CN115927390B - Cymbidium organ development gene CsPI1 and encoding protein and application thereof - Google Patents
Cymbidium organ development gene CsPI1 and encoding protein and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The invention discloses an cymbidium organ development gene CsPI1 and a coding protein and application thereof. The invention clones gene CsPI1 from cymbidium sinense, the nucleotide sequence is shown in 93 rd to 725 rd bases of SEQ ID NO.1, and the coded amino acid sequence is shown in SEQ ID NO. 2. The gene CsPI1 is overexpressed in arabidopsis thaliana, and can inhibit the vegetative growth under long sunlight, promote the flowering of plants and lead the flowering period to be advanced; can change the shape of flower organs to shorten the flower buds, and the epidermis has no or little hair; the shape of the plant sepals can be changed, so that the tips of the sepals are round and smooth, and the sepals are petal-shaped; changing the shape of plant petals to make the tips of the petals round and smooth and to be blossom-shaped; changing the shape of the horn fruit to change the slender horn fruit into a short cylinder. Therefore, the cymbidium gene CsPI1 provided by the invention has important theoretical and application values in the aspects of regulating and controlling plant vegetative growth, reproductive growth and flower organ development.
Description
Technical Field
The invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to an cymbidium organ development gene CsPI1 and a coding protein and application thereof.
Background
Mo Lanshi plant of the genus orchid of the family Orchidaceae has wide and thick green leaves, various flowers, fragrance and hearts, and is popular with consumers when the flowers meet the traditional festival spring festival of China, namely 'newspaper year round', and is the national orchid with the largest industrial production standard. As one of the traditional Chinese famous flowers, the flower shape of cymbidium is one of important qualities determining economic value. Under natural conditions, the flower shape of the cymbidium sinense is evolved into a rich variety, each flower shape can show typical shape characteristics in the evolution process, and according to the flower shape evolution rule of the cymbidium sinense variety, the cymbidium sinense has typical single-petal types, namely 7 flower part structures, such as bamboo leaf types, narcissus types, plum types, core butterfly types and lily types, and the number of multi-petal types refers to more than 7 flower parts, and mainly comprises a cluster core multi-lingual type and a peony type (Deng Yinxia, 2008).
Orchid is composed of seven flower organs to form a unique structure: three calyx of outer wheel, two petals of inner wheel and one specialized lip, and one stamen and pistil synbiotic coleus. The highly specialized flower structure of orchid, one of the largest families of angiosperms (CHRISTENHUSZ and BYNG, 2016) and one of the most evolutionary groups (Tian Min et al, 2011), provides excellent material for the study of flower organ development and flower morphogenesis of orchid plants and even whole angiosperms. MADS-box is an ancient gene family (M and F,2001;CHRISTENHUSZ and BYNG,2016) and plays an important role in regulating and controlling the development of orchid organs. Studies have shown that in classical flower organ development models, the function of class B MADS-box genes is primarily to regulate petal and stamen development (S and M, 1991), and that it was subsequently found that class B gene functions extend to the outer round to convert sepals into petal-like organs (Akira et al, 2003). In recent years, a scholars proposed an "orchid code" model, and considered that the B-class MADS-box genes determine the development of flower organs by differential expression of different combinations.
The flower type is an important index for evaluating the value of the cymbidium, the shape of the sepals directly determines the flower type of the cymbidium, and the ornamental value and the price of cymbidium varieties with different flower types are far from each other. Various unique singular flower types widely popular in the market in recent years are popular, but the excellent variety is still bred under natural variation at present. How to utilize molecular biology technology to deeply research the morphological variation mechanism of flower organs, and has important application value in the industrialized development of orchid germplasm improvement and variety optimization.
Disclosure of Invention
The first object of the invention is to provide a gene CsPI1 expressed in cymbidium, which can be used for regulating the flowering time and the flower organ morphology of plants, and the nucleotide sequence of the gene CsPI1 is shown as 93 rd to 725 rd bases of SEQ ID NO. 1.
