BnACO1 gene, protein and application of BnACO1 gene and protein in regulating and controlling plant height of brassica napus

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a BnACO1 gene, a protein and application of the BnACO1 gene in regulating and controlling the plant height of brassica napus. According to the invention, the plant height related gene BnACO1 is separated from the cabbage type rape rolled leaf mutant Bnuc1. The subcellular localization result shows that BnACO1 is localized in cytoplasm and cell membranes. According to the invention, a BnACO1 overexpression vector is constructed, and double-11 (ZS11) in wild type of brassica napus is transformed. Compared with the control ZS11, the transgenic positive plant has the advantages that the plant height is obviously reduced, the leaf is shrunk, and the flowering period is delayed by 5-7 days. The BnACO1 disclosed by the invention can be used for controlling the height of a rape plant and has an important utilization value for dwarf breeding of the rape.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and in particular relates to a BnACO1 gene, a protein and an application thereof in regulating the growth of Brassica napus plants. Background Art

[0002] Brassica napus is native to my country and is one of the country's important oil crops. Rapeseed has a high oil content, generally around 40%. Plant height is one of the important agronomic traits and the basis for the formation of excellent plant type. It is closely related to crop yield, light energy utilization efficiency and mechanized operation efficiency. At present, rapeseed varieties widely cultivated across the country are easy to cultivate and manage with their tall plant characteristics. However, this feature also invisibly leads to problems of reduced yield and increased difficulty in harvesting. In order to effectively respond to these challenges, rapeseed breeding experts have actively invested in cultivating rapeseed mutants with more compact plant height. Their in-depth research aims to explore and utilize the key gene resources behind the dwarfing trait, in order to achieve the stable inheritance and application of this trait in rapeseed breeding practice. This process not only helps to alleviate the yield loss caused by excessively tall plants, but also significantly improves the mechanized operation efficiency of rapeseed fields, thereby reducing production costs and improving overall economic benefits.

[0003] Plant height is a complex trait that is regulated by multiple genes. These genes may be distributed on different chromosomes and affect plant height in the form of quantitative trait loci (QTLs). For example, the plant height trait of wheat is regulated by multiple genes, and its quantitative trait loci are widely distributed on 21 chromosomes of wheat. Reducing plant height not only increases yield, but also improves wheat's resistance to lodging and fertilizer resistance, while also having wide adaptability and yield stability. Apple MdCo31 encodes a 2OG-Fe(II)-dependent oxygenase. Overexpression of this gene leads to phenotypes such as reduced tobacco growth, shortened internodes, and reduced plant height, proving that MdCo31 is a gene that controls apple dwarfing growth. With the development of molecular biology techniques, more and more genes that control plant height have been cloned and their functions have been verified. The discovery of these genes provides an important basis for revealing the genetic mechanism of plant height.

[0004] Plant height is also regulated by a variety of hormones. It has been found that a variety of hormones are involved in the regulation of plant height, including gibberellin, brassinosteroid, and strigolactone. These hormones affect the elongation and division of plant cells through complex signal transduction pathways, thereby regulating plant height. Studies on dwarf materials have shown that the plant hormones that affect plant height mainly include gibberellin (GA), brassinosteroid (BR), and auxin (IAA).

[0005] With the continuous deepening of genomic research, more new genes that control plant height will be discovered and cloned in the future. The discovery of these new genes will provide new perspectives and ideas for revealing the genetic mechanism of plant height, and will help increase crop yields, enhance stress resistance and promote sustainable agricultural development. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides BnACO1 gene, protein and application thereof in regulating the height of Brassica napus plants, so that the transgenic plants become shorter, the number of branches is reduced and the flowering period is delayed.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a Brassica napus BnACO1 protein, wherein the Brassica napus BnACO1 protein comprises the amino acid sequence described in any one of the following (1) and (2):

[0009] (1) the amino acid sequence shown in SEQ ID NO.3;

[0010] (2) A sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.3.

