CN114957424A - Petunia PhSPL2 and rPhSPL2 transcription factors and application thereof - Google Patents
Petunia PhSPL2 and rPhSPL2 transcription factors and application thereof Download PDFInfo
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- CN114957424A CN114957424A CN202210754901.4A CN202210754901A CN114957424A CN 114957424 A CN114957424 A CN 114957424A CN 202210754901 A CN202210754901 A CN 202210754901A CN 114957424 A CN114957424 A CN 114957424A
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- phspl2
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Abstract
The invention provides petunia PhSPL2 and rPhSPL2 transcription factors and application thereof, and belongs to the technical field of genetic engineering, wherein the nucleotide sequence of the petunia PhSPL2 transcription factor is shown as SEQ ID NO.1, and the amino acid sequence of encoded protein is shown as SEQ ID NO. 2. The nucleotide sequence of the rPhSPL2 transcription factor obtained after point mutation is carried out on the miR156/157 target site corresponding to the petunia PhSPL2 transcription factor is shown as SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4. The invention obtains transgenic plants PhSPL2, rPhSPL2 and amiRNA-PhSPL2, and carries out molecular identification and phenotype observation on the transgenic plants so as to evaluate the application prospect of the gene in regulating and controlling plant flowering and flower organ size.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to petunia PhSPL2 and rPhSPL2 transcription factors and application thereof.
Background
Petunia hybrida (Petunia hybrida) is a herbaceous flower of Petunia of solanaceae, has rich flower color, various plant types and long flowering phase, is vegetarian with the reputation of 'king of flower bed', and is widely applied to landscape architecture and home gardening. The petunia has a short growth period, a genetic transformation system is mature and easy to transform, and the petunia becomes mature technologies of model plants (Vandebusche et al, 2016) of molecular biological research, genomic data publication and omics analysis of the petunia and the like, and provides great convenience for molecular mechanism research of important functional genes of the petunia (Bombary et al, 2016).
Flowering is an important stage of plant transformation from vegetative to reproductive growth, accurate flowering time is crucial for reproductive success, and floral organ size is an important factor affecting plant mating success and subsequent genetic quality. Research on plant flowering is helpful for solving the problem of flowering sterility in the breeding process, and is also helpful for cultivating new and excellent flower varieties with appropriate flowering periods so as to meet the requirements of various festivals in the market and greatly improve the economic value (Xu et al, 2016). In addition, the research on the size of the flower organ is not only beneficial to the improvement of the ornamental value of ornamental plants, but also has close correlation between the size of the flower organ and the economic value of economic plants taking flowers and fruits as main products, which has important guiding significance for the improvement of the quality of the plants and has wide application prospect and economic value in production practice (Zhang vivid Jia, etc., 2014; Muhammad et al, 2022). Therefore, the research on plant flowering and the size of flower organs not only has important practical significance for improving ornamental characters, quality and reproductive capacity of plants, but also is closely related to economic value and has important production practice significance.
There are many measures for regulating the size of flowering and floral organs of plants, such as regulating the temperature by traditional cultivation measures, changing the growing environment of plants by regulating the temperature and applying plant growth regulators (such as auxin, gibberellin, etc.). However, the application of these measures requires great investment of manpower, material resources and financial resources in production, and the influence on plants is mostly temporary and limited. Therefore, the genotype of the plant is fundamentally improved through genetic engineering, so that the plant with better florescence and proper floral organ size and higher ornamental value is cultivated. Many genes regulating the size of flowering and floral organs of plants have been cloned successively and their functions have been studied intensively, so that the improvement of flowering and floral organ size of plants by transgenic technology has become a fast and effective technical means (Ahmed et al, 2020; David et al, 2021; Wang et al, 2021; Zhao et al, 2021).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a petunia PhSPL2 transcription factor and application thereof in controlling plant flowering and flower organ size; the application is the cloning of nucleotide coding sequences of PhSPL2 and rPhSPL2 genes (namely PhSPL2 transcription factors) and amiRNA-PhSPL2 stem-loop structure sequences, the construction of an expression vector, the complete translation regions of PhSPL2 and rPhSPL2 genes and the amiRNA-PhSPL2 stem-loop structure sequences are combined with a cauliflower mosaic virus promoter and then are transferred into plants (host arabidopsis thaliana and petunia) to obtain transgenic plants PhSPL2, rPhSPL2 and amiRNA-PhSPL2, and the molecular identification and phenotype observation are carried out on the transgenic plants to evaluate the application prospect of the genes in regulating and controlling plant flowering and flower organ size: after PhSPL2 is over-expressed, the transgenic plant is promoted to bloom in advance, the flower organs are enlarged, miR156/157 target sites corresponding to PhSPL2 are mutated (rPhSPL2), after the overexpression, the transgenic plant is also promoted to bloom in advance, the flower organs are enlarged, and after PhSPL2 interference (amiRNA-PhSPL2), the transgenic plant is delayed to bloom, and the flower organs are reduced.