The second object of the present invention is to provide the protein encoded by the gene CsPI1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
A third object of the present invention is to provide a recombinant plant expression vector containing the gene CsPI1.
The expression vector is any binary vector which can be used for agrobacterium transformation plants or can be used for plant microprojectile attack, such as pCAMBIA series vectors, pBI series vectors, pBin series vectors or Gateway TM A series of vectors.
The fourth object of the present invention is to provide the application of the CsPI1 gene, the protein encoded by the CsPI1 gene and the recombinant plant expression vector containing the CsPI1 gene in inhibiting vegetative growth, promoting reproduction, shortening flowering time, and changing the morphology of flower organs.
The flowering phase of the plant is regulated and controlled by improving the expression of the gene CsPI1, inhibiting the vegetative growth, promoting the reproductive growth, promoting the flowering of the plant and shortening the flowering time.
The improved plant flower organ morphology is realized by improving the expression of the gene CsPI1, changing the plant sepal morphology, and enabling the sepal tips to be round and moist and to be converted into petals; changing the shape of plant petals to make the tips of the petals round and smooth and to be blossom-shaped; changing the shape of the flower buds to make the flower buds shorter and round and have no hair; changing the shape of the horn fruit to change the slender horn fruit into a short cylinder.
A fifth object of the present invention is to provide a method for advancing the flowering phase of a plant, which is characterized in that the CsPI1 gene is introduced into a plant cell, tissue or organ, and the transformed plant cell, tissue or organ is cultivated into a plant, so that the CsPI1 gene is overexpressed in the plant.
A sixth object of the present invention is to provide a method for improving the morphology of a plant flower organ, which is characterized in that the CsPI1 gene is introduced into a plant cell, tissue or organ, and the transformed plant cell, tissue or organ is cultivated into a plant, so that the gene CsPI1 is overexpressed in the plant.
Preferably, the CsPI1 gene is introduced into a plant cell, tissue or organ by a plant expression vector, and the plant is arabidopsis thaliana.
The invention clones in the black orchid to obtain the floral organ development gene CsPI1, and the transgenic function identification proves that the excessive expression of the CsPI1 gene in the arabidopsis can inhibit the nutritional growth of the arabidopsis, promote reproduction and lead the flowering phase to be advanced; can change the shape of flower organs to round flower buds, and the epidermis has no or little hair; the shape of the plant sepals can be changed, so that the tips of the sepals are round and smooth, and the sepals are petal-shaped; changing the shape of plant petals to make the tips of the petals round and smooth and to be blossom-shaped; changing the shape of the horn fruit to change the slender horn fruit into a short cylinder. Therefore, the cymbidium sinense CsPI1 gene provided by the invention has very important theoretical and application values in regulating plant vegetative growth, reproductive growth and flower organ development.
Drawings
FIG. 1 shows the measurement of the expression level of the CsPI1 gene in each organ (root, pseudobulb, leaf) in the vegetative growth phase and each organ (root, stem, leaf, bud) in the early differentiation phase, the small-row bell phase and the full-flower phase of cymbidium by Real-time PCR, and the ordinate represents the relative expression level.
FIG. 2 is a sequence of PCR detection of the CsPI1 gene during 5 stages of development of cymbidium sinense by Real-time PCR: expression levels in buds at the early differentiation stage, the small-setting stage, the large-setting stage, the initial flowering stage and the full-setting stage are represented by the ordinate.
FIG. 3 shows that the expression level of the CsPI1 gene in each flower organ (sepals, petals, labial petals and coleus) in the initial flowering stage is detected by Real-time PCR, sepal represents sepals, petal represents petals, lip represents labial petals, column represents coleus, and the ordinate represents the relative expression level.