[0011] The present invention also provides a Brassica napus BnACO1 gene encoding the Brassica napus BnACO1 protein of claim 1, wherein the full-length genome sequence of the Brassica napus BnACO1 gene is the nucleotide sequence of any one of the following (1) and (2):

[0012] (1) the nucleotide sequence shown in SEQ ID NO.1;

[0013] (2) A DNA molecule having a nucleotide sequence with more than 90% homology to the sequence shown in SEQ ID NO.1 and encoding the amino acid sequence shown in SEQ ID NO.3.

[0014] The present invention also provides application of the above protein or gene in regulating the plant height or flowering period of Brassica napus.

[0015] The present invention also provides a method for regulating the plant height of Brassica napus, constructing a BnACO1 overexpression vector to obtain a Brassica napus dwarf plant; the full-length genome sequence of the BnACO1 is shown in SEQ ID No.1; the full-length cDNA sequence of the BnACO1 is shown in SEQ ID No.2.

[0016] The present invention also provides a method for regulating the flowering period of Brassica napus, constructing a BnACO1 overexpression vector to obtain a late-flowering Brassica napus plant; the full-length genome sequence of the BnACO1 is shown in SEQ ID No.1; the full-length cDNA sequence of the BnACO1 is shown in SEQ ID No.2.

[0017] Furthermore, the BnACO1 overexpression vector is obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

[0018] Further, the method comprises the following steps: inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector to obtain an overexpression vector, then transforming Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into a Brassica napus receptor, and screening the offspring of the transformed plants.

[0019] Furthermore, the homologous sequences of two enzymes, Kpn I and BamH I, were introduced into the two ends of the upstream and downstream primers of the gene cloning to form new primers;

[0020] Upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3'; downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3'.

[0021] Further, the detection primers are:

[0022] Upstream primer P9: 7′-GAGGCTTACGCAGCAGGTCTCA-3′;

[0023] Downstream primer P10: 5′-TGAGGCAATACATCAGAATCAG-3′.

[0024] Furthermore, the primer sequences for real-time fluorescence quantitative PCR analysis are:

[0025] Upstream primer P11: 5′-ATTACTGGCGTTAGCACTT-3′;

[0026] Downstream primer P12: 5′-TATTCCTTTCGACGGATC-3′.

[0027] The BnACO1 of the present invention is localized in the cytoplasm and cell membrane of tobacco mesophyll.

[0028] The overexpression of BnACO1 in the present invention reduces plant height, the number of branches, and delays flowering time by 5-7 days.

[0029] The method for overexpressing BnACO1 of the present invention is to construct the BnACO1 gene from the dwarf material M176 into the PBI121 overexpression vector, then transform the Brassica napus Zhongshuang 11, and obtain the Brassica napus overexpressing BnACO1 after screening the transgenic positive plants.

[0030] The plant overexpression vector of the present invention is obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

[0031] The Agrobacterium tumefaciens containing the plant overexpression vector of the present invention is GV3101.

[0032] Application of overexpression of BnACO1 in reducing the height of Brassica napus plants.

[0033] A method for obtaining short-stem, late-flowering Brassica napus, comprising the following steps: inserting a sequence shown in SEQ ID No. 2 between a CaMV35S promoter and an OCS terminator of a PBI121 vector to obtain a vector for overexpressing BnACO1, then transforming Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into a double 11 receptor in Brassica napus, and screening transgenic plants to obtain short-stem, late-flowering Brassica napus.

[0034] Preferably, the Brassica napus receptor is Shuang 11, a typical variety of Brassica napus.

[0035] Beneficial Effects

[0036] The present invention provides a full-length genome, cDNA sequence, protein sequence and the like of the BnACO1 gene, and confirms that BnACO1 can affect plant height by transforming Zhongshuang 11. The plant height of the transgenic plant is reduced from 173 cm to 35 cm, a decrease of 80%; the stem diameter is increased from 22.7 cm to 27.6 cm; the number of siliques on the whole plant is reduced from 290 to 94; the number of angular grains is reduced from 31 to 12; and the flowering period is delayed by 5-7 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the electrophoresis diagram of the full-length DNA and cDNA amplification of the BnACO1 gene in Brassica napus.