The technical scheme of the invention is realized as follows:
the invention provides a petunia PhSPL2 transcription factor, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The invention further protects the coding protein of the petunia PhSPL2 transcription factor, and the amino acid sequence of the coding protein is shown in SEQ ID NO. 2.
The invention further protects a primer pair used for preparing the petunia PhSPL2 transcription factor, wherein the primer pair is a full-length amplification primer and respectively consists of an upstream primer PhSPL2-F and a downstream primer PhSPL2-R, wherein:
the nucleotide sequence of the upstream primer PhSPL2-F is shown as SEQ ID NO. 5;
the nucleotide sequence of the downstream primer PhSPL2-R is shown in SEQ ID NO. 6.
The invention further provides a petunia rPhSPL2 transcription factor, wherein the rPhSPL2 transcription factor is obtained by mutation of miR156/157 target sites of the PhSPL2 transcription factor in claim 1, and the nucleotide sequence of the rPhSPL2 transcription factor is shown in SEQ ID NO. 3.
The invention further protects the coding protein of the petunia rPhSPL2 transcription factor, and the amino acid sequence of the coding protein is shown in SEQ ID NO. 4.
The invention further protects a primer pair used for preparing the petunia rPhSPL2 transcription factor, wherein the primer pair is a partial amplification primer and a full-length amplification primer, and the partial amplification primer is respectively composed of a first section of upstream primer rPhSPL2-F1, a downstream primer rPhSPL2-R1, a second section of upstream primer rPhSPL2-F2 and a downstream primer rPhSPL 2-R2; the full-length amplification primer consists of an upstream primer rPhSPL2-F1 and a downstream primer rPhSPL2-R2, wherein:
the nucleotide sequence of the upstream primer rPhSPL2-F1 is shown as SEQ ID NO. 7;
the nucleotide sequence of the downstream primer rPhSPL2-R1 is shown as SEQ ID NO. 8;
the nucleotide sequence of the upstream primer rPhSPL2-F2 is shown as SEQ ID NO. 9;
the nucleotide sequence of the downstream primer rPhSPL2-R2 is shown in SEQ ID NO. 10.
The invention further provides a method for obtaining a transcription factor by using the primer pair, which comprises the following steps:
carrying out PCR amplification by using cDNA of different tissues of petunia as a template and the primer pair of claim 3 or 6, and purifying to obtain a transcription factor; wherein,
and (3) PCR system:
conditions of PCR: performing pre-denaturation at 98 ℃ for 2 min; at 98 deg.C, 10s, 55 deg.C, 15s, 72 deg.C, 1min, 35 cycles; extension at 72 ℃ for 10 min.
The invention further protects a recombinant expression vector which contains the plant expression vector of the transcription factor, wherein the plant expression vector is pCAMBIA 2300.
The invention further provides a construction method of the recombinant expression vector, which comprises the following steps: respectively introducing the target fragments into cloning vectors
18-T, and then carrying out cloning vector containing target gene
18-T and/or
Carrying out enzyme digestion on 18-T and pCAMBIA2300 by using Sal I and Kpn I double enzyme digestion, and respectively connecting the obtained target fragment to the pCAMBIA2300 vector subjected to enzyme digestion to obtain a recombinant expression vector pCAMBIA2300-PhSPL2 and/or pCAMBIA2300-rPhSPL 2;
the invention further protects a construction method of the recombinant expression vector, which comprises the following steps: by utilizing a microRNA inhibition technology, taking a coding region sequence of PhSPL2 as a template, and combining with a fragment to be interfered to synthesize a specific primer, wherein the nucleotide sequence of an upstream primer amiRNA-PhSPL2-F is shown as SEQ ID NO.11, and the nucleotide sequence of a downstream primer amiRNA-PhSPL2-R is shown as SEQ ID NO. 12; amplifying to obtain an amiRNA-PhSPL2 stem-loop structure sequence, and then connecting into a B/C intermediate vector; and (3) carrying out enzyme digestion on the B/C intermediate vector carrying the amiRNA-PhSPL2 stem-loop structure sequence by using KpnI and SalI restriction enzymes, and connecting the target fragment to the corresponding site of the pCAMBIA2300 vector to obtain the recombinant expression vector pCAMBIA2300-amiRNA-PhSPL 2.
The invention further protects a host cell containing the recombinant expression vector, and the host cell is agrobacterium GV3101 and AGL 0.
