WO2023065966A1 - Bfne基因在番茄株型改良和生物产量提高中的应用 - Google Patents

Bfne基因在番茄株型改良和生物产量提高中的应用 Download PDF

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WO2023065966A1
WO2023065966A1 PCT/CN2022/120881 CN2022120881W WO2023065966A1 WO 2023065966 A1 WO2023065966 A1 WO 2023065966A1 CN 2022120881 W CN2022120881 W CN 2022120881W WO 2023065966 A1 WO2023065966 A1 WO 2023065966A1
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tomato
protein
sequence
amino acid
yield
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李宁
余庆辉
贺强
王柏柯
王娟
杨涛
艾斯木托拉帕提古丽
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新疆农业科学院园艺作物研究所
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention belongs to the field of plant genetic engineering, and in particular relates to the application of BFNE gene in tomato plant type improvement and biological yield improvement.
  • Tomato Solanum lycopersicum
  • my country is the largest producer of fresh tomatoes and the third largest producer of processed tomatoes, playing a pivotal role in the world tomato market. Due to climate reasons, tomatoes can only be grown once a year in Xinjiang. Tomato plant type and fruit number are important factors affecting tomato yield. Analyzing the genetic basis of plant type and yield will undoubtedly provide important genetic resources for molecular breeding.
  • the present invention firstly provides a protein, the name of the protein provided by the present invention is BFNE, which is the protein shown in the following a1) or a2) or a3) or a4):
  • amino acid sequence is the protein shown in Sequence 1;
  • the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein.
  • the tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
  • the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
  • the identity includes having 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher with the amino acid sequence shown in Sequence 1 of the present invention High, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher amino acid sequence homology.
  • the protein described in a1) or a2) or a3) or a4) above can be synthesized artificially, or its coding gene can be firstly synthesized and then biologically expressed.
  • the present invention also provides a biological material related to the BFNE protein, which is any one of the following A1) to A12):
  • A1 a nucleic acid molecule encoding a BFNE protein
  • A2) an expression cassette containing the nucleic acid molecule of A1);
  • A3 a recombinant vector containing the nucleic acid molecule of A1);
  • A5 a recombinant microorganism containing the nucleic acid molecule of A1);
  • A7 A recombinant microorganism containing the recombinant vector described in A3);
  • A9 a transgenic plant cell line containing the nucleic acid molecule of A1);
  • A10 a transgenic plant cell line containing the expression cassette described in A2);
  • A11 a transgenic plant cell line containing the recombinant vector described in A3);
  • a transgenic plant cell line containing the recombinant vector described in A4) A transgenic plant cell line containing the recombinant vector described in A4).
  • nucleic acid molecule described in A1) is the gene shown in 1) or 2) or 3) as follows:
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding the BFNE protein of the present invention can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the BFNE protein of the present invention.
  • Those artificially modified nucleotides with 75% or higher identity of the nucleotide sequence encoding the BFNE protein, as long as they encode the BFNE protein and have the same function, are all derived from the nucleotide sequence of the present invention and are equivalent to Sequences of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 of the present invention highly identical nucleotide sequences. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
  • the identity of 75% or more may be 80%, 85%, 90% or more.
  • the stringent condition is in a solution of 2 ⁇ SSC, 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5 ⁇ SSC, 0.1% SDS, Hybridize and wash the membrane twice at 68°C, 15 min each time; or hybridize and wash the membrane at 65°C in a solution of 0.1 ⁇ SSPE (or 0.1 ⁇ SSC) and 0.1% SDS.
  • the expression cassette (BFNE gene expression cassette) described in A2) that contains the nucleic acid molecule encoding the BFNE protein refers to the DNA that can express the BFNE protein in the host cell, and the DNA can not only include the promoter that starts BFNE transcription, A terminator to terminate transcription of BFNE may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters.
  • Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • CaMV 35S terminator cauliflower mosaic virus CaMV 35S terminator
  • tml terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synthase terminator.
  • the existing expression vector can be used to construct the recombinant vector containing the expression cassette of the BFNE gene.
  • the plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc.
  • the plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression.
  • the polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions.
  • Agrobacterium crown gall tumor induction (Ti) plasmid gene such as nopaline synthase gene Nos
  • plant gene such as soybean
  • the untranslated region transcribed at the 3′ end of the storage protein gene has similar functions.
  • enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc.
  • the reading frames of the sequences are identical to ensure correct translation of the entire sequence.
  • the sources of the translation control signals and initiation codons are extensive and can be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene.
  • the transformed plants can be screened directly by
  • the vector can be a plasmid, a cosmid, a phage or a viral vector.
  • the microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium.
  • the present invention also provides a new application of the above-mentioned BFNE protein or the above-mentioned biological material.
  • the present invention provides the application of the above-mentioned BFNE protein or the above-mentioned biological material in any one of the following 1)-5):
  • the tomato plant type includes tomato branches.