[0038] Figure 2 This is the gene structure of BnACO1.

[0039] Figure 3 This is the domain distribution of BnACO1.

[0040] Figure 4This is a multiple sequence alignment of BnACO1 and other proteins such as Arabidopsis AT1G34390.1.

[0041] Figure 5 Subcellular localization of BnACO1.

[0042] Figure 6 To identify the DNA level of transgenic plants.

[0043] Figure 7 To identify the transcription level of transgenic plants.

[0044] Figure 8 This is the flowering phenotype of the transgenic plants. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. The experimental methods for which specific conditions are not specified in the preferred embodiments shall be carried out in accordance with the relevant product production instructions or molecular cloning experimental guidelines. Plant materials used in the embodiments: Brassica napus Zhongshuang No. 11 was provided by the Oil Crops Research Institute of the Chinese Academy of Sciences. Zhongshuang No. 11 is a publicly available double-low, excellent conventional variety (approval number is Guoshenyou 2008030). The upper leaf roll mutant Bnuc1 was created in this experiment through EMS mutagenesis, and the upper leaf roll near-isogenic line ZS11-uc1 was created through marker-assisted selection.

[0046] Example 1: Cloning of BnACO1 full-length DNA and cDNA

[0047] 1. Extraction of Total DNA and Total RNA from Brassica napus

[0048] Tender leaves of Brassica napus ZS11-uc1 were taken, and the total gene DNA was extracted by the modified CTAB method. The concentration and quality of the DNA samples were evaluated by 1% agarose gel electrophoresis and UV spectrophotometer. The DNA samples were stored in a -20 ℃ refrigerator. Leaves of Zhongshuang No. 11 and BnACO1 overexpressing materials were collected at the seedling stage, and RNA was extracted using the plant total RNA extraction kit of TIANGEN Company and dissolved in pure water. The samples were tested for RNA quality and purity by 1% agarose gel electrophoresis and UV spectrophotometer, respectively. The electrophoresis results showed three complete RNA bands (28S, 18S, 5S), without obvious degradation and DNA contamination, which could meet the requirements of subsequent experiments. RNA samples were stored in a -80 ℃ refrigerator.

[0049] 2. Synthesis of the First Strand of Brassica napus cDNA

[0050] After equal amounts of RNA extracted in 1 above were mixed, reverse transcription was performed using Takara's PrimeScript™ 1stStrand cDNA Synthesis Kit according to its instructions, and the reverse transcription product was stored in a -20°C refrigerator for later use.

[0051] 3. Cloning of full-length DNA and cDNA of BnACO1 in Brassica napus

[0052] The extracted DNA was used as template, and the upstream primer P1: 5'-CAGAGGTTGCTTTGTTGCCTGA-3' (SEQ ID NO.4) and the downstream primer P2: 5'-CAGTGCAGTAGCAACTACACCA-3' (SEQ ID NO.5); the reverse transcribed cDNA was used as template, and the upstream primer P3: 5'-ATGGCGAGTGATCAAATCATG-3' (SEQ ID NO.6) and the downstream primer P4: 5'-CTAATTGTTGTTGACATCTGGTGG-3' (SEQ ID NO.7) were used to amplify the full-length DNA and cDNA sequences of BnACO1, respectively. PCR amplification was performed using KOD enzyme (TOYOBO, Japan). The PCR reaction system was added according to the instructions of the enzyme, and the reaction program was pre-denaturation at 94℃ for 2 min, and then 35 cycles were set according to the program of 94℃: 15 s; 52℃: 30 s; 68℃: 3 min. The PCR product was consistent with the expected size by agarose gel electrophoresis. After gel recovery, ligation of the cloning vector to transform Escherichia coli, PCR and sequencing, the full-length DNA of BnACO1 was determined to be 2054 bp ( Figure 1 ), named BnACO1DNA, the sequence is shown in SEQID NO.1; the full-length cDNA of BnACO1 was determined to be 1011 bp in length ( Figure 1 ), named it BnACO1mRNA, the sequence is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.3.