The invention further protects the use of one of the following for promoting flowering and floral organ size in plants, which comprises
(1) PhSPL2 as described above and/or rPhSPL2 transcription factors as described above;
(2) the interference vector amiRNA of PhSPL2, described above, PhSPL 2;
(3) the above recombinant expression vector;
(4) the host cell described above.
As a further improvement of the invention, the plants are Arabidopsis thaliana and petunia.
The raw materials of the invention are as follows:
the invention introduces PhSPL2 and rPhSPL2 genes and amiRNA-PhSPL2 (namely PhSPL2 and rPhSPL2 transcription factors and amiRNA-PhSPL2) into plant cells or tissues by an agrobacterium-mediated conventional biotechnology method, and cultivates the transformed plant tissues into plants. When the gene fragment of the present invention is used to construct a plant expression vector, any one of an enhancer promoter and an inducible promoter may be added in front of the transcription initiation nucleotide. To facilitate the identification and selection of transgenic plant cells or plants, the vectors used may be processed, for example, by the addition of antibiotic markers which confer resistance (e.g., kanamycin or ampicillin, etc.). The hosts transformed were Arabidopsis thaliana and petunia.
Transgenic Arabidopsis thaliana and petunia were obtained by transforming PhSPL2 and rPhSPL2 transcription factors and amiRNA-PhSPL 2. Through observing the plant phenotype and the expression analysis of related genes, the florescence and the flower organ size of the transgenic plant are obviously changed. The results of all experiments are combined to show that PhSPL2 promotes the plant to bloom and changes the size of the flower organ.
The invention has the following beneficial effects: the miR156/157 target site corresponding to the biological PhSPL2 transcription factor is mutated and named as rPhSPL2 transcription factor. After the PhSPL2 was interfered by miRNA inhibition technology, the fragment was named amiRNA-PhSPL 2. The overexpression vectors pCAMBIA2300-PhSPL2 and pCAMBIA2300-rPhSPL2 are constructed and introduced into arabidopsis thaliana and petunia, and the interference expression vector pCAMBIA2300-amiRNA-PhSPL2 is constructed and introduced into petunia, so that a transgenic plant is obtained. After overexpression of petunia, the transgenic plant has obvious early blossoming and enlarged floral organs compared with a control plant, the expression of related genes is detected, and the expression quantity of the genes in the transgenic plant is obviously increased. After the interference expression in petunia, the transgenic plant has obvious late flowers and small flower organs compared with a control plant, the expression of related genes is detected, and the expression quantity of the genes in the transgenic plant is obviously reduced. The PhSPL2 transcription factor provides a molecular biological basis for regulating and controlling the flowering and the size of flower organs of horticultural plants by means of plant genetic engineering. The application of the transcription factor is favorable for promoting the plant to bloom, changing the size of a floral organ, improving the quality of the plant and cultivating a new variety with excellent characters.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a phenotypic analysis of overexpression of PhSPL2 transcription factor in Arabidopsis thaliana according to an embodiment of the present invention;
wherein: a W115 (left) and 35S, PhSPL2 transgenic line (right) early flowering phenotype, b-c35S, PhSPL2 transgenic line expression level detection, d-e transgenic line flowering time and lotus throne number isophenotype statistical analysis, scale: 10 cm;
FIG. 2 is a phenotypic analysis of overexpression of the rPhSPL2 transcription factor in Arabidopsis according to an embodiment of the present invention;
wherein: a W115 (left) and 35S, rPhSPL2 transgenic line (right) early flowering phenotype, b-c35S, rPhSPL2 transgenic line expression level detection, d-e transgenic line flowering time and rosette leaf number isophenotype statistical analysis, scale: 10 cm;
FIG. 3 is a phenotypic analysis of overexpression of the PhSPL2 transcription factor in petunia according to an embodiment of the present invention;
wherein: a W115 (left) and 35S PhSPL2 transgenic line (right) early flowering phenotype, b W115 (left) and 35S PhSPL2 transgenic line (right) floral organ enlargement phenotype, c-d 35S PhSPL2 transgenic line expression amount detection, e-f transgenic line flowering time and flower diameter isophenotype statistical analysis, scale: 10 cm;
FIG. 4 is a phenotypic analysis of overexpression of the rPhSPL2 transcription factor in petunia according to an embodiment of the invention;
wherein: a W115 (left) and 35S rPhSPL2 transgenic line (right) early flowering phenotype, b W115 (left) and 35S rPhSPL2 transgenic line (right) floral organ enlargement phenotype, c-d 35S rPhSPL2 transgenic line expression level detection, e-f transgenic line flowering time and flower diameter isophenotype statistical analysis, scale: 10 cm;
FIG. 5 is a phenotypic analysis of PhSPL2 transcription factor after intervention in petunia using microRNA suppression technology according to an embodiment of the present invention;
wherein: a W115 (left) and 35S, amiRNA-PhSPL2 transgenic line (right) late-flowering phenotype, b W115 (left) and 35S, amiRNA-PhSPL2 transgenic line (right) floral organ reduction phenotype, c-e35S amiRNA-PhSPL2 transgenic line expression amount detection, f-i transgenic line flowering time, plant height, internode distance, branch number and other phenotypes are analyzed, and a ruler: 10 cm.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: isolated cloning of the PhSPL2 Gene
Total RNA was extracted from petunia samples by the trizol method, and a novel homologous gene of SPL2 was obtained from the genome and transcriptome data of petunia and named as PhSPL 2. The gene codes 466 amino acids, which are composed of 4 exons and 3 introns, and the length of the coding region is 1404 bp.