FIG. 4 is a diagram showing PCR identification of agrobacterium transformed with the expression vector pCAMBIA1301-35s-CsCOL4, wherein M is Marker2000, 1 is the gene specific primer identification for the screened positive clones, 2 is the gene specific primer and carrier primer identification for the same screened positive clones, and 3 is the carrier universal primer identification for the same screened positive clones.
FIG. 5 is a PCR identification of transgenic (CsPI 1) Arabidopsis, where M is Marker2000, 1 is wild type Arabidopsis, and 2-4 are CsPI1 transgenic Arabidopsis lines.
FIG. 6 is a graph comparing flowering of CsPI1 transgenic Arabidopsis and wild type Arabidopsis under long sun exposure, wherein WT is wild type Arabidopsis and 1-3 are CsPI1 transgenic Arabidopsis lines.
FIG. 7 is a graph comparing leaves of CsPI1 transgenic Arabidopsis with wild type Arabidopsis under long sunlight, wherein WT is wild type Arabidopsis, csPI1-1, csPI1-2, csPI1-3 is three CsPI1 transgenic Arabidopsis lines.
FIG. 8 is a graph comparing the morphology of organs of CsPI1 transgenic Arabidopsis and wild type Arabidopsis under long sun exposure, wherein WT is wild type Arabidopsis and CsPI1-OE is a CsPI1 overexpressing transgenic Arabidopsis strain.
Detailed Description
The invention is further illustrated below in connection with specific examples. It should be understood that these examples are for illustration of the invention and are not intended to limit the scope of the invention. The following examples, in which no particular experimental procedure is noted, may be carried out in accordance with conventional methods. Conditions as described in J.Sam Brooks et al, guidelines for molecular cloning experiments, F.Osbert et al, guidelines for precision-compiled molecular biology experiments, or according to the instructions of the manufacturer of the product used.
Example 1: cloning of the cuttlefish CsPI1 Gene
The Cymbidium species "penguin Bai Mo" (Cymbidium sinense. 'Qi Jian Bai Mo') was used as the test material and plant material was grown under normal greenhouse conditions (L/D, 12h/12h;25-30 ℃).
RNA extraction: total RNA extraction of the flower buds of the test material was performed by Trizol reagent (purchased from Invitrogen company), and the whole procedure was strictly described according to the RNA extraction procedure of the Trizol reagent.
Reverse transcription: reverse transcription of mRNA into cDNA first strand using a reverse transcription kit of Norpran was performed according to the instructions of the reagents.
Cloning of the Gene: the first strand of the reverse transcribed cymbidium bud cDNA is used as a template, and CsPI1-F1 is used as a template: ATGGGACGTGGAAAGATAGAGATC and CsPI1-R1: CTTGTTTCCCTGCAAGTTGG as primers, conventional PCR amplification was performed and the full length of CsPI1 was cloned according to the Invirazan high fidelity DNA polymerase reagent (Phanta Max DNA Polymerase) product instructions. The sample addition system was referred to the enzyme instructions and the PCR reaction procedure was as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 1min, a total of 34 cycles, and then extension at 72℃for 5min. The PCR products were detected by 1.5% agarose gel electrophoresis and the target bands were recovered, and the method was performed according to the column type PCR product purification kit (SanPrep Column PCR Product Kit) product instructions (Biotechnology Co., ltd.). The target fragment was recovered and ligated to the T vector by referring to the product instructions of the T vector kit (pClone 007Blunt Simple Vector Kit) from the ProteUK Biotech Co., ltd, to construct a T vector ligation product with the target fragment. Mu.l of the ligation product was transferred to E.coli DH 5. Alpha. Competent cells (Plasmodium falciparum), and the mixture was resuscitated by adding 800ml of LB and plated on LB plates containing 50mg/L kana antibiotic (Kan) overnight at 37 ℃. White clones were picked up, grown in LB+Kan (50 mg/L) liquid medium, and submitted to sequencing. The nucleotide sequence is shown as SEQ ID NO.1, the open reading frame is from 93 rd to 725 th bases, the length is 633bp, the open reading frame is named as CsPI1 gene, the encoding number of the open reading frame is 211 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.