  • the regulating tomato plant type is regulating tomato branching.
  • the tomato yield includes tomato fruit number and/or tomato fruit weight.
  • the regulating tomato yield is regulating tomato fruit quantity and/or regulating tomato fruit weight.
  • the regulation of tomato plant type is to increase tomato branching
  • the regulation of tomato yield is to increase tomato yield. It is embodied as follows: the higher the content and/or activity of BFNE protein in tomato or the higher the expression level of BFNE gene, the more the number of branches (number of side branches) of tomato, the more fruit quantity of tomato, and the more fruit weight of tomato .
  • the purpose of tomato breeding is to breed tomato varieties with increased branch number and/or increased yield.
  • the tomato is wild tomato. Described wild tomato specifically can be Micro Tom small tomato.
  • the invention also provides a method for cultivating transgenic tomato with altered plant type and/or increased yield.
  • the method for cultivating transgenic tomato with plant type change and/or yield increase provided by the invention comprises the steps of: increasing the content and/or activity of BFNE protein in recipient tomato to obtain transgenic tomato; number) and/or higher yield than the recipient tomato.
  • the yield of the transgenic tomato is higher than that of the recipient tomato, which means that the number of fruits of the transgenic tomato is higher than that of the recipient tomato and/or the weight of the fruit of the transgenic tomato is higher than that of the recipient tomato .
  • the method for increasing the content and/or activity of the BFNE protein in the recipient tomato is to overexpress the BFNE protein in the recipient tomato.
  • the overexpression method is to introduce the gene encoding the BFNE protein into the recipient tomato.
  • the gene encoding the BFNE protein is shown as sequence 2 in the sequence listing.
  • the gene encoding the BFNE protein is introduced into the recipient tomato through the pCAMBIA1300-BFNE recombinant expression vector.
  • the recipient tomato is wild tomato. Described wild tomato specifically can be Micro Tom small tomato.
  • the transgenic tomato with changed plant type and/or increased yield bred by the above method also belongs to the protection scope of the present invention.
  • the present invention finally provides a method for identifying or distinguishing between wild tomato and cultivated tomato.
  • the method for identifying or distinguishing wild tomato and cultivated tomato comprises the steps of: detecting whether the tomato to be tested contains BFNE protein or its coding gene: if the tomato to be tested contains BFNE protein (or contains the protein shown in sequence 1) or its Encoding gene (or containing the gene shown in sequence 2 or sequence 3), then the tomato to be tested is a wild tomato; if the tomato to be tested does not contain BFNE protein (or contains the protein shown in sequence 4) or its coding gene (or contains the sequence 5), the tomato to be tested is a cultivated tomato.
  • the method for detecting whether the tomato to be tested contains BFNE protein or its coding gene comprises the following steps: extract the genomic DNA of the tomato to be tested, and use the single-stranded DNA shown in sequence 6 and the single-stranded DNA shown in sequence 7 to carry out PCR amplification to obtain the amplified product, and then electrophoresis detection of the amplified product, if the amplified amplified product band with a size of 777bp is obtained, the tomato to be tested is a wild tomato, and if the amplified amplified product with a size of 534bp product band, the tomato to be tested is a cultivated tomato.
  • the variety of the wild tomato can be any of the following varieties: class tomato Solanum lycopersicoides, hairy tomato Solanum habrochaites, Pennelli tomato Solanum pennellii, Chilean tomato Solanum chilense, Peruvian tomato Solanum peruvianum, polyglandular tomato Solanum corneliomulleri, Solanum neorickii, Solanum chmielewskii, Solanum pimpinellifolium, Solanum galapagense.
  • the variety of the cultivated tomato can be any one of the following varieties: cherry tomato Solanum lycopersicum var.cerasiforme, cultivated tomato M82Solanum lycopersicum, cultivated tomato Heinz-1706 Solanum lycopersicum.
  • the present invention utilizes tomato pan-genome data to directly mine a new gene BFNE by performing multi-genome comparison. This operation is time-saving and efficient, and avoids the time-consuming and labor-intensive shortcomings of traditional QTL gene mapping methods.
  • the present invention reports for the first time that the BFNE gene is a pleiotropic gene that can regulate tomato plant type and biological yield. It is highly original and lays the foundation for the improvement of tomato varieties and the cultivation of ideal tomato varieties.
  • Figure 1 is a schematic diagram of the presence/absence variant PAV pleiotropic gene BFNE mined from the tomato pan-genome.
  • Fig. 2 is the expression level of BFNE gene in each tissue of wild tomato and cultivated tomato.
  • Fig. 3 is the identification of transgenic BFNE tomato.
  • A is PCR identification.
  • M 2000bp; 1500bp; 1000bp; 750bp; 500bp; 250bp; 100bp. 1-5 are Galapagos Solanum galapagense, floret tomato Solanum neorickii, cultivated tomato M82Solanum lycopersicum, Micro Tom tomato (Micro Tom tomato) and transgenic BFNE tomato plants.