[0053] Example 2: Bioinformatics analysis of the BnACO1 gene

[0054] The gene structure of BnACO1 was analyzed using the online website http: / / gsds.gao-lab.org / ; the protein structure was analyzed using the website https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. The full-length DNA of BnACO1 is 2054 bp, containing 11 exons and 10 introns ( Figure 2The full-length cDNA is 1011 bp long and encodes 336 amino acid residues. The amino acid sequence of BnACO1 protein was compared with that of the homologous proteins ACO in Arabidopsis and rice, and it was found that BnACO1 has two typical domains of ACO protein: DIOX_N domain at the N-terminus and 2OG-Fe(II)_Oxy domain at the C-terminus. Figure 3 and Figure 4 ).

[0055] Example 3: Subcellular localization analysis of BnACO1

[0056] The cloned full-length cDNA plasmid of BnACO1 was used as a template, and primers with restriction sites were designed to amplify the target fragment. The upstream primer was P5: 5'-GAACGATAGGGTACCATGGCGAGTGATCAAATCATG-3' (SEQ ID NO.8), and the downstream primer was P6: 5'-GCTCACCATGGATCCATTGTTGTTGACATCTGGTGG-3' (SEQ ID NO.9). At the same time, the plasmid pCAMBIA1305-GFP was digested with two enzymes (Kpn I and Sal I, Takara). The target gene fragment recovered from the gel and the restriction-cut plasmid fragment were connected using the homologous recombination ClonExpress IIOne Step Cloning Kit (Vazyme, Nanjing). The reaction system was referred to the instructions of the enzyme. After the recombinant plasmid was transformed into Escherichia coli, the bacterial liquid was detected and sequenced for verification.

[0057] Take a tube (100 μL) of Agrobacterium GV3101 (Weidi Biotechnology) competent cells and melt them on ice, add 4 μL of 35S::BnACO1-GFP recombinant plasmid, mix gently and place on ice for 5 min, freeze in liquid nitrogen for 5 min, then quickly place in a 37°C water bath for 5 min, ice bath for 2 min, add 700 μL of YEB liquid medium without antibiotics, culture at 28°C and 200 rpm for 3-4 h, evenly spread the bacterial liquid on YEB solid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin, culture at 28°C upside down for 48 h, and pick a single clone for PCR detection. Save the positive Agrobacterium for later use.

[0058] Take 200 μL of the above positive Agrobacterium culture and add it to 20 mL of YEB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin, and culture it at 28 ℃ and 200 rpm for 12-16 h; take 1 mL of the culture to 100 mL of fresh YEB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin, and culture it with homologous shaking for 8-12 h until OD 600 Centrifuge at 4000 rpm for 5 min, discard the supernatant, and resuspend twice with 10 mM MES, 10 mM MgCl2, 100 µM AS suspension to make the OD 600 =0.5, and stand in the dark for 1-3 h. Water the tobacco thoroughly one day in advance, inject the bacterial solution into the lower epidermis of the tobacco leaves through a syringe, and culture in the dark at 23 ℃ for 24 h before taking out and culturing normally. In this experiment, the empty pCAMBIA1305-GFP Agrobacterium was used as a control. Use a laser confocal microscope to observe the location of fluorescence in the epidermal cells of tobacco leaves in the control combination experimental group and take pictures. Figure 5 The results showed that the control group GFP empty vector had fluorescence in the nucleus, cytoplasm and cell membrane of tobacco mesophyll cells, and the BnACO1 of the present invention was localized in the cytoplasm and cell membrane of tobacco mesophyll cells.

