CN109988230B - Application of nucleoporin Nup98a and Nup98b in regulating and controlling flowering time of plants - Google Patents
Application of nucleoporin Nup98a and Nup98b in regulating and controlling flowering time of plants Download PDFInfo
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Abstract
The invention relates to the technical field of plant molecular biology, in particular to application of nucleoporin Nup98a and Nup98b in regulating and controlling plant flowering time. The invention identifies the function of the nucleoporin Nup98 in regulating and controlling the flowering time and the growth cycle of plants, and provides a method for changing the flowering time of plants by using nucleoporin Nup 98. The simultaneous inactivation of Nup98a and Nup98b can lead to early flowering and shortened growth period of Arabidopsis. Due to the high conservation of the Nup98 gene in plants, the application and the method of the nucleoporin Nup98 in the aspects of regulating and controlling the flowering time and the growth cycle of the plants have general popularization and application values in different plants, and provide a method and a basis for solving the problems of flowering asynchronism, growth period control of various plants, photoperiod sensitivity and introduction in plant cross breeding.
Description
Technical Field
The invention relates to the technical field of plant molecular biology, in particular to application of nucleoporin Nup98a and Nup98b in regulating and controlling plant flowering time.
Background
Nuclear Pore Complex (NPC) is the largest protein Complex on the Nuclear membrane of eukaryotic cells, and it is widely involved in many growth and development processes of organisms by regulating the transport of mRNA and protein between the nucleus and cytoplasm. The research on the function of the nucleoporin in the process of regulating and controlling the growth and development of plants and the development of the nucleoporin with important functions on the growth and development of the plants have important significance on the genetic breeding and the crop planting of the plants. At present, only a few NPC components in plants are preliminarily analyzed in function, and the function research of Nucleoporin 98 (Nup98) is not reported. The present invention finds that the Nup98 protein has very high homology in main crops such as soybean, corn, rice, etc. There are two highly homologous Nup98 genes a and b in arabidopsis thaliana, having the same protein domain as human Nup98 protein, namely the N-terminal FG repeat domain and the C-terminal autoproteolytic domain (APD).
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide application of nucleoporins Nup98a and Nup98b in regulating and controlling flowering time of plants.
In order to achieve the purpose, the technical scheme of the invention is as follows:
according to the invention, through the analysis of the promoters of an arabidopsis Nup98a gene (the sequence is shown as SEQ ID NO.3, and the sequence of a coded protein is shown as SEQ ID NO. 1) and a Nup98b gene (the sequence is shown as SEQ ID NO.4, and the sequence of the coded protein is shown as SEQ ID NO. 2), the promoters of the two genes contain a plurality of identical and specific cis-acting elements. Through protein localization experiments, the Nup98a and Nup98b proteins are not only localized on a nuclear membrane, but also widely distributed in a nuclear cytoplasm. Through inactivation experiments on Nup98a and Nup98b genes, T-DNA insertion single mutants of Nup98a and Nup98b genes are found to have no obvious development phenotype, however, a double mutant (Nup98) of Nup98a Nup98b shows an obvious early flowering phenotype, and the Arabidopsis Nup98a and Nup98b are proved to be functionally redundant and jointly regulate the plant flowering process.
Specifically, the invention provides an application of nucleoporin Nup98 in regulating and controlling the flowering time of plants.
The invention provides application of nucleoporin Nup98 in regulation and control of plant growth cycle.
The invention provides application of nucleoporin Nup98 in plant genetic breeding or transgenic plant construction.
The sequence of the nucleoporin Nup98 is any one of the following sequences:
(1) an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;
(2) the amino acid sequence of the protein with the same function is obtained by deleting, replacing or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO. 2;
(3) an amino acid sequence of a protein which has at least 40 percent of homology with the amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and has the same function.
According to the invention, bioinformatics analysis shows that the Nup98 protein has very high homology in arabidopsis thaliana and main crops such as soybean, corn and rice, so that the Nup98 protein of the main crops such as soybean, corn and rice has the same functions as the arabidopsis thaliana Nup98 protein according to the conservation of the protein sequence and functions.
Therefore, the proteins having the amino acid sequence of the same functional protein obtained by deletion, substitution or insertion of one or more amino acids of the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 in (2) or (3) having at least 40% homology to the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 and having the amino acid sequence of the same functional protein include, but are not limited to, nucleopore protein Nup98 of soybean (2 nucleopore proteins Nup98, glyma.12g235000 and glyma.13g26410 are present in soybean), nucleopore protein Nup98 of corn (2 nucleopore proteins Nup98, GRMZM2G 0515 and GRMZM2G 3424 are present in corn), nucleopore protein Nup98 of rice (2 nucleopore proteins Nup98, LOC _ Os12G06890 and grmz _ Os12G 905634), and nucleopore protein Nup 3598 or mutant having the same function as LOC p 3598 or LOC 3598 in rice (2).
Further, the invention provides an application of the coding gene of the nucleoporin Nup98 or the biological material containing the coding gene in regulating and controlling the flowering time of plants, regulating and controlling the growth cycle of the plants, carrying out genetic breeding on the plants or constructing transgenic plants.
Preferably, the nucleotide sequence of the coding gene of Nup98 is any one of the following:
(1) a nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO. 4;
(2) the nucleotide sequence of the protein with the same function is obtained by deleting, replacing or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO. 4;
(3) nucleotide sequence which can be hybridized with the nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO.4 under strict conditions.