  • B is the identification of BFNE gene expression level.
  • Fig. 4 is a comparison diagram of the plant type of transgenic BFNE tomato and wild plants.
  • Fig. 5 is a comparative graph of the biological yield of transgenic BFNE tomato and wild plants.
  • Fig. 6 is the red fruit weight comparison (unit is gram) of transgenic BFNE tomato and wild plant.
  • Fig. 7 is the amplification result of PAV-F/PAV-R primer pair in wild tomato and cultivated tomato.
  • M 2000bp; 1500bp; 1000bp; 750bp; 500bp; 250bp; 100bp.
  • 1 Solanum lycopersicoides
  • 2 Solanum habrochaites
  • 3 Solanum pennellii
  • 4 Solanum chilense
  • 5 Solanum peruvianum
  • 6 Solanum corneliomulleri
  • 7 Solanum florets neorickii
  • 8 Solanum chmielewskii
  • 9 Solanum pimpinellifolium
  • 10 Solanum galapagense
  • 11 cherry tomato Solanum lycopersicum var.cerasiforme
  • 12 cultivated tomato M82 Solanum lycopersicum
  • 13 cultivated Tomato Heinz-1706Solanum lycopersicum.
  • test methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
  • Tomato class Solanum lycopersicoides, hairy tomato Solanum habrochaites, Pennelli tomato Solanum pennellii, Chilean tomato Solanum chilense, Peruvian tomato Solanum peruvianum, polyglandular tomato Solanum corneliomulleri, floret tomato Solanum neorickii, Kemeliuski Solanum in the following examples chmielewskii, gooseberry Solanum pimpinellifolium, and Galapagos Solanum galapagense are all recorded in the literature "Spooner DM, Peralta IE, Knapp S. Comparison of AFLPs with Other Markers for Phylogeneti: Inference in Wild Tomatoes [Solanum L.
  • the cultivated tomato Heinz-1706 Solanum lycopersicum in the following examples is recorded in the document "Aureliano B, Naama M, Tecle I Y, et al. The Sol Genomics Network (solgenomics.net): growing tomatoes using Perl [J]. Nucleic Acids Research, 2011, 39 (Database issue): 1149-55.”, the public can obtain from the Horticultural Crops Research Institute of Xinjiang Academy of Agricultural Sciences, and this biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • the plant expression vector pCAMBIA1300 in the following examples is described in the document "Das S S, Sanan M N.A direct method for genetically transforming rice seeds modeled with FHVB2, a suppressor of RNAi [J]. Plant Cell Tissue & Organ Culture, 2015, 120 (1 ):277-289.”, the public can obtain from Horticultural Crops Research Institute of Xinjiang Academy of Agricultural Sciences, this biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Embodiment 1 the cloning of BFNE gene and its expression level analysis in each tissue of wild tomato and cultivated tomato
  • the present invention utilizes the tomato pan-genome data to directly mine and clone the BFNE gene by performing multi-genome comparison, and the specific steps are as follows:
  • the genome sequence of the unique gene BFNE in wild tomato is shown in sequence 3 in the sequence listing, the CDS sequence is shown in sequence 2 in the sequence listing, and the amino acid sequence of the BFNE protein encoded by the BFNE gene is shown in sequence 1 in the sequence listing.
  • Tissue samples of roots, stems, leaves, and seedlings of wild tomato Galapagos and cultivated tomato M82 were collected, RNA was extracted for reverse transcription, and quantitative RT-PCR was performed using LightCycler96 real-time PCR system. Tomato Actin was used as an internal reference, and in the qRT-PCR experiment, three samples (biological replicates) were processed for each treatment.
  • the primer sequences are as follows:
  • RTBF4-F GTGATCATCGCGTTGCTGTT (SEQ ID NO: 8);
  • RTBF4-R TCCAAGATTTGGTGTTGCTGC (SEQ ID NO: 9);
  • Actin-F GGTGTTATGGTCGGAATGGG (SEQ ID NO: 10);
  • Embodiment 2 the application of BFNE gene in tomato plant type improvement and biological yield improvement
  • the BFNE gene shown in Sequence 2 was integrated into the XbaI and KpnI restriction sites of the plant expression vector pCAMBIA1300 by restriction digestion and ligation, and the other sequences of the plant expression vector pCAMBIA1300 were kept unchanged to obtain the pCAMBIA1300-BFNE recombinant expression vector.
  • the T 0 generation BFNE gene transgenic tomato plants were planted in the greenhouse, and the T 1 generation BFNE gene transgenic tomato plants were obtained by selfing and identified.