Example 2: analysis of the expression Pattern of the Vanilla CsPI1 Gene
Root, stem and leaf of black orchid in vegetative growth stage, root, stem, leaf and flower bud of flower bud in early differentiation stage, small-row-bell stage and full-bloom stage are taken, total RNA of these materials is extracted with Trizol reagent (Invitrogen) respectively, and genomic DNA is removed by treatment with DNase I (Takara). After OD260 was measured, 2. Mu.g of RNA was quantitatively removed, and then reverse transcription was performed using a reverse transcription kit for Norflua (methods reference). The cDNA was diluted 5-fold and was quantified according to the fluorescent quantitative qPCR kit (ChamQ TM Universal SYBR qPCR Mix) product instructions for useqPCR detection was performed by a fluorescent quantitative PCR instrument (LightCycler 480 II, roche), with primer sequences CsPI1-F2: TTGCACCATCAGCA ATTGGC and CsPI1-R2: TCATTGGCATCTGAGCTGCA, internal reference primer ACT-F: TTTATGAGGGTT ATGCGCTTCC, ACT-R: ATTTCCCGTTCCGCAGTAGTT real-time fluorescent quantitative PCR detection is carried out on the expression quantity of CsPI1 in different flower organs of different periods of the development of the cymbidium sinense.
qPCR reaction System (20. Mu.l) was as follows:
the reaction procedure was as follows: (1) pre-denaturation at 95 ℃ for 30sec; (2) the PCR reaction was carried out for 40 cycles: 95℃for 5sec,56℃for 10sec and 72℃for 2min; (4) 95 ℃ for 2sec and 56 ℃ for 15sec; (5) continuous at 95 ℃; (6) 30sec at 40 ℃.
The resulting data were analyzed using ABI 7500Real-time PCR system software. The results show that the expression level of CsPI1 in pseudobulb is highest and the expression level of roots and leaves is relatively low in the vegetative growth period; after entering the reproductive phase, the expression level of CsPI1 was greatly reduced in roots, stems, and leaves, whereas CsPI1 was significantly higher than vegetative organs in the buds at different phases and reached the peak in the small-row bell phase (fig. 1). Further studying the expression level of CsPI1 in flower buds in the initial differentiation stage, the small-row bell stage, the large-row bell stage, the initial flowering stage and the full-bloom stage, it was found that the gene developed from the flower buds to flowering in a continuous upward trend, the expression level was highest in the initial flowering stage, and the expression level was drastically decreased in the full-bloom stage (fig. 2). The expression profile of CsPI1 in different flower organs of "penguin Bai Mo" was analyzed, and the results showed that CsPI1 was expressed at high levels in the labial lobes, followed by the pistil column, with small amounts in the calyx and petals (fig. 3). It can be seen that CsPI1 plays an important regulatory role in floral organ development.
Example 3: overexpression of the cuttlefish CsPI1 Gene in Arabidopsis and phenotypic identification of transgenic plants
(1) Construction of target gene CsPI1 super-expression vector
The cDNA obtained by the reverse transcription is used as a template, and is amplified by using a high-fidelity DNA polymerase reagent (Phanta Max DNA Polymerase) of the Northenzan, and the amplification primers are as follows according to the product specification:
CsPI1-F3：atttggagaggacagggtaccATGGGACGTGGAAAGATAGAGATC；
CsPI1-R3：caccatggtactagtgtcgacCTTGTTTCCCTGCAAGTTGG。
the reaction procedure is pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 15s, annealing at 56℃for 15s, extension at 72℃for 1min, followed by 34 cycles and then extension at 72℃for 5min.