It will be understood by those skilled in the art that, given the amino acid sequence of the Nup98 protein, different nucleotide sequences encoding genes encoding proteins having the same function as the Nup98a or Nup98b protein may be obtained according to the degeneracy of the codon.
Preferably, the biological material comprises an expression cassette, a vector, a transposon, an engineered bacterium, a host cell or a transgenic cell line.
Experiments prove that nucleoporin Nup98 is a negative control factor for plant flowering, namely, inactivated nucleoporin Nup98 can advance the flowering time of plants or shorten the growth period.
Therefore, the application of the nucleoporin Nup98 of the invention is to lead the flowering time of plants to be advanced or the growth period to be shortened by inactivating the nucleoporin Nup98 or reducing the expression of the nucleoporin Nup 98.
In the invention, the plants comprise corn, soybean, rice, wheat and arabidopsis thaliana.
As an embodiment of the present invention, when the plant is Arabidopsis thaliana, the flowering-time or the growth-period of Arabidopsis thaliana is advanced by inactivating both Nup98a having a sequence shown in SEQ ID NO.1 and Nup98b having a sequence shown in SEQ ID NO. 2.
Furthermore, the invention also provides a method for regulating and controlling the flowering time of the plant, regulating and controlling the growth cycle or constructing the transgenic plant, which is realized by regulating and controlling the expression of nucleoporin Nup98 of the plant.
Preferably, the method for regulating the flowering time of plants, regulating the growth cycle or constructing transgenic plants leads the flowering time of plants to be advanced or the growth period to be shortened by inactivating nucleoporin Nup98 or reducing the expression of nucleoporin Nup 98.
The invention has the beneficial effects that: the invention firstly identifies the function of the nucleoporin Nup98 in regulating and controlling the flowering time and the growth cycle of plants, and provides a method for changing the flowering time of plants by using the mutation of the nucleoporin Nup 98. Experiments prove that Nup98 double-mutant arabidopsis thaliana with Nup98a and Nup98b inactivated simultaneously shows development phenotypes such as early flowering and shortened growth period. Because the Nup98 gene is highly conserved in plants, especially in main crops such as corn, rice, soybean and the like, the application and method of the nucleoporin Nup98 in the aspects of regulating and controlling the flowering time and the growth cycle of the plants have general popularization and application values in different plants, and a method and basis are provided for solving the problem of flowering asynchronism in plant cross breeding, the problem of growth period control of various crops, vegetables, fruits and flowers, the problem of photoperiod sensitivity and the problem of introduction.
Drawings
FIG. 1 is a structural pattern diagram of Nup98 protein and the alignment result of the structural domain sequence in example 1 of the present invention; wherein, A is a structural pattern diagram of arabidopsis Nup98 and human Nup98-96 precursor; b is the alignment of arabidopsis Nup98 and human Nup98 protein autozymolysis structural domain sequences; c is the similarity analysis of the autoproteolytic structure sequences of arabidopsis Nup98 and human Nup 98; d is the sequence similarity analysis between Arabidopsis Nup98 and human Nup98 protein.
FIG. 2 is a schematic structural diagram of a cloning intermediate vector pGWCm according to example 3 of the present invention.
FIG. 3 is a schematic structural diagram of the cloning intermediate vector FU79-GFP of example 4 of the present invention.
FIG. 4 is a schematic structural diagram of an intermediate carrier FU76-35S according to embodiment 4 of the present invention.
FIG. 5 is a schematic structural view of a plant expression vector FU39-2(intron) according to example 4 of the present invention.
FIG. 6 is the subcellular localization of Nup98 protein in example 5 of the present invention, in which GFP represents that Nup98 protein localizes to the nucleus, PI represents cell wall staining, Merge represents the superposition of the first two pictures, and Bright Field represents the Bright Field.
FIG. 7 is the identification of T-DNA insertion mutant of Nup98 gene of Arabidopsis thaliana in example 6 of the present invention, wherein a is the phenotype of T-DNA insertion mutant of Nup98 gene and the scale is 2 cm; b is a structural schematic diagram of the T-DNA insertion of the Nup98 gene, the inverted triangle represents the T-DNA insertion site, the black and gray boxes represent the exon and 5 'or 3' untranslated region, respectively, the black line represents the intron, and the horizontal line below the gene structure represents the RT-PCR product position; c is the transcription level of the Nup98 gene in wild type and mutant analyzed by RT-PCR, and ACTIN2 is an internal reference gene; d is the expression level detection of Nup98a protein in wild type and mutant, and HSP90 is reference protein.
FIG. 8 is a phenotype of flowering of wild type and mutant in example 7 of the present invention, a is a phenotype of flowering of wild type (Col), Nup98a, Nup98b gene single mutant (Nup98a1, Nup98a2, Nup98b1) and double mutant (Nup98a1Nup98b1 and Nup98a2Nup98b1) and transgenic plant (35S: GFP: Nup98b Nup98a1Nup98b1) complemented with Nup98b gene, the scale is 2 cm; b is the statistics of the flowering time of the wild type and mutant plants shown in a, expressed as the number of rosette leaves at the time of flowering (n.gtoreq.30), and error bars indicate standard deviation, significance level (P < 0.01).