  • Genomic DNA was extracted from Galapagos Solanum galapagense, Solanum neorickii, cultivated tomato M82Solanum lycopersicum, Micro Tom tomato (Micro Tom tomato) and T1 generation BFNE gene transgenic tomato plants, using BFNE-F: GCTGCTAAACAACATCCAGAAGAG and BFNE-R: TCCGCAGACGAGACAATGA for PCR amplification, and electrophoresis detection of PCR amplification products.
  • BFNE gene expression levels in the roots, stems and leaves of T1 generation BFNE gene-transferred tomato plants identified as positive by PCR were detected by RT-PCR, and Micro Tom was used as a control.
  • the sequences of BFNE gene-specific primers are as follows: RTBF1-F: TAGTTGCAGCAATGGGCACT (SEQ ID NO: 12); RTBF1-R: TTCCATTGCCTCGTGAGGTG (SEQ ID NO: 13).
  • the primer sequences of internal reference genes are as follows: Actin-F: GGTGTTATGGTCGGAATGGG (SEQ ID NO: 10); Actin-R: CAGGGTGTTTCTCAGGAGCAA (SEQ ID NO: 11).
  • the T1 generation BFNE gene-transferred tomato plants and wild-type plants were planted in the greenhouse at the same time, and the phenotype was observed at the maturity stage, and the number of branches and the number and weight of fruits were counted.
  • Repeat 1, repeat 2, and repeat 3 represent three repeat single plants selected from WT and BFNE-transgenic tomatoes, respectively.
  • BFNE-1, BFNE-2 and BFNE-3 are T1 transgenic BFNE gene tomato plants; WT-1, WT-2 and WT-3 are wild tomato (Micro Tom); 1, 2 and 3 represents three replicate plants selected from the BFNE gene-transferred tomato plants of the T1 generation and the wild tomato (Micro Tom).
  • BFNE has important application value in molecular breeding for improving tomato plant type and improving tomato biomass.
  • Embodiment 3 the application of BFNE gene in identifying or distinguishing wild tomato and cultivated tomato
  • numbers 1-10 are all wild tomatoes; numbers 11-13 are all cultivated tomatoes.
  • Experimental method extract the genomic DNA of the tomato to be tested, amplify it with PAV-F: CTTGCTTTGCTATCAGACACAC (SEQ ID NO: 6) and PAV-R: GCAAGTCAAGTCAGCATTCA (SEQ ID NO: 7) to obtain the amplified product, and then detect the size of the amplified product by electrophoresis.
  • PAV-F CTTGCTTTGCTATCAGACACAC
  • PAV-R GCAAGTCAAGTCAGCATTCA
  • the tomato species to be tested can be identified as wild tomato or cultivated tomato according to the following method: extract the genomic DNA of the tomato to be tested, and use PAV-F/PAV-R primers to amplify to obtain the amplified product. Then the amplified product is detected by electrophoresis. If the amplification product band with a size of 777bp is amplified, the tomato to be tested is a wild tomato, and if the amplified product band with a size of 534bp is amplified, the tomato to be tested is a cultivated tomato. .