And (3) carrying out agarose electrophoresis on the amplified product, and carrying out gel recovery sequencing to determine the CsPI1 gene. The expression vector pCAMBIA1301 is digested by KpnI and SalI, and then the target gene and the linearized vector are recombined and connected according to the product specification of a homologous recombination connection kit (ClonExpress One Step Cloning Kit), and the pCAMBIA1301 recombination reaction system (20 μl) is as follows:
and (3) reacting for 30min at 37 ℃, then converting the connection product into escherichia coli DH5 alpha, identifying positive monoclonal, and constructing an expression vector pCAMBIA1301-35s-CsPI1 containing the target gene CsPI1.
(2) Transformation of recombinant plasmid pCAMBIA1301-35s-CsPI1 into Agrobacterium GV3101
Mu.l of recombinant plasmid expression vector pCAMBIA1301-35s-CsPI1 was taken and mixed with competent cells of Agrobacterium GV3101, and placed on ice for 5min. Then quickly freezing with liquid nitrogen for 5min, and quickly transferring to 37 ℃ and standing for 5min. Mu.l of LB medium without antibiotics was added and incubated at 28℃for 3h at 180 rpm. More than the supernatant was aspirated off by centrifugation at 6000rpm at room temperature for 1min, 100. Mu.l of the supernatant was left for resuspension, the plates were spread evenly on LB solid medium plates containing Kan (50 mg/L) with a spreader, and the plates were placed in an incubator at 28℃for cultivation until colonies grew (about 2 days). Colony PCR identification is carried out by picking monoclonal, 3 pairs of primers are used for PCR primer identification, as shown in FIG. 4, 1 is a target gene primer, and CsPI1-F1: ATGGGACGTGGAAAGATAGAGATC and CsPI1-R1: CTTGTTTCCCTGCA AGTTGG, total amplified length 633bp;2 is chimeric primer on the vector and target gene, csPI1-F2: atttggagag gacagggtaccATGGGACGTGGAAAGATAGAGATC and M13F: ACTGGCCGTCGTTTTAC, the total amplification length is 1332bp;3 is a universal primer M13F on a carrier: ACTGGCCGTCGTTTTAC and M13R: CAGG AAACAGCTATGAC, total amplified length 2201bp.
From this, it was confirmed that the expression vector pCAMBIA1301-35s-CsPI1 was transferred into Agrobacterium GV3101 of 1. Positive clones were selected for subsequent infection experiments.
(3) Transformation of Arabidopsis thaliana by inflorescence infection
Selecting a positive clone, inoculating into LB+Kan (kanamycin) 50mg/L+Genta 25mg/L liquid culture medium, shake culturing at 28deg.C 200rpm for 24-36 hr to make OD 600 =0.8 or so.
The bacterial solution was transferred into a sterile 50ml centrifuge tube, the bacterial strain was collected by centrifugation at 5000rpm for 15min, the Agrobacterium was resuspended in an equal volume of permeation medium (1/2MS+0.5 g/L MES+5% Superose, pH 5.7), and the surfactant Silwet was added to a final concentration of 0.02% (200. Mu.l/L). The MS medium is a medium commonly used in the art, and the formula is referred to (Murashige T and Skoog F, 1962).
The Arabidopsis plants (buds) which have just flowering are taken and soaked in the solution for 1min. And (5) obliquely airing redundant bacterial liquid.
Covering the membrane of the arabidopsis inflorescence of the dip-dyeing bacteria liquid for moisturizing, culturing in the dark for about 8 hours, then uncovering the membrane, and normally managing under illumination until the seeds are ripe and harvested.
(4) Molecular characterization of transgenic plants
Collection of contemporary transgenic plant seeds (T) 0 Generation), sterilized (10% naclo 10min, 6 times of sterilized water rinse) and then sown on 1/2MS solid medium containing 30mg/L hygromycin for screening. The green resistant plants growing for about 10 days are transplanted into a pot, and the matrix is peat soil and vermiculite with the ratio of 3:1 (volume ratio). Single plant collection T 1 Seed generation. The seeds are planted on a 1/2MS culture medium containing 30mg/L hygromycin, the separation ratio is counted, and transgenic plant lines which meet the separation ratio of 3 survival and 1 death are selected and transplanted into a pot for cultivation. Collect T 2 Seed generation, planting the seed on 1/2MS culture medium containing 30mg/L hygromycinThe seeds are all green seedlings on the culture medium, then T 2 The seed is homozygous.