[0059] Example 4: Construction and genetic transformation of BnACO1 gene overexpression vector

[0060] 1. Construction of BnACO1 gene overexpression vector

[0061] The plant overexpression vector PBI121 was digested with restriction endonucleases Kpn I and BamH I (Takara), and the linearized vector fragment was recovered by 1% gel electrophoresis. The homologous sequences of Kpn I and BamH I were introduced into the upstream and downstream primers of the gene cloning to form new primers (upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3' (SEQ ID NO.10), downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3' (SEQ ID NO.11)), and the target gene fragment with homology to both ends of the linearized vector was obtained by PCR amplification. The target gene fragment was connected to the linearized vector using the ClonExpressII One Step Cloning Kit (Vazyme, Nanjing), and the reaction system was carried out according to the instructions. After reacting at 37℃ for 30 min, the ligation product was transformed into Escherichia coli, and a single clone was picked for bacterial liquid PCR detection, and the positive single clone was sent for sequencing for further verification. The sequencing results were consistent with the results of gene cloning, indicating that the BnACO1 gene was successfully inserted into the overexpression vector PBI121. The recombinant vector was named 35S::BnACO1.

[0062] 2. Agrobacterium-mediated transformation of rapeseed

[0063] The 35S::BnACO1 vector constructed in Example 3 was transformed into Agrobacterium GV3101 competent cells by freeze-thaw method, and 700 μL YEB liquid medium was added for shaking culture for 3 h. The cells were collected by brief centrifugation and spread on solid YEB plates containing Kan and Rif antibiotics. The cells were incubated at 28 °C for about 48 h, and Agrobacterium single clones were picked for PCR detection and sequencing verification. The correctly sequenced Agrobacterium single clones were transferred to 400 mL YEB liquid medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and cultured at 28 °C with shaking until OD 600 Centrifuge at 5000 rpm for 15 min at 0.8-1.0, 4 ℃, collect the precipitate and suspend the bacteria in 1 / 2 MS liquid medium. Add 6-BA and 0.05% Silwet-L-77 at a final concentration of 0.01 mg / L when soaking flowers. Take Zhongshuang No. 11 as the receptor material. Before soaking flowers, remove the blooming flowers and put the remaining flower buds into the prepared Agrobacterium suspension. Depending on the temperature and weather conditions, soak once every 5-7 days, for a total of 3 times. Collect seeds after the rapeseed matures and record them as T0 generation.

[0064] 3. Identification of genetically modified rapeseed

[0065] Identification of transgenic positive strains: The harvested T0 generation transgenic seeds were sown at the Baima base of Nanjing Agricultural University. Rapeseed leaves were collected at the 3-5 leaf stage to extract DNA. The DNA quality was tested by electrophoresis and used as a template for PCR detection. PCR used 2×easy mix (Vazyme, Nanjing), and the system and procedures were designed according to the instructions. The upstream primer used for DNA molecular detection of materials transformed with overexpression vectors came from the 35S promoter sequence on the vector (upstream primer P9: 7'-GAGGCTTACGCAGCAGGTCTCA-3' (SEQ ID NO.12)), and the downstream primer came from the sequence of the target gene (primer sequence P10: 5'-TGAGGCAATACATCAGAATCAG-3' (SEQ ID NO.13)). The screened positive materials were subjected to routine field management ( Figure 6 ), bagged and self-pollinated during the flowering period, and the seeds were collected for later use, which were recorded as T1 generation. The additional generation experiment was carried out in an artificial climate incubator. The T3 generation positive plants with consistent phenotype and genotype were used as materials for subsequent experiments.