  • a wild tomato gene is first excavated by the pan-genome, and it is named BFNE (branch and fruit number enhancer gene) gene, and then the BFNE gene is overexpressed in wild tomato (Micro Tom) to obtain the trans BFNE gene tomato.
  • BFNE branch and fruit number enhancer gene
  • the present invention also finds that the BFNE gene can also be used to identify or distinguish wild tomato and cultivated tomato, and can be used for wild tomato and cultivated tomato hybrid breeding and tomato variety improvement.

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Abstract

提供了BFNE基因在番茄株型改良和生物产量提高中的应用。BFNE基因的CDS序列如序列表中序列2所示，基因组序列如序列表中序列3所示，其编码的氨基酸序列如序列表中序列1所示。还提供了BFNE蛋白质或其相关生物材料在如下1)-5)任一种中的应用：1)调控番茄株型；2)调控番茄产量；3)培育株型改变和/或产量增加的转基因番茄；4)鉴定或区分野生番茄和栽培番茄；5)番茄育种。

Description

BFNE基因在番茄株型改良和生物产量提高中的应用 技术领域
本发明属于植物基因工程领域，具体涉及BFNE基因在番茄株型改良和生物产量提高中的应用。
背景技术
农业是我国的基础产业，粮食生产更是重中之重。粮食是人类生存的基本条件，随着人口的不断增加，耕地的逐渐减少，粮食问题、农业发展问题不断突出。利用高新技术育种农业科技攻关，大力培养优良品种，提高单位面积作物的产量，是当前增加产量最直接最有效的途径。地球的耕地是有限的。随着世界人口的不断增长，人均耕地越来越少了。近50年来，世界人均耕地的数量竟减少了一半以上。全国的人均耕地面积都不乐观，18亿亩红线非常重要。相比内地，新疆的耕地面积算大的，但是耕地面积不一定就代表产量，受限于各种各样的原因，如种植技术、土地盐碱度大、作物品种等原因。
番茄(Solanum lycopersicum)是重要的蔬菜和经济作物，在世界范围内广受欢迎。我国是鲜食番茄第一大生产国，也是加工番茄的第三大生产国，在世界番茄市场中具有举足轻重的地位。由于气候原因，新疆一年番茄只能种一季。番茄株型和果实数量均为影响番茄产量的重要因子，解析株型和产量的遗传基础无疑为分子育种提供重要基因资源。
发明公开
本发明首先提供了一种蛋白质，本发明提供的蛋白质的名称为BFNE，其为如下a1)或a2)或a3)或a4)所示的蛋白质：
a1)氨基酸序列是序列1所示的蛋白质；
a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番茄株型或产量相关的蛋白质。
上述a2)所述的蛋白质中，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述a3)所述的蛋白质中，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述a4)所述的蛋白质中，所述同一性包括与本发明序列1所示的氨基酸序列具有90％或更高，或91％或更高，或92％或更高，或93％或更高，或94％或更高，或95％或更高，或96％或更高，或97％或更高，或98％或更高，或99％或更高同源性的氨基酸序列。
上述a1)或a2)或a3)或a4)所述的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
本发明又提供了与BFNE蛋白质相关的生物材料，该生物材料为下述A1)至A12)中的任一种：
A1)编码BFNE蛋白质的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物；
A9)含有A1)所述核酸分子的转基因植物细胞系；
A10)含有A2)所述表达盒的转基因植物细胞系；
A11)含有A3)所述重组载体的转基因植物细胞系；
A12)含有A4)所述重组载体的转基因植物细胞系。
上述生物材料中，A1)所述核酸分子为如下1)或2)或3)所示的基因：
1)其编码序列是序列2或序列3所示的DNA分子；
2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码BFNE 蛋白质的DNA分子；
3)在严格条件下与1)或2)限定的核苷酸序列杂交，且编码BFNE蛋白质的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码BFNE蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有编码BFNE蛋白质的核苷酸序列75％或者更高同一性的核苷酸，只要编码BFNE蛋白质且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明编码序列1所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述生物材料中，所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或在0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述生物材料中，A2)所述含有编码BFNE蛋白质的核酸分子的表达盒(BFNE基因表达盒)是指能够在宿主细胞中表达BFNE蛋白质的DNA，该DNA不但可包括启动BFNE转录的启动子，还可包括终止BFNE转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子；组织、器官和发育特异的启动子及诱导型启动子。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子。