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1Nup98 protein structural Pattern map and Domain sequence alignment
There is a pair of Nup98 proteins with very high homology in the arabidopsis genome: nup98a and Nup98 b. They are encoded by the Nup98a (At1g10390) and Nup98b (At1g59660) genes, respectively. The above gene sequences can be obtained from The Arabidopsis Information Resource database (The Arabidopsis Information Resource, TAIR, http:// www.arabidopsis.org /). The software DNAMAN used for sequence alignment analyzed Nup98a and Nup98b for amino acid sequence similarity of 60.8%. Alignment in The conserved domain database in The international Center for Biotechnology Information (NCBI) database shows that arabidopsis Nup98a and Nup98b proteins have The same protein domains as human Nup98 protein, i.e., N-terminal FG repeat domain and C-terminal autoproteolytic domain (APD) (fig. 1 a). However, in studies of vertebrate nucleoporin function, Nup98 was found to result from HFS cleavage of the Nup98-Nup96 precursor protein via the autoproteolytic site at the C-terminus of the APD domain, with cleavage occurring between F and S; whereas in arabidopsis, Nup98a and Nup98b are both encoded by separate genes. Notably, comparing the APD domains of arabidopsis Nup98a and Nup98b with the APD structure of human Nup98, respectively, the domain similarities were 37.6% and 44.2%, respectively, while the similarity between the APD domains of arabidopsis Nup98a and Nup98b was as high as 78.5%, and in arabidopsis Nup98 protein, HFS (B, C, D of fig. 1) remained at the autoproteolytic site at the C-terminus of the APD domain, indicating that Nup98 may have a common ancestor evolutionarily. The results show that Nup98 domain retains high conservation, although it undergoes different differentiation courses in animals and plants.
EXAMPLE 2 cloning of the Nup98 Gene
Using the forward primer 1: ATGTTTGGCTCATCTAATCCTTT (shown as SEQ ID NO. 5), reverse primer 1: CTAAACTCCATCTTCTTCATCTTC (shown as SEQ ID NO. 6) and forward primer 2: ATGTTCGGTTCTTCTAATAATAAT (shown as SEQ ID NO. 7), reverse primer 2: CTACACATCATATTCATCTCCAAG (shown as SEQ ID NO. 8), obtaining Nup98a and Nup98b from Arabidopsis thaliana by cloning respectively and sequencing, wherein the gene sequences of Nup98a and Nup98b are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively; the amino acid sequences of the protein coded by the protein are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The PCR reaction program of the above-mentioned clone Nup98a and Nup98b genes was pre-denaturation at 95 ℃ for 5min, 30s at 94 ℃, 35s at 55 ℃, 1min at 72 ℃ for 30s, 25 cycles, and extension at 72 ℃ for 10 min.
EXAMPLE 3 construction of cloning vector for Arabidopsis Nup98 Gene
The PCR product amplified in example 2 was directly cloned into pGWGCm (shown in FIG. 2) according to the TA cloning method. Before cloning, pGWGCm was previously hydrolyzed with Ahd I endonuclease to obtain a T vector. The ligation product is transformed into escherichia coli DH5 alpha, and is propagated in the escherichia coli DH5 alpha, and the sequences of Nup98a and Nup98b shown in SEQ ID NO.3 and SEQ ID NO.4 are obtained by sequencing and screening the positive clone.
Example 4 construction of expression vector for Arabidopsis Nup98 Gene
Nup98a gene was digested and ligated to FU79-GFP vector (shown in FIG. 3) using Nup98a cloning vector obtained in example 3 as a template, FU79-GPF-Nup98a, vector FU76-35S (shown in FIG. 4) and plant expression vector FU39-2(intron) (shown in FIG. 5) were mixed at equal ratio and then subjected to LR reaction (reaction system: 50ng each of plasmid, 1. mu.l of LR enzyme, and H supplementation2O to a final volume of 5. mu.l; reaction conditions are as follows: after mixing evenly, reacting for more than 6 hours at 25 ℃), constructing and obtaining the plant expression vector FU39-2-35S, GFP and Nup98 a. FU39-2-35S GFP Nup98b was constructed as described above. The two expression vectors described above were used to overview the localization and phenotypic analysis of Nup98a and Nup98b in plants.
Example 5 subcellular localization of the Arabidopsis Nup98 Gene
By using the expression vector constructed in example 4, over-expression Arabidopsis plants of 35S, GFP, Nup98a and 35S, GFP, Nup98b were obtained by the Arabidopsis flower dipping method, respectively. The localization conditions of GFP-Nup98a and GFP-Nup98b proteins in transgenic plants are observed by using a laser confocal microscope, and the results are shown in figure 6, wherein Nup98a and Nup98b proteins in Arabidopsis are not only localized on nuclear membranes, but also widely distributed in nuclear cytoplasm, and the localization conditions are consistent with the subcellular distribution characteristics of Nup98 of metazoans reported in the prior art.
Example 6 identification of T-DNA insertion mutants of the Arabidopsis Nup98 Gene
Single mutants of T-DNA insertion were ordered from the Arabidopsis Biological Resource Center (ABRC) nup98a and nup98b, respectively: and they were named Nup98a-1 (SALK-015016), Nup98a-2 (SALK-103803) and Nup98b-1 (GABI-288A 08), respectively, in which T-DNA was inserted in the 3 rd and 2 nd exons of Nup98a gene, respectively, in the Nup98a-1 and Nup98a-2 mutants, and in the Nup98b-1 mutant, in the last intron of Nup98b (b of FIG. 7). After screening for homozygous mutants by PCR, the resulting single mutants of each of Nup98a and Nup98b were found to be devoid of any significant difference in developmental phenotype compared to the wild type by phenotypic analysis (a of figure 7).