[0066] The gene expression level of the transgenic materials was analyzed. With reference to the quantitative primers in the https: / / brassica.biodb.org / database (upstream primer P11: 5'-ATTACTGGCGTTAGCACTT-3' (SEQ ID NO.14) and downstream primer P12: 5'-TATTCCTTTCGACGGATC-3') (SEQ ID NO.15), real-time fluorescence quantitative PCR was performed to analyze the expression level of the BnACO1 gene in Zhongshuang No. 11 and genetic materials. Before real-time fluorescence PCR, the designed quantitative primers and the internal reference gene BnActin primers (upstream primer P13: 5'-ATTCAGCCCCTTGTTTGTG-3' (SEQ ID NO.16) and downstream primer P14: 5'-GTAAGCGTCTTTTTGACCCAT-3' (SEQ ID NO.17)) were tested to analyze the most suitable annealing temperature of the primers (60°C), and the template cDNA was appropriately diluted by the same multiple according to the Ct value. qRT-PCR was performed on a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument using the SuperReal PreMix Plus (TIANGEN) kit and the two-step method according to the instructions. The expression level of the BnACO1 gene in the transgenic material and the Zhongshuang No. 11 material was analyzed. The results of qRT-PCR showed that the expression level of the BnACO1 gene in the transgenic material was significantly higher than that in the control group ( Figure 7The two materials were cultured under the same conditions, and the differences in plant height were observed and photographed at the seedling, flowering and mature stages. The results of genetic transformation showed that the plant height of the 35S-driven BnACO1 overexpressing transgenic plants was significantly reduced, which was consistent with the expected phenotype ( Figure 8 The height of the transgenic plants was reduced from 172 cm to 37 cm, a decrease of 80%, and the stem diameter was increased from 22.7 cm to 27.6 cm; the number of siliques per plant was reduced from 290 to 94; the number of pods per pod was reduced from 31 to 12; and the flowering period was delayed by 5-7 days.

[0067] The above-mentioned embodiments are only preferred implementation modes for fully illustrating the present invention. It should be pointed out that equivalent substitutions or improvements made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