可用现有的表达载体构建含有所述BFNE基因表达盒的重组载体。所 述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因，和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
上述生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中，所述微生物可为酵母、细菌、藻或真菌，如农杆菌。
本发明还提供了上述BFNE蛋白质或上述生物材料的新用途。
本发明提供了上述BFNE蛋白质或上述生物材料在如下1)-5)任一种中的应用：
1)调控番茄株型；
2)调控番茄产量；
3)培育株型改变和/或产量增加的转基因番茄；
4)鉴定或区分野生番茄和栽培番茄；
5)番茄育种。
进一步的，所述番茄株型包括番茄分枝。所述调控番茄株型为调控番茄分枝。
所述番茄产量包括番茄果实数量和/或番茄果实重量。所述调控番茄产量为调控番茄果实数量和/或调控番茄果实重量。
更进一步的，所述调控番茄株型为增加番茄分枝，所述调控番茄产量为提高番茄产量。具体体现为：番茄中的BFNE蛋白质的含量和/或活性越高或BFNE基因表达水平越高，番茄的分枝数(侧枝数)越多，番茄的果实数量越多，番茄的果实重量越多。
上述应用中，所述番茄育种的目的为培育分枝数增加和/或产量增加的番茄品种。
上述应用中，所述番茄为野生番茄。所述野生番茄具体可为Micro Tom小番茄。
本发明还提供了一种培育株型改变和/或产量增加的转基因番茄的方法。
本发明提供的培育株型改变和/或产量增加的转基因番茄的方法包括如下步骤：提高受体番茄中BFNE蛋白质的含量和/或活性，得到转基因番茄；所述转基因番茄的分枝数(侧枝数)和/或产量高于所述受体番茄。
上述方法中，所述转基因番茄的产量高于所述受体番茄体现为所述转基因番茄的果实数量高于所述受体番茄和/或所述转基因番茄的果实重量高于所述受体番茄。
上述方法中，所述提高受体番茄中BFNE蛋白质的含量和/或活性的方法为在受体番茄中过表达BFNE蛋白质。
进一步的，所述过表达的方法为将BFNE蛋白质的编码基因导入受体番茄中。
更进一步的，所述BFNE蛋白质的编码基因如序列表中序列2所示。
在本发明的具体实施例中，所述BFNE蛋白质的编码基因通过pCAMBIA1300-BFNE重组表达载体导入受体番茄中。
上述方法中，所述受体番茄为野生番茄。所述野生番茄具体可为Micro Tom小番茄。
由上述方法培育得到的株型改变和/或产量增加的转基因番茄也属于本发明的保护范围。
本发明最后提供了一种鉴定或区分野生番茄和栽培番茄的方法。
本发明提供的鉴定或区分野生番茄和栽培番茄的方法包括如下步骤：检测待测番茄是否含有BFNE蛋白质或其编码基因：若待测番茄含有BFNE蛋白质(或含有序列1所示的蛋白质)或其编码基因(或含有序列2或序列3所示的基因)，则待测番茄为野生番茄；若待测番茄不含有BFNE蛋白质(或含有序列4所示的蛋白质)或其编码基因(或含有序列5所示的基因)，则待测番茄为栽培番茄。
进一步的，所述检测待测番茄是否含有BFNE蛋白质或其编码基因的方法包括如下步骤：提取待测番茄的基因组DNA，采用序列6所示的单链DNA和序列7所示的单链DNA进行PCR扩增，得到扩增产物，然后电泳检测所述扩增产物，若扩增得到大小为777bp的扩增产物条带，则待测番茄为野生番茄，若扩增得到大小为534bp的扩增产物条带，则待测番茄为栽培番茄。
更进一步的，所述野生番茄的品种可为如下品种中的任一种：类番茄Solanum lycopersicoides、多毛番茄Solanum habrochaites、潘那利番茄Solanum pennellii、智利番茄Solanum chilense、秘鲁番茄Solanum peruvianum、多腺番茄Solanum corneliomulleri、小花番茄Solanum neorickii、克梅留斯基Solanum chmielewskii、醋栗Solanum pimpinellifolium、加拉帕戈斯Solanum galapagense。
所述栽培番茄的品种可为如下品种中的任一种：樱桃番茄Solanum lycopersicum var.cerasiforme、栽培番茄M82Solanum lycopersicum、栽培番茄Heinz-1706 Solanum lycopersicum。
本发明的有益效果如下：
(1)本发明利用番茄泛基因组数据，通过进行多基因组比对，直接挖掘得到一个新基因BFNE，该操作省时高效，避免了传统的QTL基因定位方法耗时耗力的缺点。
(2)本发明首次报道BFNE基因是一个可调控番茄株型和生物产量的多效基因，具有极高的首创性，为番茄品种的改良及理想番茄品种的培育 奠定基础。
附图说明
图1为番茄泛基因组挖掘到的存在/缺失变异PAV多效性基因BFNE示意图。
图2为BFNE基因在野生番茄和栽培番茄各个组织的表达量。
图3为转BFNE基因番茄的鉴定。A为PCR鉴定。M：2000bp；1500bp；1000bp；750bp；500bp；250bp；100bp。1-5分别为加拉帕戈斯Solanum galapagense、小花番茄Solanum neorickii、栽培番茄M82Solanum lycopersicum、麦克汤姆小番茄(Micro Tom小番茄)和转BFNE基因番茄植株。B为BFNE基因表达水平鉴定。
图4为转BFNE基因番茄和野生植株的株型对照图。
图5为转BFNE基因番茄和野生植株的生物产量对照图。
图6为转BFNE基因番茄和野生植株的红果果重对比(单位为克)。