The three single mutants were subjected to molecular characterization, and the results of RT-PCR showed that no transcript of the corresponding gene was detected in each single mutant (no transcript of nup98a was detected in both the nup98a-1 and nup98a-2 mutants, and no transcript of nup98b was detected in the nup98b-1 mutant). Moreover, the expression level of the Nup98a gene in the wild type is obviously higher than that of the Nup98b gene, and when the Nup98a gene is mutated, the expression level of the Nup98b gene in vivo is increased; after the Nup98b gene is mutated, the expression level of the Nup98a gene is obviously improved (c of figure 7).
Protein level detection using the Nup98a protein specific monoclonal antibody revealed that no accumulation of Nup98a protein was detected in both Nup98a-1 and Nup98a-2 single mutants, whereas the expression level of Nup98a protein was significantly increased in the Nup98b-1 mutant (fig. 7 d). The above results indicate that the Nup98a gene and the Nup98b gene may be functionally redundant to jointly regulate the growth and development process of Arabidopsis thaliana.
In order to further research the functions of the Nup98a gene and the Nup98b gene, two simultaneous-inactivation Nup98 double mutants of the Nup98a gene and the Nup98b gene, namely Nup98a-1Nup98b-1(Nup98-1) and Nup98a-2Nup98b-1(Nup98-2), are respectively constructed through hybridization.
The method used for the hybridization was as follows: removing the male bud of the female parent of Arabidopsis thaliana to be bloomed, taking down the stamen of the bloomed male parent, and artificially pollinating the pollen of the male parent onto the stigma of the female parent. And (3) harvesting hybrid seeds after the siliques are mature, namely F1 generation, selfing F1 generation to obtain F2 generation, and identifying the genotype of the hybridized plants by using a PCR method in F2 generation.
Example 7 flowering time statistics of 7 nup98 mutants
As a result of analyzing the flowering-time of each of the Nup98 mutants in which Nup98a gene and Nup98b gene were individually and simultaneously inactivated, as shown in FIG. 8, under long-day conditions, the flowering-time of three single mutants, Nup98a-1, Nup98a-2 and Nup98b-1, did not significantly change compared to the number of rosette leaves (11.7. + -. 0.5 pieces) at the time of wild-type flowering, and the number of rosette leaves at the time of flowering was 12. + -. 0.7 pieces, 11.3. + -. 1.1 pieces and 11.3. + -. 0.5 pieces, respectively. However, Nup98 double mutants of which the Nup98a and Nup98b genes are simultaneously inactivated, namely Nup98-1(9.8 +/-0.4 tablets) and Nup98-2(9.1 +/-0.8 tablets), show early flowering phenotype. In the double-mutant background, the early-flowering phenotype of the double mutant can be complemented by using a 35S promoter to regulate the expression of the Nup98b gene, and the number of rosette leaves at the time of flowering is 10.8 +/-1.1.
The results indicate that Nup98 acts as a negative regulator of flowering in arabidopsis, and that the early flowering phenotype of the arabidopsis Nup98 double mutant is indeed caused by loss of Nup98 function (simultaneous inactivation of the Nup98a gene and the Nup98b gene).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of crop science of Chinese academy of agricultural sciences
Application of <120> nucleoporin Nup98a and Nup98b in regulating and controlling flowering time of plants
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Thr Arg Glu Val Ile Val Tyr Met Asp Glu Ser Lys Lys Pro Ala Val
945 950 955 960
Gly Gln Gly Leu Asn Lys Pro Ala Glu Val Thr Leu Leu Asn Ile Lys
965 970 975
Cys Ile Asp Lys Lys Thr Gly Lys Gln Phe Thr Glu Gly Glu Arg Val
980 985 990
Glu Lys Tyr Lys Met Met Leu Lys Lys Lys Ala Glu Ala Gln Gly Ala
995 1000 1005
Glu Phe Val Ser Phe Asp Pro Val Lys Gly Glu Trp Lys Phe Arg Val