[0068] Sequence Listing

[0069] SEQ ID NO.1

[0070] 1 atggagaatc acacgaaaga ggagctgcaa gacccaacca tgcaagtttc atctcttaat

[0071] 61 tgcatcgatc tcgccaacga tgatcttcat cattctgttg tttccctcaa acaggtcccc

[0072] 121 tctcctttct atttaattaa tctgaaaatt agattttctt taaaaatata tatatatata

[0073] 181 tatatattca aatcaagcgt gagaattgtt gtttcaggca tgtttggatt gtggattttt

[0074] 241 ctatgtgatc aatcacggta taaacgagga gttcatggat gatgttttcg agcagagcaa

[0075] 301 gaagtttttc gctcttcctt tagaggagaa gatgaaagtt ctgagaaacg aaaagcaccg

[0076] 361 aggctataca ccagttcttg atgaaatctt agatcctgag aatcaagtta atggttcgtg

[0077] 421 catgtgttat cagtttaaaa tcaaaaggtg ttttgttgat tgattgagtt tttttttagg

[0078] 481 agatcataaa gagggttatt acattggat tgaagttcct aaagatgatc ctgattatga

[0079] 541 taaccattc tatggcccca acccttggcc tgattctggt tagtgttttt ctcctttgtt

[0080] 601 ttgtctttgt taatttcact ttctttgaat aaactttcgt tgattattgg cttagatgta

[0081] 661 ttgcctcagt ggcgagaaac catggagaaa tatcaccaag aagcattgta agtcctcgtc

[0082] 721 tgaggtttag aatttctgat tcgacaggat tagttgtgtt atgctccatc tctgttcttg

[0083] 781 acaatcaccc aaagtttcgt tttgtatatc aggagggttt ctaaggatat tggaagatta

[0084] 841 ctggcgttag cacttgattt ggatgcgaat tactttgata ccccggagat gcttggaaag

[0085] 901 cctattgcga ctatgcgatt actgcattat gaaggtcatt tattttatta tgtaacatta

[0086] 961 cagcggaatg tgattctta agctttgtat gtcactaaca ttttttcacc tttttttat

[0087] 1021ctttaggggt ttctgatccg tcgaaaggaa tatatgcttg tggagcacat actgattacg

[0088] 1081gaatgatgac tctattagcc actgatggtg taatgggcct ccaggtacta aaagcatcac

[0089] 1141agtttttt tttatcctat cctttatacc ttttgacact actagggtta gttacgagaa

[0090] 1201attgcgaaaa cttggtgaca gatatgcaaa gataagaacg ccaagcctca gaagtgggaa

[0091] 1261tatgtaccgg cgattaaagg gtaactaaca actctgttta tagtcttcac tgggtccatc

[0092] 1321atccattaac catgtcatga gtgaatgata tattattggc ttaagactgt ttgcctaatt

[0093] 1381acatgttttc ttgtctgagc atgtgatcag tattagtatt tgtcaatttg gtggatttct

[0094] 1441ttattttctt tgtctacaga gcatatatag tgaatcttgg agatatgctg gaacgttgga

[0095] 1501gcaatggctt tttcaagtaa tacttctaat caactattga ttgtgatttg tgagtcgtat

[0096] 1561tttctagttg gtgcttttct cacattcatt tcgactctac agatccacgt tacatcgggt

[0097] 1621tcttgggaac ggtcaggaac gatatctgt aaggctctta ctaatctacc tgtgtctgat

[0098] 1681cagtacaatc tttgtggacg tgatggctat agggccttat gcttcaatgc tttgcttatg

[0099] 1741acatgttcag aaccaggtct agcgtataac atcgacgttg catcctatgt tttctgaatg

[0100] 1801cagattccgt ttttcgtgga accaaatcat gaatgtctgg tggagtgtct cccaacttgc

[0101] 1861aagtccgtaa gcaacgttcc caagtaagac ttttatttta tctttgtgtt actgagacgt

[0102] 1921ctgagtggta aacaaatgtt tataaatgat tttgatgctt gtatagatat ccagcgatca

[0103] 1981aatcttgac atacctcacg caacgttaca aggaaacaca tgcagagtta agcatttaca

[0104] 2041atcaagaaat horse

[0105] SEQ ID NO.2

[0106] 1 atggagaatc acacgaaaga ggagctgcaa gacccaacca tgcaagtttc atctcttaat

[0107] 61 tgcatcgatc tcgccaacga tgatcttcat cattctgttg tttccctcaa acaggcatgt

[0108] 121 ttggattgtg gattttcta tgtgatcaat cacggtata acgaggagtt catggatgat

[0109] 181 gttttcgagc aggregate gtttttcgct cttcctttag aggregate aggregate

[0110] 241 agaaacgaaa agcaccgagg ctatacacca gttcttgatg aaatcttaga tcctgagaat

[0111] 301 caagttaatg gagatcataa agagggttat tacattgga ttgaagttcc taaagatgat

[0112] 361 cctgattatg ataaaccatt ctatggcccc aacccttggc ctgattctga tgtattgcct

[0113] 421 cagtggcgag aaaccatgga gaaatatcac caagaagcat tgagggtttc taaggatatt

[0114] 481 ggaagatc tggcgttagc acttgatttg gatgcgaatt actttgatac cccggagatg

[0115] 541 cttggaaagc ctattgcgac tatgcgatta ctgcattatg aaggggttc tgatccgtcg

[0116] 601 aaaggaatat atgcttgtgg agcacatact gattacgga tgatgactct attagccact

[0117] <h2 style=";text-align:left;direction:ltr">661 gatggtgtaa tgggcctcca gatatgcaaa gataagaacg ccaagcctca gaagtgggaa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0118] <h2 style=";text-align:left;direction:ltr"> 721 tatgtaccgg cgattaaagg agcatatata gtgaatcttg gagatatgct ggaacgttgg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0119] <h2 style=";text-align:left;direction:ltr"> 781 agcaatggct ttttcaaatc cacgttacat cgggttcttg ggaacggtca ggaacgatat<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0120] <h2 style=";text-align:left;direction:ltr"> 841 tctattccgt ttttcgtgga accaaatcat gaatgtctgg tggagtgtct cccaacttgc<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0121] <h2 style=";text-align:left;direction:ltr"> 901 aagtccgtaa gcaacgttcc caaatatcca gcgatcaaat cttcgacata cctcacgcaa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0122] <h2 style=";text-align:left;direction:ltr"> 961 cgttacaagg aaacacatgc agagttaagc atttacaatc aagaaatatg a<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0123] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0124] <h2 style=";text-align:left;direction:ltr"> 1 MENHTKEELQ DPTMQVSSLN CIDLANDDLH HSVVSLKQAC LDCGFFYVIN HGINEEFMDD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0125] <h2 style=";text-align:left;direction:ltr"> 61 VFEQSKKFFA LPLEEKMKVL RNEKHRGYTP VLDEILDPEN QVNGDHKEGY YIGIEVPKDD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0126] <h2 style=";text-align:left;direction:ltr"> 121 PDYDKPFYGP NPWPDSDVLP QWRETMEKYH QEALRVSKDI GRLLALALDL DANYFDTPEM<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0127] <h2 style=";text-align:left;direction:ltr"> 181 LGKPIATMRL LHYEGVSDPS KGIYACGAHT DYGMMTLLAT DGVMGLQICK DKNAKPQKWE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0128] 241 YVPAIKGAYI VNLGDMLERW SNGFFKSTLH RVLGNGQERY SIPFFVEPNH ECLVECLPTC