图7为PAV-F/PAV-R引物对在野生番茄和栽培番茄中的扩增结果。M：2000bp；1500bp；1000bp；750bp；500bp；250bp；100bp。1：类番茄Solanum lycopersicoides；2：多毛番茄Solanum habrochaites；3：潘那利番茄Solanum pennellii；4：智利番茄Solanum chilense；5：秘鲁番茄Solanum peruvianum；6：多腺番茄Solanum corneliomulleri；7：小花番茄Solanum neorickii；8：克梅留斯基Solanum chmielewskii；9：醋栗Solanum pimpinellifolium；10：加拉帕戈斯Solanum galapagense；11：樱桃番茄Solanum lycopersicum var.cerasiforme；12：栽培番茄M82 Solanum lycopersicum；13：栽培番茄Heinz-1706Solanum lycopersicum。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的试验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的类番茄Solanum lycopersicoides、多毛番茄Solanum habrochaites、潘那利番茄Solanum pennellii、智利番茄Solanum chilense、秘鲁番茄Solanum peruvianum、多腺番茄Solanum  corneliomulleri、小花番茄Solanum neorickii、克梅留斯基Solanum chmielewskii、醋栗Solanum pimpinellifolium、加拉帕戈斯Solanum galapagense均记载于文献“Spooner DM,Peralta IE,Knapp S.Comparison of AFLPs with Other Markers for Phylogeneti:Inference in Wild Tomatoes[Solanum L.Section Lycopersicon(Mill.)Wettst.][J].Taxon,2005,54(1):43-61.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的樱桃番茄Solanum lycopersicum var.cerasiforme记载于文献“Ranc N,
Figure PCTCN2022120881-appb-000001
S,Santoni S,et al.A clarified position for solanum lycopersicum var.cerasiforme in the evolutionary history of tomatoes(solanaceae)[J].BMC Plant Biology,2008,8.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的栽培番茄M82 Solanum lycopersicum记载于文献“Carter J D,Pereira A,Veilleux D.An active ac/ds transposon system for activation tagging in tomato cultivar m82 using clonal propagation.[J].Plant Physiology,2013,162(1):145-156.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的栽培番茄Heinz-1706 Solanum lycopersicum记载于文献“Aureliano B,Naama M,Tecle I Y,et al.The Sol Genomics Network(solgenomics.net):growing tomatoes using Perl[J].Nucleic Acids Research,2011,39(Database issue):1149-55.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的Micro Tom小番茄记载于文献“Chetty V J,Ceballos N,Garcia D,et al.Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato(Solanum lycopersicum L.)cultivar Micro-Tom[J].Plant Cell Reports, 2013,32(2):239-247.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
下述实施例中的植物表达载体pCAMBIA1300记载于文献“Das S S,Sanan M N.A direct method for genetically transforming rice seeds modelled with FHVB2,a suppressor of RNAi[J].Plant Cell Tissue&Organ Culture,2015,120(1):277-289.”中，公众可从新疆农业科学院园艺作物研究所获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
实施例1、BFNE基因的克隆及其在野生番茄和栽培番茄各个组织的表达量分析
一、BFNE基因的获得
本发明利用番茄泛基因组数据，通过进行多基因组比对，直接挖掘克隆得到BFNE基因，具体步骤如下：
1、采用PacBio+Hi-C策略，组装了番茄属11个番茄(9个野生番茄和2个栽培番茄)高质量染色体级的基因组序列。
2、应用四种从头预测软件，包括RepeatScout、LTR-FINDER、MITE-hunter和PILER-DF，形成一个重复序列数据库。通过使用RepeatMasker对最终合并的数据库进行同源搜索，识别重复序列注释。
3、核心基因与非核心基因分析，包括功能富集分析、保守性分析、重复序列区域富集等。
4、基于基因组序列比对进行基因组SVs的检测，对SVs特征进行分析，如对SVs类型、长度分布、基因组分布、重复含量进行比较等。
5、最终找到野生番茄特有基因BFNE，结果如图1所示。
野生番茄中的特有基因BFNE的基因组序列如序列表中序列3所示，CDS序列如序列表中序列2所示，BFNE基因编码的BFNE蛋白的氨基酸序列如序列表中序列1所示。
二、BFNE基因在野生番茄和栽培番茄各个组织的表达量分析
采集野生番茄加拉帕戈斯和栽培番茄M82的根、茎、叶、幼苗组织样品，提取RNA反转录，使用LightCycler96实时PCR***进行定量RT-PCR。 以番茄Actin作为内参，在qRT-PCR实验中，每个处理三个样本(生物重复)。引物序列具体如下：
RTBF4-F：GTGATCATCGCGTTGCTGTT(序列8)；
RTBF4-R：TCCAAGATTTGGTGTTGCTGC(序列9)；
