTACAAATTGGGTATCACCAGAATGATCCGATACAAGTTGTGACGGCCGGGCCGAGCGTGAATTACATGGGAGGATGGTTGCCATGA；
the amino acid sequence of the protein RhSEP3 is shown as SEQ ID No. 2:
MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSNRGKLYEFCSSSSMLKTLERYQKCNYGAPETNVSTREALELSSQQEYLKLKARYEALQRNQRNLLGEDLGPLSSKELESLERQLDMSLKQIRSTRTQCMLDQLTDLQRKEHMLNEANRTLKQRLFEGYNVHQLQLNANAEDVGYGRQQAHHQPQGEGFFQPLECEPTLQIGYHQNDPIQVVTAGPSVNYMGGWLP
the method uses China rose 'kala' flower bud cDNA as a template to amplify the required fragment, and comprises the following specific steps:
designing a primer pair of a RhSEP3 gene according to a RhSEP3 sequence shown in SEQ ID No. 1:
RhSEP3 forward primer: 5'-GAGAGAAGAGAAATGGGGAGGGG-3' the number of the individual pieces of the plastic,
RhSEP3 reverse primer: 5'-GGCTTCACCACACAGAGTTGCTT-3'.
And PCR is carried out by taking cDNA after reverse transcription of RNA extracted from China rose 'kala' flower buds as a template and a primer pair of the RhSEP3 gene,
PCR system:
| T3 Super PCR Mix | 22μl |
| forward primer | 1μl |
| Reverse primer | 1μl |
| Template | 1μl |
| Totalizing | 25μl |
PCR conditions:
| pre-denaturation at 94 ℃C | 3min |
| 94℃ | 30sec |
| 60℃ | 30sec |
| 72℃ | 1min |
| Extension at 72 DEG C | 5min |
Purifying to obtain amplified product, namely RhSEP3 gene;
the amplified RhSEP3 gene is connected to pClone007 vector (purchased from the Optimum of the family of the engine) to screen positive clone and sequence to obtain the required full-length gene; this clone was designated pClone007-RhSEP3 plasmid.
Example 2: rhSEP3 gene expression condition detection in China rose 'kala' malformation flower development
The method for extracting total RNA of China rose adopts a CTAB method to extract sample RNA of the flower buds in normal and bent stem plants in different development periods. Reverse transcription was performed using Evo M-MLV PIus cDNA synthesis kit, see the instruction manual (Code No. AG11615) for specific procedures. And detecting the expression condition of the RhSEP3 gene by using a qRT-PCR method. qRT-PCR reactions were performed using a TAKARA fluorescent quantitative PCR kit (RR 430A, taKaRa) using an instrument model of iQ5 RT-PCR detection system (Bio-Rad, USA). Three technical replicates were performed per sample, using 2 -△△CT The relative expression is calculated by the method, and the SPSS software is used for carrying out statistical analysis on the original data.
The specific primer sequences of the qRT-PCR reaction are as follows:
qP-RhSEP3-F：5’-AAATGCTGAGGATGTGG-3’；
qP-RhSEP3-R：5’-CCATCCTCCCATGTAAT-3’；
the RcGAPDH gene is selected as an internal reference gene, and the primer sequence is as follows:
qGAPDH-F：5’-CTCAGACTCCTCCTTGATAGC-3’；
qGAPDH-R：5’-TTCTGCCTGCTCTCAATGG-3’。
the real-time fluorescent quantitative detection shows that the expression quantity of RhSEP3 in the sepals is higher than that of petals, the expression quantity of falling sepals is obviously higher than that of normal sepals, and the expression quantity in the variable sepals is obviously lower than that of normal petals.
Example 3: rhSEP3 gene expression condition detection in rose leaf variation
In order to further investigate the expression mechanism of RhSEP3 in China rose malformation, especially in sepals, RNA at different positions of 6 China rose "flower-changing leaf" varieties was extracted, and qRT-PCR reaction was performed on RhSEP3 with reference to the expression of the internal reference gene RhGAPDH, using an instrument model of iQ5 RT-PCR detection system (Bio-Rad, USA). Three technical replicates were performed for each sample, the relative expression was calculated using the 2-delta-CT method, and statistical analysis was performed on the raw data using SPSS software.
The specific primer sequences of the qRT-PCR reaction are as follows:
qP-RhSEP3-F：5’-AAATGCTGAGGATGTGG-3’；
qP-RhSEP3-R：5’-CCATCCTCCCATGTAAT-3’；
the RcGAPDH gene is selected as an internal reference gene, and the primer sequence is as follows:
qGAPDH-F：5’-CTCAGACTCCTCCTTGATAGC-3’；
qGAPDH-R：5’-TTCTGCCTGCTCTCAATGG-3’。
the real-time fluorescent quantitative detection shows that the expression quantity of the RhSEP3 in the flower stems is highest in the rose leaf-changing varieties, and the expression quantity of the RhSEP3 in sepals is secondary except for 'green sepals'. In addition, the expression level of RhSEP3 in the 'four lovely' petals is higher than that in the carpel, and the expression level of the other varieties in the carpel is higher than that in the petals. Taken together, it is shown that RhSEP3 gene is expressed in China rose flower organs and possibly participates in the flower organ change process of plants.