The nucleotide sequence of the PhSPL2 gene is shown as SEQ ID NO.1, and the amino acid sequence of the PhSPL2 gene coding protein is shown as SEQ ID NO. 2. The primers used are shown in Table 1.
TABLE 1
| Name (R) | Sequence of |
| PhSPL2-F | AGATGGAGTGGAATGTGAAGTG |
| PhSPL2-R | AGCTCAACAGGTCTTGAAATCC |
The PCR reaction condition is 98 ℃ for 2 min; 10s at 98 ℃, 15s at 55 ℃ and 1min at 72 ℃ (35 cycles); 10min at 72 ℃.
Example 2: isolated cloning of the rPhSPL2 Gene
Total RNA is extracted from petunia samples by a trizol method, and a new SPL2 homologous gene is obtained according to the genome and transcriptome data of the petunia and is named as PhSPL 2. PhSPL2 is divided into two sections at the corresponding miR156/157 target site for amplification so as to mutate the target site, and two sections of PCR products obtained by amplification are purified and then are used as templates for carrying out overlap extension PCR so as to obtain rPhSPL 2. The gene codes 466 amino acids, which are composed of 4 exons and 3 introns, and the length of the coding region is 1404 bp.
The nucleotide sequence of the rPhSPL2 gene is shown as SEQ ID NO.3, and the amino acid sequence of the PhSPL2 gene coding protein is shown as SEQ ID NO. 4. The primers used are shown in Table 2.
TABLE 2
| Name (R) | Sequence of |
| rPhSPL2-F1 | AGATGGAGTGGAATGTG |
| rPhSPL2-R1 | GGAAAGCAGTGATAGCGCTCGAGGAAATTCCGGTCCTGTACCC |
| rPhSPL2-F2 | CGAGCGCTATCACTGCTTTCCACTAATTCCTGGGGTTCATCT |
| rPhSPL2-R2 | GCTCAACAGGTCTTGAAATCC |
The PCR reaction condition is 98 ℃ for 2 min; 10s at 98 ℃, 15s at 55 ℃ and 1min at 72 ℃ (35 cycles); 10min at 72 ℃.
Example 3: vector construction
Will carry the full-length coding sequence of PhSPL2 and rPhSPL2
The 18-T vector is cut by Sal I and Kpn I restriction enzymes, the target fragments are respectively connected to corresponding sites of the pCAMBIA2300 vector, and the vector contains a CaMV35S promoter and a Nos 3' transcription terminator to construct an overexpression vector 35S: PhSPL2 and 35S: rPhSPL 2.
Fragments to be interfered are obtained on http:// psams. carringontlab. org/amiRNA/designer # start (Carbonell et al 2014) by using the sequence of the coding region of PhSPL2 as a template by utilizing a microRNA inhibition technology, and primers for amplifying the sequence of the stem-loop structure of amiRNA-PhSPL2 are synthesized, wherein the used primers are shown in Table 3. The B/C intermediate vector carrying the amiRNA-PhSPL2 stem-loop structure sequence is cut by KpnI and SalI restriction enzyme, and the target fragment is connected to the corresponding site of the pCAMBIA2300 vector to construct an expression vector 35S, namely, amiRNA-PhSPL 2. All constructed plasmids were verified by PCR and double digestion, and the plasmids were finally transformed into Agrobacterium GV3101 and AGL0, respectively.