1010 1015 1020
Glu His Phe Ser Ser Tyr Lys Leu Gly Asp Glu Asp Glu Glu Asp Gly
1025 1030 1035 1040
Val
<210> 2
<211> 997
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Phe Gly Ser Ser Asn Asn Asn Pro Phe Gly Gln Ser Ser Ile Ser
1 5 10 15
Ser Pro Phe Gly Thr Gln Thr His Ser Leu Phe Gly Gln Thr Asn Asn
20 25 30
Asn Ala Ser Asn Asn Pro Phe Ala Thr Lys Pro Phe Gly Thr Ser Thr
35 40 45
Pro Phe Gly Ala Gln Thr Gly Ser Ser Met Phe Gly Gly Thr Ser Thr
50 55 60
Gly Val Phe Gly Ala Pro Gln Thr Ser Ser Pro Phe Gly Ala Ser Pro
65 70 75 80
Gln Ala Phe Gly Ser Ser Thr Gln Ala Phe Gly Ala Ser Ser Thr Pro
85 90 95
Ser Phe Gly Ser Ser Asn Ser Pro Phe Gly Gly Thr Ser Thr Phe Gly
100 105 110
Gln Lys Ser Phe Gly Leu Ser Thr Pro Gln Ser Ser Pro Phe Gly Ser
115 120 125
Thr Thr Gln Gln Ser Gln Pro Ala Phe Gly Asn Ser Thr Phe Gly Ser
130 135 140
Ser Thr Pro Phe Gly Ala Ser Thr Thr Pro Ala Phe Gly Ala Ser Ser
145 150 155 160
Thr Pro Ala Phe Gly Val Ser Asn Thr Ser Gly Phe Gly Ala Thr Asn
165 170 175
Thr Pro Gly Phe Gly Ala Thr Asn Thr Thr Gly Phe Gly Gly Ser Ser
180 185 190
Thr Pro Gly Phe Gly Ala Ser Ser Thr Pro Ala Phe Gly Ser Thr Asn
195 200 205
Thr Pro Ala Phe Gly Ala Ser Ser Thr Pro Leu Phe Gly Ser Ser Ser
210 215 220
Ser Pro Ala Phe Gly Ala Ser Pro Ala Pro Ala Phe Gly Ser Ser Gly
225 230 235 240
Asn Ala Phe Gly Asn Asn Thr Phe Ser Ser Gly Gly Ala Phe Gly Ser
245 250 255
Ser Ser Thr Pro Thr Phe Gly Ala Ser Asn Thr Ser Ala Phe Gly Ala
260 265 270
Ser Ser Ser Pro Ser Phe Asn Phe Gly Ser Ser Pro Ala Phe Gly Gln
275 280 285
Ser Thr Ser Ala Phe Gly Ser Ser Ser Phe Gly Ser Thr Gln Ser Ser
290 295 300
Leu Gly Ser Thr Pro Ser Pro Phe Gly Ala Gln Gly Ala Gln Ala Ser
305 310 315 320
Thr Ser Thr Phe Gly Gly Gln Ser Thr Ile Gly Gly Gln Gln Gly Gly
325 330 335
Ser Arg Val Ile Pro Tyr Ala Pro Thr Thr Asp Thr Ala Ser Gly Thr
340 345 350
Glu Ser Lys Ser Glu Arg Leu Gln Ser Ile Ser Ala Met Pro Ala His
355 360 365
Lys Gly Lys Asn Met Glu Glu Leu Arg Trp Glu Asp Tyr Gln Arg Gly
370 375 380
Asp Lys Gly Gly Gln Arg Ser Thr Gly Gln Ser Pro Glu Gly Ala Gly
385 390 395 400
Phe Gly Val Thr Asn Ser Gln Pro Ser Ile Phe Ser Thr Ser Pro Ala
405 410 415
Phe Ser Gln Thr Pro Val Asn Pro Thr Asn Pro Phe Ser Gln Thr Thr
420 425 430
Pro Thr Ser Asn Thr Asn Phe Ser Pro Ser Phe Ser Gln Pro Thr Thr
435 440 445
Pro Ser Phe Gly Gln Pro Thr Thr Pro Ser Phe Arg Ser Thr Val Ser
450 455 460
Asn Thr Thr Ser Val Phe Gly Ser Ser Ser Ser Leu Thr Thr Asn Thr
465 470 475 480
Ser Gln Pro Leu Gly Ser Ser Ile Phe Gly Ser Thr Pro Ala His Gly
485 490 495
Ser Thr Pro Gly Phe Ser Ile Gly Gly Phe Asn Asn Ser Gln Ser Ser
500 505 510
Pro Leu Phe Gly Ser Asn Pro Ser Phe Ala Gln Asn Thr Thr Pro Ala
515 520 525
Phe Ser Gln Thr Ser Pro Leu Phe Gly Gln Asn Thr Thr Pro Ala Leu
530 535 540
Gly Gln Ser Ser Ser Val Phe Gly Gln Asn Thr Asn Pro Ala Leu Val
545 550 555 560
Gln Ser Asn Thr Phe Ser Thr Pro Ser Thr Gly Phe Gly Asn Thr Phe
565 570 575
Ser Ser Ser Ser Ser Leu Thr Thr Ser Ile Ser Pro Phe Gly Gln Ile
580 585 590
Thr Pro Ala Val Thr Pro Phe Gln Ser Ala Gln Pro Thr Gln Pro Leu
595 600 605
Gly Ala Phe Gly Phe Asn Asn Phe Gly Gln Thr Gln Ile Ala Asn Thr
610 615 620
Thr Asp Ile Ala Gly Ala Met Gly Thr Phe Ser Gln Gly Asn Phe Lys
625 630 635 640
Gln Gln Pro Ala Leu Gly Asn Ser Ala Val Met Gln Pro Thr Pro Val
645 650 655
Thr Asn Pro Phe Gly Thr Leu Pro Ala Leu Pro Gln Ile Ser Ile Ala
660 665 670
Gln Gly Gly Asn Ser Pro Ser Ile Gln Tyr Gly Ile Ser Ser Met Pro
675 680 685
Val Val Asp Lys Pro Ala Pro Val Arg Val Ser Pro Leu Leu Thr Ser
690 695 700
Arg His Leu Leu Gln Arg Arg Val Arg Leu Pro Thr Arg Lys Tyr Arg
705 710 715 720
Pro Ser Asp Asp Gly Pro Lys Val Pro Phe Phe Ser Asp Glu Glu Glu
725 730 735
Asn Ser Ser Thr Pro Lys Ala Asp Ala Phe Phe Ile Pro Arg Glu Asn
740 745 750
Pro Arg Ala Leu Phe Ile Arg Pro Val Glu Arg Val Lys Ser Glu His
755 760 765
Pro Lys Asp Ser Pro Thr Pro Leu Gln Glu Asn Gly Lys Arg Ser Asn
770 775 780