The expression of the gene of interest in the single inserted strain was detected by PCR. The primer sequences were as follows: csPI1-F1: ATGGGACGTGGAAAGATAGAGATC, csPI1-R1: CTTGTTTCCCTGCAAGTTGG.
The identification results are shown in FIG. 5, wherein M is Marker2000, 1 is wild type Arabidopsis thaliana control, and 2-4 are CsPI1 transgenic Arabidopsis thaliana lines. The result shows that pCAMBIA1301-35s-CsPI1 recombinant plasmid has been inserted into the genome of Arabidopsis thaliana, thus obtaining transgenic Arabidopsis thaliana transformed into CsPI1 overexpression
(5) Phenotype identification of transgenic plants
Transgenic Arabidopsis thaliana T 2 The generation plant and the wild type Arabidopsis are cultured under the same condition, and under long sunlight (16 hours of illumination/8 hours of darkness), the phenotype of the Arabidopsis plant over-expressing the CsPI1 is obviously different from that of the wild type Arabidopsis plant, as shown in figure 6, the transgenic 35S is that the Arabidopsis plant strain of the CsPI1 shows early flowering and flowers about 6 days earlier than the control under the long sunlight condition, which indicates that the over-expression of the CsPI1 can promote the flowering of the Arabidopsis. It was also found that the vegetative growth of CsPI1 Arabidopsis was inhibited and that the transgenic 35S, csPI1 Arabidopsis rosette leaves were smaller and fewer than the wild-type leaves (FIG. 7). The transgenic 35S is that the morphology of the flower organ of CsPI1 Arabidopsis thaliana is obviously changed compared with that of a wild type Arabidopsis thaliana flower bud is oblong and has a small quantity of single hairs; the sepal is green, oval, blunt at the top and tightly wrapped petals in a saccular shape at the base; the petals of the wild arabidopsis thaliana are long round, the tips are round, and the bases are linear; the horn fruit is slender. The transgenic 35S is that CsPI1 Arabidopsis flower bud is in a round sphere shape, and the epidermis hair is less and less obvious than that of a wild type; the sepals are changed into white green and elongated petal-shaped sepals, the top ends are round, the base parts are in blossom shape, and petals are not wrapped; the tips of the petals are round and moist, and the basal parts are blossom-shaped; the horn was cylindrical (fig. 8).
The analysis of the results shows that the CsPI1 gene is a floral organ development gene, and the excessive expression of the gene in Arabidopsis thaliana under long sunlight inhibits vegetative growth, promotes reproductive growth and ensures that plants bloom in advance; the over-expression of the gene also changes the morphology of flower buds, sepals, petals and horns, thereby affecting the development of flower organs of plants.