TABLE 3
Example 4: overexpression of PhSPL2 and rPhSPL2 genes in Arabidopsis thaliana
The invention respectively obtains 46 and 37 35S: PhSPL2 and 35S: rPhSPL2 transgenic arabidopsis T 1 Generation positive strains. Respectively selecting 3 phenotypic strains, taking AtEF1 alpha as an internal reference gene, and detecting the expression level of PhSPL2 by semi-quantitative and quantitative detection to find that the expression level of 3 phenotypic strains in 35S: PhSPL2 and 35S: rPhSPL2 transgenic strains is higher than that of wild Arabidopsis. Selecting T's corresponding to 3:1 separation ratio by chi-square detection 2 Transgenic lines of generation were planted in a climatic chamber under long-day conditions (16/8h, day/day) at 22 ℃ and the flowering time and morphological characteristics of rosette leaves and cauline leaves of 35 transgenic positive plants were counted for each line. As shown in FIG. 1 and Table 4, after PhSPL2 is over-expressed in Arabidopsis, phenotypic observation and data statistics show that lines 5, 23 and 31 have obvious early-flowering phenotype, particularly line 23, flowering time is about 7d earlier than that of wild type, and the number of rosette leaves is obviously reduced, but the number of cauline leaves has no obvious difference. After the rPhSPL2 is over-expressed in Arabidopsis, as shown in figure 2 and table 5, phenotype observation and data statistics show that strains No.4, 16 and 27 have obvious early flowering phenotypes, particularly the strain No. 27, the flowering time is about 9 days earlier than that of a wild type, meanwhile, the number of rosette leaves is remarkably reduced, but the number of cauline leaves has no remarkable difference. There is a positive correlation between the phenotype and expression level of 35S: PhSPL2 and 35S: rPhSPL2 transformed Arabidopsis thaliana. Values are mean ± sd, asterisks represent significant differences (. P < 0.05,. P < 0.01).
TABLE 4 analysis of phenotypic data in Arabidopsis after overexpression of PhSPL2
TABLE 5 analysis of phenotypic data in Arabidopsis thaliana after overexpression of rPhSPL2
Example 5: overexpression of PhSPL2 and rPhSPL2 genes in petunia
The W115 wild type petunia was transformed with 35S: PhSPL2 and 35S: rPhSPL2 overexpression vectors. The petunia transformed plant is obtained by the steps of collecting tender leaves at the top of the plant, carrying out disinfection on an explant by mercuric chloride, infecting with agrobacterium liquid, co-culturing the explant, carrying out selective culture, cutting buds, rooting, hardening seedlings, transplanting and the like. The transgenic positive lines were determined by PCR detection. Extracting W115 and T by Trizol method 1 Generating total RNA of transgenic line, and reverse transcription. The expression level of PhSPL2 in the transgenic petunia lines is identified by taking PhEF1 alpha as an internal reference gene.
The invention respectively obtains 32 and 28 35S PhSPL2 and 35S rPhSPL2 transgenic petunia T 1 And (3) generation positive strains are respectively selected, and the expression level of PhSPL2 is semi-quantitatively and quantitatively detected, so that the expression level of 3 phenotypic strains in 35S: PhSPL2 and 35S: rPhSPL2 transgenic strains is higher than that of wild petunia. T-treatment of 3 phenotypic lines 2 Generation analysis, 25T strains were selected each 2 Phenotypic observations and data statistics were performed. After the PhSPL2 was overexpressed in petunias, as shown in fig. 3 and table 6, it was found that lines 5, 9 and 28 had a significant early flowering phenotype, particularly line 9, which showed a flowering time that was about 8 days earlier than the wild type, while flower diameter was larger than the wild type, and plant height, leaf length-to-width ratio, pitch spacing and branch number were not significantly different from the wild type. After the over-expression of rPhSPL2 in petunia, as shown in FIG. 4 and Table 7, it was found that lines No.12, No. 15 and No. 19 had significant early flowering phenotype, especially line No.12, which showed a flowering time 14d earlier than the wild type, while the flower diameter was larger than the wild type, and there was no significant difference in plant height, leaf length-to-width ratio, pitch spacing and branch number compared with the wild type. The phenotype and the expression quantity of 35S: PhSPL2 and 35S: rPhSPL2 transformed petunia are in positive correlation. Values are mean ± standard deviation, asterisks represent significant differences compared to CK group (. P < 0.05,. P < 0.01).
TABLE 6 analysis of phenotypic data in petunias following overexpression of PhSPL2
TABLE 7 analysis of phenotypic data in petunias following overexpression of rPhSPL2
Example 6: interference expression of PhSPL2 gene in petunia
The 35S: amiRNA-PhSPL2 vector was transformed into W115 wild-type petunia. The method is the same as the above, and the petunia transformed plant, the positive strain detection and the expression quantity detection are obtained.