Gly Val Thr Asn Gly Ala Asn His Glu Thr Lys Asp Asn Gly Ala Ile
785 790 795 800
Arg Glu Ala Pro Pro Val Lys Val Asn Gln Lys Gln Asn Gly Thr His
805 810 815
Glu Asn His Gly Gly Asp Lys Asn Gly Ser His Ser Ser Pro Ser Gly
820 825 830
Ala Asp Ile Glu Ser Leu Met Pro Lys Leu His His Ser Glu Tyr Phe
835 840 845
Thr Glu Pro Arg Ile Gln Glu Leu Ala Ala Lys Glu Arg Val Glu Gln
850 855 860
Gly Tyr Cys Lys Arg Val Lys Asp Phe Val Val Gly Arg His Gly Tyr
865 870 875 880
Gly Ser Ile Lys Phe Leu Gly Glu Thr Asp Val Cys Arg Leu Asp Leu
885 890 895
Glu Met Val Val Gln Phe Lys Asn Arg Glu Val Asn Val Tyr Met Asp
900 905 910
Glu Ser Lys Lys Pro Pro Val Gly Gln Gly Leu Asn Lys Pro Ala Val
915 920 925
Val Thr Leu Leu Asn Ile Lys Cys Met Asp Lys Lys Thr Gly Thr Gln
930 935 940
Val Met Glu Gly Glu Arg Leu Asp Lys Tyr Lys Glu Met Leu Lys Arg
945 950 955 960
Lys Ala Gly Glu Gln Gly Ala Gln Phe Val Ser Tyr Asp Pro Val Asn
965 970 975
Gly Glu Trp Thr Phe Lys Val Glu His Phe Ser Ser Tyr Lys Leu Gly
980 985 990
Asp Glu Tyr Asp Val
995
<210> 3
<211> 3126
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtttggct catctaatcc ttttggacag tcatctggta ccagcccatt tgggtctcaa 60
tctttgtttg gtcagaccag caatacaagt agtaataatc cctttgctcc agctacaccg 120
tttggcacgt ctgctccttt tgctgcacag tcgggaagtt caatattcgg aagcacttca 180
accggtgttt ttggtgcacc tcaaacatcc tctccctttg cctccacccc aacttttgga 240
gcttcttcat caccagcatt tgggaattct actccggcct ttggagcatc tccagcatct 300
tcaccttttg gtggatcttc tggttttggg cagaaacctt tgggattttc aacgcctcag 360
tcaaatcctt ttggaaactc cacgcagcaa tcccaacctg catttggaaa cacctctttt 420
ggttcgtcta caccttttgg tgccactaac acgcctgcct ttggcgctcc aagcacgcct 480
tcttttggcg ccacaagcac accctcgttt ggtgcatcaa gcacgcctgc ctttggtgcc 540
acaaacacgc ctgccttcgg tgcctcaaac tctccctcct ttggcgccac aaacacacct 600
gcatttggcg cgtcaccgac tccagctttt ggtagcactg gcaccacgtt tggtaacact 660
ggctttggtt ctggaggtgc ttttggagct tcaaacaccc ctgcttttgg tgcatcaggc 720
actccggctt ttggtgcatc aggcactcct gccttcggtg catcaagtac tcctgccttt 780
ggggcatcca gtactcctgc ctttggggca tccagtactc ctgcctttgg gggatctagt 840
actccttctt ttggtgcgtc aaatacctca tcctttagtt ttggctcgtc tccagctttt 900
ggccagtcta catcagcctt tggtagcagc gcctttggct ctacaccatc tccctttgga 960
ggtgcgcagg cttcaacccc tacatttggg ggttctggtt ttggtcaatc aactttcggt 1020
ggtcaacaag ggggcagtcg agctgtgcct tatgcaccaa ctgttgaggc agacacaggg 1080
accggcactc agcctgctgg aaagttggaa tctatttctg ccatgccagc atacaaagag 1140
aaaaactatg aggagctgag gtgggaagat taccagcgag gagataaagg tggaccactt 1200
cctgctggcc agtctcctgg caatgccgga tttggtatat caccttctca accaaatcca 1260
ttttctccat caccggcgtt tggtcagaca tcagcaaatc caacaaaccc tttttcgagc 1320
tcaacatcta ccaacccttt tgcccctcaa accccaacaa ttgctagttc gagttttgga 1380
acggctacct caaattttgg ttcctcgccc tttggtgtaa catcttcttc caatcttttt 1440
ggttcaggtt catcaactac cacatccgtt tttggatcat cctccgcttt tggaacaacg 1500
acaccatcac cgttatttgg ttcatcaagc actcctggat ttggctcttc ttcatcaatc 1560
tttggttcag ctcctggtca gggggcaact ccagcatttg gcaacagtca gccatctact 1620
ctgttcaatt caaccccttc gaccgggcag actggttctg cgtttggaca gactggttct 1680
gcttttggac agttcggaca gagtagtgct cctgcatttg gccaaaatag catttttaat 1740
aaaccttcca ctggatttgg aaacatgttt tccagttctt caacattaac cacgtctagc 1800
agctctccct ttggacaaac aatgcctgct ggtgtgacac cctttcaatc agctcagcca 1860
ggtcaagcat caaatggttt tggtttcaac aacttcggtc aaaatcaagc agctaatact 1920
actggaaatg ctggtggact ggggattttt ggtcaaggca acttcgggca atcgcctgct 1980
ccgctgaact ctgttgtttt gcaaccagtt gctgtgacaa acccatttgg aacacttcct 2040
gctatgcctc agatttcaat aaatcaaggt ggaaattcac cttccattca atatggaatc 2100
tccagcatgc ctgttgttga caaacctgct cctgttagaa tatcatctct cctgacttct 2160
cgacacctat tacacaggcg ggtcaggctg ccggcaagaa agtaccgtcc tggcgagaat 2220
ggtccaaagg ttccattttt cactgatgac gaagagagct caagtacacc aaaggcggat 2280
gctcttttca ttccaaggga gaaccctaga gctctagtta tacgcccagt tcaacagtgg 2340
tcttcaaggg ataaatcaat acttccaaag gaacaacgcc cgactgctcc attacatgac 2400