Example 4: construction of RhSEP3 gene over-expression vector and transformation
To elucidate the function of the gene, the cloned gene fragment was overexpressed in tobacco and its function was verified from transgenic tobacco phenotypes. The method comprises the following specific steps:
firstly, the positive clone pClone007-RhSEP3 plasmid obtained in the example 1 is digested with SmaI and XbaI to recover target fragment; simultaneously, the overexpression vector pCAMBIA2300s was digested in the same way (FIG. 1). After the completion of the cleavage, E.coli DH 5. Alpha. Was transformed by ligation using a vector comprising the cleavage fragment of RhSEP3 gene and the cleavage pCAMBIA2300s (FIG. 1) (E.coli strain was purchased from Optimago Co., ltd.). Positive clones were screened by PCR amplification to obtain a transformation vector, which was designated pCAMBIA2300s-RhSEP3 (FIG. 5).
Introducing pCAMBIA2300s-RhSEP3 into wild tobacco by agrobacterium-mediated genetic transformation method, infecting, co-culturing, screening to obtain transformed seedling with resistance,
and obtaining the transgenic plant through conventional steps such as rooting, seedling training, transplanting and the like.
The main steps and application reagents for the above genetic transformation are as follows:
abbreviations for the phytohormones used for the reagents and solution abbreviation medium are shown below: 6-BA (6-Benzylaminoprone, 6-BenzylaminoPurine); NAA (Naphthalene acetic acid, naphthylacetic acid); kan (Kanamycin ); cef (cefotaxin, cephalosporin).
1) Culture medium formula for genetic transformation of tobacco
The composition of the medium for genetic transformation of tobacco and its amounts are listed in table 1.
2) And (3) infecting tobacco tissues and obtaining resistant seedlings by agrobacterium:
a. cutting young leaves of the wild tobacco aseptic seedling, which are fully unfolded at the upper part, cutting the leaves into small pieces with the size of 1cm x 1cm, and placing the small pieces into an aseptic beaker;
b. pouring the prepared bacterial liquid into a beaker, and gently shaking the beaker. Soaking the leaves in the bacterial liquid for 8min;
c. taking out the leaves in the step b, and transferring the leaves to sterilized filter paper for drying; then placing the mixture on a co-culture medium as shown in table 1 for dark culture for three days at 28 ℃;
d. three days later, the leaves are transferred to a bud selection medium as shown in Table 1, and alternate culture is carried out by adopting illumination and dark culture (the illumination intensity is 1000-1500lx, the illumination time is 16h/d, the dark time is 8 h/d), and the screening differentiation of the resistant buds is carried out, wherein the culture temperature is 28 ℃;
e. after the formation of the resistant buds, cutting the resistant buds, transferring the cut resistant buds to a strong seedling selection medium as shown in table 1, and alternately culturing the resistant buds by adopting illumination and dark culture (the illumination intensity is 1000-1500lx, the illumination time is 16h/d, the dark time is 8 h/d), wherein the screening of the resistant seedlings is carried out, and the culture temperature is 28 ℃;
f. the selected resistant seedlings are transferred to rooting selection medium in Table 1 to root, and alternately cultured by light and dark (light intensity 1000-1500lx, light time 16h/d, dark time 8 h/d) at 28deg.C.
Example 5: transgenic tobacco RhSEP3 flower phenotype observation
Floral phenotype observations were performed on control and transgenic tobacco grown to flowering (fig. 4). The flowering time of RhSEP3 transgenic tobacco is obviously shortened, and the period from inheritance to flowering lasts for more than 8 months, while compared with common tobacco, the flowering time of RhSEP3 can be obviously shortened as compared with the period of about one year. Meanwhile, in the over-expression plant OE-5, sepals and petals of the RhSEP3 transgenic tobacco are differentiated into 6, and the sepals and petals are respectively increased by 1 compared with those of the control tobacco. It can be seen that RhSEP3 is involved in the formation of sepals and petal primordia. Meanwhile, in the over-expression plant OE-2, the petal of the RhSEP3 transgenic tobacco is deepened relative to the contrast, the color of the contrast tobacco is pink compared with that of the contrast tobacco, the petal of the RhSEP3 transgenic tobacco is light pink, and the RhSEP3 possibly participates in synthesis or accumulation of anthocyanin. At the same time, it was observed in tobacco stamens and pistils that the filaments and columns of the over-expressed plant OE-2 were shortened, and the anthers were also less plump than the control tobacco, indicating that RhSEP3 may be involved in the development of stamens and pistils.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A Chinese rose RhSEP3 gene has a nucleotide sequence shown in SEQ ID No. 1.
2. A protein RhSEP3 encoded by the gene of claim 1, the amino acid sequence of which is shown in SEQ ID No. 2.
3. A primer pair for obtaining the RhSEP3 gene according to claim 1, characterized in that: the primer pair is as follows:
RhSEP3 forward primer: 5'-GAGAGAAGAGAAATGGGGAGGGG-3' the number of the individual pieces of the plastic,
RhSEP3 reverse primer: 5'-GGCTTCACCACACAGAGTTGCTT-3'.
4. A recombinant expression vector, characterized in that: the recombinant expression vector contains the plant expression vector of the RhSEP3 gene as claimed in claim 1, wherein the plant expression vector is pCAMBIA2300s.
5. A host cell comprising the recombinant expression vector of claim 4, wherein: the host cell is Agrobacterium GV3101.
6. The application of one of the following in regulating the development of plant flower organs is characterized in that: it comprises the following steps:
(1) The RhSEP3 gene of claim 1;
(2) The recombinant expression vector of claim 4;
(3) The host cell of claim 5.
7. The use according to claim 6, characterized in that: the plant is tobacco or China rose.
8. The use of one of the following in the cultivation of a new plant variety of the new flower type, characterized in that: it comprises the following steps:
(1) The RhSEP3 gene of claim 1;
(2) The recombinant expression vector of claim 4;
(3) The host cell of claim 5.
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