The invention obtains 30 35S amiRNA-PhSPL2 transformed petunia T 1 And selecting 3 phenotypic strains from the generation positive strains, and detecting the expression level of amiRNA-PhSPL2 through semi-quantitative and quantitative detection to find that the expression level of the 3 phenotypic strains is higher than that of the wild petunia. T-Generation of 3 phenotypic lines 2 Generation analysis, 25T were selected each 2 Phenotypic observations and statistics were performed on the generations and as shown in FIG. 5 and Table 8, it was found that lines 10, 17 and 25 had significant late-flowering phenotypes, particularly line 25, with flowering time delayed by about 20 days from wild-type. Meanwhile, the plant height of the transgenic line becomes short, the pitch interval becomes short, the number of branches is increased, the diameter of the flower becomes small, and the length-width ratio of the leaves has no significant difference compared with the wild type. The research result shows that the phenotype and the expression quantity of 35S amiRNA-PhSPL2 transformed petunia are in positive correlation. Values are mean ± sd, asterisks indicate significant differences compared to CK group (. P < 0.05,. P < 0.01).
TABLE 8 analysis of phenotypic data in petunias following interferometric expression of PhSPL2
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> research institute for garden plants and flowers in Zhejiang province (research institute for Xiaoshan cotton and hemp in Zhejiang province)
Guangzhou Academy of forestry and landscape science
<120> petunia PhSPL2 and rPhSPL2 transcription factors and application thereof
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gctggatcct tcaatttgtc aagtggtggc agtggcactg gtggttatgg ctctgatgtg 180
gggcgtggct cttcgatcaa aagctcaatg tctgcttcga ctgattcttc accaagggag 240
agcttgaaag catccaattt tgcttttgaa gcatttgatg gttctcctga ggatcttagt 300
aagaaaatgg ggccttgcag tgctgagctc tttagaaatt ctccagcaat ggaggcttca 360
gtaggctctg ttgagccatt gataggtctg aaactaggta agcggacatg tgaggccatc 420
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aggctttctt cgttgtttta tgatagcagg caacctatga gtcttttgct caacagcgcc 840
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aaattcagta taacaagaga atttacctta aagcctgaaa gagctggcag cacaaatggg 960
cagtcactgc tggcagggaa ccagctttca cgggtagtgg gtgcacacag taatacacct 1020
aacttgctct tgccctccaa gggcactgca gccgaggttt tcaatcgagg tgccaaggaa 1080
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aattcctggg gttcatctga gcctgaatct gtttcactca accgcccaac acatgcaagt 1200
catatcagca tggccgagcc aattatgcat gcaattcctc agggcattcc gcatttgtcc 1260
tgtgactact ggcaagctgg acaaaattca tcctatcctc gaaaccatac attggctgct 1320
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ttttatctca atgcattgaa ctga 1404
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<213> Petunia hybrida)
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Ser Ser Lys Val Ser Ser Phe Pro Gln Asn Pro Ile Ser Ser Ala Ala
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Ala Leu Asn
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<213> Petunia hybrida)
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tctgatcaca atgcacgacg ccgcaaaccg cagcaggaaa ctatccagtt caactcagga 780
aggctttctt cgttgtttta tgatagcagg caacctatga gtcttttgct caacagcgcc 840
caacttatcc actccaaagc tgctgctgca agttcttcct gggaaagcac tcaagacact 900
aaattcagta taacaagaga atttacctta aagcctgaaa gagctggcag cacaaatggg 960
cagtcactgc tggcagggaa ccagctttca cgggtagtgg gtgcacacag taatacacct 1020
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tccatgttca atatgggtac aggaccggaa tttcctcgag cgctatcact gctttccact 1140
aattcctggg gttcatctga gcctgaatct gtttcactca accgcccaac acatgcaagt 1200
catatcagca tggccgagcc aattatgcat gcaattcctc agggcattcc gcatttgtcc 1260
tgtgactact ggcaagctgg acaaaattca tcctatcctc gaaaccatac attggctgct 1320
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<210> 4
<211> 467
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<213> Petunia hybrida)
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Met Glu Trp Asn Val Lys Trp Asp Trp Gly Asn Leu Val Met Phe Asp
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35 40 45
Gly Gly Ser Gly Thr Gly Gly Tyr Gly Ser Asp Val Gly Arg Gly Ser
50 55 60
Ser Ile Lys Ser Ser Met Ser Ala Ser Thr Asp Ser Ser Pro Arg Glu
65 70 75 80
Ser Leu Lys Ala Ser Asn Phe Ala Phe Glu Ala Phe Asp Gly Ser Pro
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Glu Asp Leu Ser Lys Lys Met Gly Pro Cys Ser Ala Glu Leu Phe Arg