aatgggaaga gtcccgacat ggccactgat gcagcaaacc atgacagaaa tggtaacgga 2460
gaacttggtg ctactgggga aagaattcat acatcagtca atgcaaatca gaaaccgaat 2520
ggcacaacac gctcagatca ggctagcgag aaagagcgtc cctacaaaac tctaagtgga 2580
cacagagcag gagaagccgc aattgtttat gagcacggtg cagatatcga ggctcttatg 2640
ccgaagctac gacaatctga ttatttcaca gagccacgta tccaagaact agcagctaag 2700
gaaagagcgg atccgggtta ctgcaggcgg gtaagagact ttgtagtggg acgacacggt 2760
tatggtagca taaagttcat gggagagaca gatgtgcgta ggctagactt ggaatcactg 2820
gttcagttca atacacggga agtgattgta tacatggacg agagcaagaa accggctgtt 2880
gggcaaggtt tgaataaacc ggcggaagta acgttgctga acataaaatg tattgacaag 2940
aagacaggga aacagtttac tgaaggagag agagtggaga agtataagat gatgcttaag 3000
aagaaagctg aggcacaagg agctgagttt gtgtcatttg atccagtgaa aggcgaatgg 3060
aagtttcggg tcgagcattt tagttcgtat aagctgggcg acgaagatga agaagatgga 3120
gtttag 3126
<210> 4
<211> 2994
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgttcggtt cttctaataa taatcctttt ggacagtcat ctataagcag tccttttggg 60
acccaaactc attctttgtt cggacagacc aataataacg cgagcaataa tccttttgct 120
acgaaacctt ttggtacttc tactcctttt ggcgcacaga cagggagttc tatgtttggg 180
ggcacttcaa ctggtgtgtt tggtgcacct caaacttctt caccttttgg tgcttcacca 240
caagcgtttg ggagctctac tcaggctttt ggagcatctt ctaccccttc atttggcagt 300
tcaaattcac cttttggtgg cacctctacg tttggtcaga agtcttttgg cctctcaaca 360
cctcagtcaa gtccttttgg aagcaccaca caacaatcac agcctgcctt tggaaacagc 420
acttttggtt catcaacacc ttttggtgcc tcaaccacgc ctgcctttgg tgcctctagc 480
acgcctgcct ttggtgtctc aaacacttcc gggtttggag ccacaaacac acccggattt 540
ggtgccacaa acacaacagg gtttggtggc agtagcacac ctgggtttgg ggcatcaagc 600
acgcctgcct ttggctccac aaacacgcct gcttttggtg cttcaagtac tcctttattc 660
ggctcctcga gctcacctgc atttggtgcg tcacctgctc cagcctttgg tagctctggc 720
aatgcatttg gcaacaatac cttcagttct ggaggtgctt ttggttcatc aagcactcct 780
acgtttgggg catctaacac ttctgccttt ggggcatcaa gttccccatc ttttaatttc 840
ggttcttccc cagcttttgg acagtctacc tcagcatttg gtagcagttc ctttggttct 900
acacaatctt ccttaggttc tacaccatct cccttcggag cccaaggtgc acaggcttcg 960
acttcaacat ttgggggtca atcaactatc gggggtcaac aaggggggag tagggttatt 1020
ccttatgcac ctacaactga cacagcgtca ggcactgaaa gcaagtctga aagactacaa 1080
tctatatctg ccatgcctgc acacaaaggg aaaaacatgg aggagcttag gtgggaggac 1140
taccagcgag gagataaagg aggacaacgt tctactggtc aatctcctga aggtgctgga 1200
tttggtgtaa caaattctca gccaagtata ttctctacgt caccagcgtt tagccagaca 1260
cctgtgaatc caacaaaccc tttttcacag acgactccaa caagtaacac aaatttttcc 1320
ccctccttta gtcaacccac taccccatcc tttggtcaac ccactacccc atcctttagg 1380
tcaactgtat caaatactac atccgttttt ggatcatcct ctagtttaac aacaaacaca 1440
tcgcagccac ttggttcatc aatatttggt tctactcctg cacatgggtc aactccagga 1500
tttagcattg gtggatttaa caacagtcag tcatctccgc tgtttggttc aaacccctca 1560
tttgcacaaa ataccactcc tgcattcagc cagactagtc ctttgtttgg acagaatacc 1620
actcctgcat taggacagtc tagttctgtg tttggacaga ataccaatcc tgcacttgta 1680
cagagcaaca ctttcagtac accttccact ggctttggga acacgttttc aagctcttca 1740
tccttaacta caagcatctc cccctttggt caaataacgc ctgctgttac gccctttcaa 1800
tcagctcagc caactcaacc attaggtgct tttggtttca acaactttgg tcaaacccaa 1860
atagctaata caactgacat tgcaggtgca atgggaactt ttagccaagg aaactttaag 1920
caacagcctg cattgggtaa ctctgctgtt atgcaaccaa ctcctgttac aaacccattt 1980
ggaacacttc ctgctttgcc tcagatttca attgctcaag gtggaaattc accttcgatt 2040
cagtatggta tctccagcat gcctgtcgtt gataagcctg ctcctgttag agtatcacca 2100
ctcctaactt ctcgacacct tttacaaagg cgggtcaggc taccaacaag aaagtaccgt 2160
cctagtgatg atggtccaaa ggttccattc ttcagcgatg aagaggagaa ttccagtaca 2220
ccaaaggctg atgcgttttt cattccaagg gagaacccta gagctttatt tatacgccca 2280
gttgaaaggg taaaatcaga acatccaaag gatagcccga ccccactgca ggagaacggt 2340
aagaggtcga atggggttac taatggagcc aaccatgaga ctaaggataa cggtgcaatt 2400
cgtgaagctc ccccggtaaa agtcaatcag aaacagaacg ggacacatga aaatcacgga 2460
ggcgacaaaa acggttcaca cagttcacca agtggagcag atattgagtc attaatgccg 2520
aagttgcatc actctgaata tttcacagag cctcggatcc aagaactggc tgcaaaagaa 2580