[0129] 301 KSVSNVPKYP AIKSSTYLTQ RYKETHAELS IYNQEI

Claims

1. A Brassica napus BnACO1 protein, characterized in that The Brassica napus BnACO1 protein comprises the amino acid sequence described in any one of the following (1) and (2): (1) the amino acid sequence shown in SEQ ID NO.3; (2) A sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO.

3.

2. A Brassica napus BnACO1 gene, characterized in that: Encoding the Brassica napus BnACO1 protein of claim 1, wherein the full-length genomic sequence of the Brassica napus BnACO1 gene is the nucleotide sequence described in any one of the following (1) and (2): (1) the nucleotide sequence shown in SEQ ID NO.1; (2) A DNA molecule having a nucleotide sequence with more than 90% homology to the sequence shown in SEQ ID NO.1 and encoding the amino acid sequence shown in SEQ ID NO.

3.

3. Use of the protein according to claim 1 or the gene according to claim 2 in regulating the plant height or flowering period of Brassica napus.

4. A method for regulating the plant height of Brassica napus, characterized in that: A BnACO1 overexpression vector was constructed to obtain dwarf plants of Brassica napus. The full-length genome sequence of BnACO1 is shown in SEQ ID No.

1. The full-length cDNA sequence of BnACO1 is shown in SEQ ID No.

2.

5. A method for regulating the flowering period of Brassica napus, characterized in that: A BnACO1 overexpression vector was constructed to obtain late-flowering plants of Brassica napus. The full-length genome sequence of BnACO1 is shown in SEQ ID No.

1. The full-length cDNA sequence of BnACO1 is shown in SEQ ID No.

2.

6. The method according to claim 4 or 5, characterized in that: The BnACO1 overexpression vector is obtained by inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector.

7. The method according to claim 6, characterized in that The method comprises the following steps: inserting the sequence shown in SEQ ID No. 2 between the CaMV35S promoter and the OCS terminator of the PBI121 vector to obtain an overexpression vector, then transforming Agrobacterium tumefaciens GV3101, transforming the obtained transgenic engineered bacteria into a Brassica napus receptor, and screening the offspring of the transformed plants.

8. The method according to claim 6, characterized in that The homologous sequences of Kpn I and BamH I were introduced into the two ends of the upstream and downstream primers of gene cloning to form new primers; Upstream primer P7: 5'-CGCGGATCCATGGAGAATCACACGAAA-3'; downstream primer P8: 5'-ACGCGTCGACTCATATTTCTTGATTGTAAATG-3'.

9. The method according to claim 8, characterized in that The detection primers are: Upstream primer P9: 7′-GAGGCTTACGCAGCAGGTCTCA-3′; Downstream primer P10: 5′-TGAGGCAATACATCAGAATCAG-3′.

10. The method according to claim 8, characterized in that The primer sequences for real-time fluorescence quantitative PCR analysis are: Upstream primer P11: 5′-ATTACTGGCGTTAGCACTT-3′; Downstream primer P12: 5′-TATTCCTTTCGACGGATC-3′.

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