100 105 110
Asn Ser Pro Ala Met Glu Ala Ser Val Gly Ser Val Glu Pro Leu Ile
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Gly Leu Lys Leu Gly Lys Arg Thr Cys Glu Ala Ile Thr Gly Gly Ser
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Ser Ser Lys Val Ser Ser Phe Pro Gln Asn Pro Ile Ser Ser Ala Ala
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Thr Lys Lys Thr Lys Ser Ser Cys Gln Asn Thr Ala Leu Ser Arg Cys
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Gln Val Glu Gly Cys Ser Leu Asp Leu Ser Ser Ala Lys Glu Tyr Tyr
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Arg Lys His Lys Val Cys Asp Ser His Ala Lys Cys Pro Lys Val Ile
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245 250 255
Phe Asn Ser Gly Arg Leu Ser Ser Leu Phe Tyr Asp Ser Arg Gln Pro
260 265 270
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275 280 285
Ala Ala Ser Ser Ser Trp Glu Ser Thr Gln Asp Thr Lys Phe Ser Ile
290 295 300
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305 310 315 320
Gln Ser Leu Leu Ala Gly Asn Gln Leu Ser Arg Val Val Gly Ala His
325 330 335
Ser Asn Thr Pro Asn Leu Leu Leu Pro Ser Lys Gly Thr Ala Ala Glu
340 345 350
Val Phe Asn Arg Gly Ala Lys Glu Ser Met Phe Asn Met Gly Thr Gly
355 360 365
Pro Glu Phe Pro Arg Ala Leu Ser Leu Leu Ser Thr Asn Ser Trp Gly
370 375 380
Ser Ser Glu Pro Glu Ser Val Ser Leu Asn Arg Pro Thr His Ala Ser
385 390 395 400
His Ile Ser Met Ala Glu Pro Ile Met His Ala Ile Pro Gln Gly Ile
405 410 415
Pro His Leu Ser Cys Asp Tyr Trp Gln Ala Gly Gln Asn Ser Ser Tyr
420 425 430
Pro Arg Asn His Thr Leu Ala Ala Asn Thr His Thr Ala Gly Phe Gln
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Glu Ile Glu Leu Phe Lys Ala Pro Phe Asp Ser Asp Phe Tyr Leu Asn
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Ala Leu Asn
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<210> 5
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<212> DNA
<213> Artificial sequence (none)
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agatggagtg gaatgtgaag tg 22
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<213> Artificial sequence (none)
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<210> 7
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gctcaacagg tcttgaaatc c 21
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aagactatta tcgga 75
<210> 12
<211> 75
<212> DNA
<213> Artificial sequence (none)
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aatgtccgat aatagtcttt agctcaaaaa atagataacg aatgtgatca tcatgagcta 60
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Claims (13)
1. A petunia PhSPL2 transcription factor is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The protein encoded by the petunia PhSPL2 transcription factor of claim 1, wherein the amino acid sequence is represented by SEQ ID No. 2.
3. Primer pair for preparing the petunia PhSPL2 transcription factor of claim 1, wherein the primer pair is a full-length amplification primer consisting of an upstream primer PhSPL2-F and a downstream primer PhSPL2-R, wherein:
the nucleotide sequence of the upstream primer PhSPL2-F is shown in SEQ ID NO. 5;
the nucleotide sequence of the downstream primer PhSPL2-R is shown as SEQ ID NO. 6.
4. The petunia rPhSPL2 transcription factor is characterized in that the rPhSPL2 transcription factor is obtained by mutation of miR156/157 target site of the PhSPL2 transcription factor of claim 1, and the nucleotide sequence of the rPhSPL2 transcription factor is shown in SEQ ID NO. 3.
5. The protein encoded by the petunia rphy rpspl 2 transcription factor according to claim 4, wherein the amino acid sequence is represented by SEQ ID No. 4.
6. The primer pair for preparing the petunia rPhSPL2 transcription factor as claimed in claim 4, wherein the primer pair is a partial amplification primer and a full-length amplification primer, and the partial amplification primer consists of a first segment of upstream primer rPhSPL2-F1, a downstream primer rPhSPL2-R1, a second segment of upstream primer rPhSPL2-F2 and a downstream primer rPhSPL 2-R2; the full-length amplification primer consists of an upstream primer rPhSPL2-F1 and a downstream primer rPhSPL2-R2, wherein:
the nucleotide sequence of the upstream primer rPhSPL2-F1 is shown as SEQ ID NO. 7;
the nucleotide sequence of the downstream primer rPhSPL2-R1 is shown as SEQ ID NO. 8;
the nucleotide sequence of the upstream primer rPhSPL2-F2 is shown as SEQ ID NO. 9;
the nucleotide sequence of the downstream primer rPhSPL2-R2 is shown as SEQ ID NO. 10.