agggttgaac agggttattg caagcgggta aaggactttg ttgttgggag acacggttac 2640
gggagcataa agtttctagg agagacggat gtgtgtagac tcgacctgga aatggtggtt 2700
cagttcaaaa accgggaagt gaatgtgtac atggacgaga gcaaaaaacc gccggtgggt 2760
caaggattga ataagcctgc ggtggtaact ttgctgaaca taaaatgtat ggacaagaag 2820
accggcacgc aagtgatgga aggcgagaga ttggacaagt acaaggaaat gctcaagagg 2880
aaagctgggg aacaaggagc tcagtttgtg tcttatgatc ctgtgaatgg cgagtggact 2940
ttcaaggtcg aacatttcag ttcctacaag cttggagatg aatatgatgt gtag 2994
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgtttggct catctaatcc ttt 23
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctaaactcca tcttcttcat cttc 24
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgttcggtt cttctaataa taat 24
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctacacatca tattcatctc caag 24
Claims (5)
1. The application of the arabidopsis thaliana nucleoporins Nup98a and Nup98b in shortening the flowering time of arabidopsis thaliana is disclosed, wherein the amino acid sequence of Nup98a is shown as SEQ ID NO.1, and the amino acid sequence of Nup98b is shown as SEQ ID NO. 2;
the application is to lead the flowering time of arabidopsis thaliana to be advanced by inactivating nucleopore proteins Nup98a and Nup98b or reducing the expression of nucleopore proteins Nup98a and Nup98 b.
2. The application of arabidopsis thaliana nucleoporins Nup98a and Nup98b in shortening the growth cycle of arabidopsis thaliana is disclosed, wherein the amino acid sequence of Nup98a is shown as SEQ ID NO.1, and the amino acid sequence of Nup98b is shown as SEQ ID NO. 2;
the application is that the growth period of arabidopsis is shortened by inactivating nucleopore proteins Nup98a and Nup98b or reducing the expression of nucleopore proteins Nup98a and Nup98 b.
3. The application of the arabidopsis nucleoporins Nup98a and Nup98b in the genetic breeding of arabidopsis with shortened early flowering or growth period or the construction of transgenic arabidopsis with shortened early flowering or growth period is disclosed, wherein the amino acid sequence of Nup98a is shown as SEQ ID NO.1, and the amino acid sequence of Nup98b is shown as SEQ ID NO. 2.
4. The application of coding genes of arabidopsis nucleopore proteins Nup98a and Nup98b or biological materials containing the coding genes in arabidopsis genetic breeding for shortening the flowering time of arabidopsis, shortening the growth cycle of arabidopsis, and shortening the early flowering or the growth period or constructing transgenic arabidopsis with shortened early flowering or the growth period, wherein the amino acid sequence of Nup98a is shown as SEQ ID NO.1, and the amino acid sequence of Nup98b is shown as SEQ ID NO. 2;
the application is that the flowering time of arabidopsis is advanced or the growth period is shortened by inactivating the coding genes of nucleopore proteins Nup98a and Nup98b or reducing the expression of the coding genes of nucleopore proteins Nup98a and Nup98 b;
the nucleotide sequence of the coding gene of Nup98a is shown in SEQ ID NO.3, and the nucleotide sequence of the coding gene of Nup98b is shown in SEQ ID NO. 4;
the biological material is an expression cassette, a vector, a transposon, an engineering bacterium, a host cell or a transgenic cell line.
5. A method for shortening flowering time and growth cycle of Arabidopsis thaliana or constructing transgenic Arabidopsis thaliana with shortened early flowering or growth period is characterized in that the flowering time of Arabidopsis thaliana is advanced or the growth period is shortened by inactivating nucleopore proteins Nup98a and Nup98b or by reducing expression of nucleopore proteins Nup98a and Nup98 b;
the amino acid sequence of Nup98a is shown in SEQ ID NO.1, and the amino acid sequence of Nup98b is shown in SEQ ID NO. 2.
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