SEQ ID NO.1 (CsPI 1 nucleotide sequence)
CCCTTCTTCGCTGTGCTCCTTTTGCGTCTTTGCGCTTCTGTTCTTGAGATCTATTGCGGTC
TTTTTTTCATCCTGCTCGGTTTTGAGTGGAGATGGGACGTGGAAAGATAGAGATCAAAC
GGATCGAGAACTCAACAAACCGGCAAGTAACCTTCTCGAAGAGACGGAATGGAATCAT
GAAGAAGGCGAAGGAGATCAGCGTGCTCTGCGACGCCCAGGTCTCGCTTGTTATCTTTT
CCAGCCTTGGAAAGATGTTTGAGTATTGTAGCCCCTCCACCACGTTGTCCAAGATACTGG
AGAAATACCAGCAAAACTCGGGCAAGAAACTCTGGGACGCAAAACACGAGAACTTGA
GCGCGGAGATCGATCGGATCAAGAAGGAAAATGATAACATGCAGATCGAGCTCAGGCAT
TTGAAAGGGGAAGATCTGAACTCTCTTAACCCAAAAGAGCTTATCCCGATTGAGGAAAC
GCTCCAGAATGGTCTTACTAGTGTTCGGAATAAACAGATGGACTTCTTGAAGATGCTAAA
AAAGAATGAAAGAATGCTGGAAGAGGAAAATAAAAGGCTCACATACCTATTGCACCATC
AGCAATTGGCAATGGAAGGGAGTATGAGAGAACTGGACATCGGGTATCACCAGAAAGAT
AGGGAATATGCAGCTCAGATGCCAATGACCTTTCGTGTTCAACCCATTCAGCCCAACTTG
CAGGGAAACAAGTAACACTGTTAACGCCTTACTACTTTCCTCTTAGTTATATGAATTATAA
TATTAGCTTTTTAGCAGTTTCATATGAATATGAAAAACTTGTGCTGATGATTATGACATATG
TGCGTGCTACTGATATTGGTATTGTAACTGCTTGTTGATTGACAATACAATGTATTTTGGC
CTACTGTCTTTGGCTTGAGCACTGTTCAATTAGTAGTGGATATATACTTGTGCAGTATTTT
TGTTAAGGGTTTTCTGCTAATTCATCTTGASEQ ID NO.2 (CsPI 1 amino acid sequence)
MGRGKIEIKRIENSTNRQVTFSKRRNGIMKKAKEISVLCDAQVSLVIFSSLGKMFEYCSPSTT
LSKILEKYQQNSGKKLWDAKHENLSAEIDRIKKENDNMQIELRHLKGEDLNSLNPKELIPIE
ETLQNGLTSVRNKQMDFLKMLKKNERMLEEENKRLTYLLHHQQLAMEGSMRELDIGYHQ
KDREYAAQMPMTFRVQPIQPNLQGNK。
Claims (4)
1. The application of the gene CsPI1 or the protein encoded by the gene CsPI1 in regulating and controlling the flowering phase of plants and improving the morphology of flowers and organs of the plants is characterized in that the nucleotide sequence of the gene CsPI1 is shown as 93 rd to 725 rd bases of SEQ ID NO.1, and the regulation and control of the flowering phase of plants is realized by improving the expression of the gene CsPI1, inhibiting the vegetative growth, promoting the reproductive growth, promoting the flowering of plants and shortening the flowering time;
the improved plant flower organ morphology is realized by improving the expression of the gene CsPI1, changing the plant sepal morphology, and enabling the sepal tips to be round and moist and to be converted into petals; changing the shape of plant petals to make the tips of the petals round and smooth and to be blossom-shaped; changing the shape of the flower buds to make the flower buds shorter and round and have no hair; changing the shape of the horn fruit to change the slender horn fruit into a short cylinder.
2. A method for advancing the flowering phase of a plant is characterized in that a CsPI1 gene is introduced into a plant cell, a tissue or an organ, the transformed plant cell, tissue or organ is cultivated into a plant, the CsPI1 is over-expressed in the plant, and the nucleotide sequence of the gene CsPI1 is shown as 93 rd to 725 th bases of SEQ ID NO. 1.
3. A method for improving the morphology of plant flower organ is characterized by introducing the CsPI1 gene into plant cells, tissues or organs, culturing the transformed plant cells, tissues or organs into plants, and enabling the CsPI1 to be over-expressed in the plants, wherein the nucleotide sequence of the gene CsPI1 is shown as 93 rd to 725 th bases of SEQ ID No. 1.
4. A method according to claims 2-3, wherein the CsPI1 gene is introduced into a plant cell, tissue or organ by means of a plant expression vector, and the plant is arabidopsis thaliana.
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| WO2015168124A1 (en) * | 2014-04-28 | 2015-11-05 | The Trustees Of The University Of Pennsylvania | Compositions and methods for controlling plant growth and development |
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