7. A method for obtaining a transcription factor by using the primer pair of claims 3 and 6, comprising the steps of:
carrying out PCR amplification by using cDNA of different tissues of petunia as a template and the primer pair of claim 3 or 6, and purifying to obtain a transcription factor; wherein,
and (3) PCR system:
conditions of PCR: pre-denaturation at 98 ℃ for 2 min; at 98 deg.C, 10s, 55 deg.C, 15s, 72 deg.C, 1min, 35 cycles; extension at 72 ℃ for 10 min.
8. A recombinant expression vector characterized by: the recombinant expression vector contains the plant expression vector of the transcription factor of claim 1 and/or 4, wherein the plant expression vector is pCAMBIA 2300.
9. A method for constructing the recombinant expression vector of claim 8, wherein: the method comprises the following steps: respectively introducing the target fragments into cloning vectors
18-T, and then carrying out cloning vector containing target gene
18-T and/or
The 18-T and the pCAMBIA2300 are subjected to enzyme digestion by using Sal I and Kpn I, and the obtained target fragments are respectively connected to the pCAMBIA2300 vector subjected to enzyme digestion to obtain a recombinant expression vector pCAMBIA2300-PhSPL2 and/or pCAMBIA2300-rPhSPL 2.
10. A method for constructing the recombinant expression vector of claim 8, wherein: the method comprises the following steps: by utilizing microRNA inhibition technology, taking a PhSPL2 coding region sequence as a template, and synthesizing a specific primer with a fragment to be interfered, wherein the nucleotide sequence of an upstream primer amiRNA-PhSPL2-F is shown as SEQ ID NO.11, and the nucleotide sequence of a downstream primer amiRNA-PhSPL2-R is shown as SEQ ID NO. 12; amplifying to obtain an amiRNA-PhSPL2 stem-loop structure sequence, and then connecting a B/C intermediate vector; and (3) carrying out enzyme digestion on the B/C intermediate vector carrying the amiRNA-PhSPL2 stem-loop structure sequence by using KpnI and SalI restriction enzymes, and connecting the target fragment to the corresponding site of the pCAMBIA2300 vector to obtain the recombinant expression vector pCAMBIA2300-amiRNA-PhSPL 2.
11. A host cell comprising the recombinant expression vector of claim 8, wherein: the host cells are Agrobacterium GV3101 and AGL 0.
12. The application of one of the following items in promoting plant flowering and flower organ size is characterized in that: it comprises
(1) PhSPL2 of claim 1 and/or rPhSPL2 transcription factor of claim 4;
(2) PhSPL2 interference vector amiRNA-PhSPL2 of claim 1;
(3) the recombinant expression vector of claim 8;
(4) the host cell of claim 11.
13. Use according to claim 12, characterized in that: the plants are Arabidopsis thaliana and petunia.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1244585A (en) * | 1998-08-07 | 2000-02-16 | 农林水产省农业生物资源研究所长 | Method for leading-in petunia transcription factor Pet SPL 2 gene to shorten inflorescence internode |
| JP2002027992A (en) * | 2001-05-28 | 2002-01-29 | National Institute Of Agrobiological Sciences | Method for shortening intergeniculum of inflorescence by transducing gene encoding transcription factor pet spl2 in petunia |
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- 2022-06-29 CN CN202210754901.4A patent/CN114957424A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1244585A (en) * | 1998-08-07 | 2000-02-16 | 农林水产省农业生物资源研究所长 | Method for leading-in petunia transcription factor Pet SPL 2 gene to shorten inflorescence internode |
| JP2002027992A (en) * | 2001-05-28 | 2002-01-29 | National Institute Of Agrobiological Sciences | Method for shortening intergeniculum of inflorescence by transducing gene encoding transcription factor pet spl2 in petunia |
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| Title |
|---|
| LIU,G. 等: "GenBank: MF580469.1，Petunia x hybrida squamosa promoter binding-like protein 2 (SPL2) mRNA, complete cds", GENBANK DATABASE * |
| QIN ZHOU 等: "miR156/157 Targets SPLs to Regulate Flowering Transition, Plant Architecture and Flower Organ Size in Petunia", PLANT CELL PHYSIOL., vol. 62, no. 5, pages 839 - 857 * |
| 周琴: "矮牵牛miR156/157和SPL基因家族的鉴定和功能研究", 华中农业大学博士学位论文, pages 43 - 87141 * |
| 金春梅: "超量表达PhMIR156g和PhSPL13对矮牵牛生长发育的影响", 中国优秀硕士学位论文全文数据库农业科技辑, pages 048 - 253 * |
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