Actin-F：GGTGTTATGGTCGGAATGGG(序列10)；
Actin-R：CAGGGTGTTCTTCAGGAGCAA(序列11)。
结果如图2所示，结果表明：BFNE基因在野生番茄加拉帕戈斯根、茎、叶、幼苗中的表达量均高于栽培番茄M82，尤其是在叶中。
实施例2、BFNE基因在番茄株型改良和生物产量提高中的应用
一、转BFNE基因番茄的制备
1、重组表达载体的构建
通过酶切和连接，将序列2所示的BFNE基因整合到植物表达载体pCAMBIA1300的XbaI和KpnI酶切位点间，且保持植物表达载体pCAMBIA1300的其它序列不变，得到pCAMBIA1300-BFNE重组表达载体。
2、转基因番茄植株的获得
将上述步骤1获得的pCAMBIA1300-BFNE重组表达载体导入GV3101农杆菌电转感受态细胞(北京博迈德基因技术有限公司，货号为BC308-01)中，经鉴定得到重组菌pCAMBIA1300-BFNE/GV3101，然后将重组菌pCAMBIA1300-BFNE/GV3101通过叶盘法转化到Micro Tom小番茄中，获得T 0代转BFNE基因番茄植株。
3、转基因番茄植株的鉴定
将T 0代转BFNE基因番茄植株种植于温室，自交获得T 1代转BFNE基因番茄植株，并对其进行鉴定。
1)PCR鉴定
分别提取加拉帕戈斯Solanum galapagense、小花番茄Solanum neorickii、栽培番茄M82Solanum lycopersicum、麦克汤姆小番茄(Micro Tom小番茄)和T 1代转BFNE基因番茄植株的基因组DNA，采用BFNE-F：GCTGCTAAACAACATCCAGAAGAG和BFNE-R：TCCGCAGACGAGACAATGA进行PCR扩增，并电泳检测PCR扩增产物。
结果如图3A所示。从图中可以看出：加拉帕戈斯Solanum  galapagense、小花番茄Solanum neorickii和转BFNE基因番茄植株中均扩增得到大小为186bp的条带，而栽培番茄M82Solanum lycopersicum和麦克汤姆小番茄(Micro Tom小番茄)中均没有扩增出条带。
2)BFNE基因表达量检测
通过RT-PCR分别检测经PCR鉴定为阳性的T 1代转BFNE基因番茄植株的根、茎和叶中的BFNE基因表达水平，同时以Micro Tom小番茄作为对照。BFNE基因特异引物序列如下：RTBF1-F：TAGTTGCAGCAATGGGCACT(序列12)；RTBF1-R：TTCCATTGCCTCGTGAGGTG(序列13)。内参基因引物序列如下：Actin-F：GGTGTTATGGTCGGAATGGG(序列10)；Actin-R：CAGGGTGTTCTTCAGGAGCAA(序列11)。
结果如图3B所示。从图中可以看出：与野生型Micro Tom小番茄相比，T 1代转BFNE基因番茄植株茎和叶中的BFNE基因表达水平均显著提高。
二、转BFNE基因番茄的株型与产量分析
将T 1代转BFNE基因番茄植株和野生型植株(Micro Tom小番茄)同时种植在温室，于成熟期观察表型，并统计分枝数和果实数量与重量。
结果表明：转BFNE基因番茄分枝(侧枝)增多，而野生型植株无分枝(表1、图4)。而且转BFNE基因番茄的单株总果数量和总果重均高于野生型番茄(表2、图5和图6)。
表1、转BFNE基因番茄与野生型番茄的分枝数
Figure PCTCN2022120881-appb-000002
注：重复一、重复二、重复三分别表示WT和转BFNE基因番茄分别选取的三株重复单株。
表2、转BFNE基因番茄与野生型番茄的果实数量与总重
Figure PCTCN2022120881-appb-000003
Figure PCTCN2022120881-appb-000004
注：BFNE-1、BFNE-2和BFNE-3均为T 1代转BFNE基因番茄植株；WT-1、WT-2和WT-3均为野生番茄(Micro Tom小番茄)；1、2和3表示T 1代转BFNE基因番茄植株和野生番茄(Micro Tom小番茄)分别选取的三株重复单株。
以上结果表明：野生番茄BFNE基因可以使Micro Tom小番茄的分枝增多，果实产量提高。BFNE在改良番茄株型及提高番茄生物产量的分子育种中具有重要应用价值。
实施例3、BFNE基因在鉴定或区分野生番茄和栽培番茄中的应用
由于野生番茄中的BFNE的基因组序列如序列表中序列3所示，其编码的BFNE蛋白的氨基酸序列如序列表中序列1所示；栽培番茄中的BFNE的基因组序列如序列表中序列5所示，其编码的BFNE蛋白的氨基酸序列如序列表中序列4所示，因此可根据野生番茄和栽培番茄中的BFNE蛋白或其编码基因序列来鉴定或区分野生番茄和栽培番茄。具体方法如下：
实验材料：
1：类番茄Solanum lycopersicoides。
2：多毛番茄Solanum habrochaites。
3：潘那利番茄Solanum pennellii。
4：智利番茄Solanum chilense。
5：秘鲁番茄Solanum peruvianum。
6：多腺番茄Solanum corneliomulleri。
7：小花番茄Solanum neorickii。
8：克梅留斯基Solanum chmielewskii。
9：醋栗Solanum pimpinellifolium。
10：加拉帕戈斯Solanum galapagense。
11：樱桃番茄Solanum lycopersicum var.cerasiforme。
12：栽培番茄M82Solanum lycopersicum。
13：栽培番茄Heinz-1706Solanum lycopersicum。
其中，编号1-10均为野生番茄；编号11-13均为栽培番茄。
实验方法：提取待测番茄的基因组DNA，采用PAV-F：CTTGCTTTGCTATCAGACACAC(序列6)和PAV-R：GCAAGTCAAGTCAGCATTCA(序列7)进行扩增，得到扩增产物，然后电泳检测扩增产物大小。
实验结果：所有野生番茄中均扩增得到大小为777bp的扩增产物条带，所有栽培番茄中均扩增得到大小为534bp的扩增产物条带(图7)。
因此，在实际应用中，可按照如下方法鉴定待测番茄品种为野生番茄还是栽培番茄：提取待测番茄的基因组DNA，采用PAV-F/PAV-R引物对进行扩增，得到扩增产物，然后电泳检测扩增产物，若扩增得到大小为777bp的扩增产物条带，则待测番茄为野生番茄，若扩增得到大小为534bp的扩增产物条带，则待测番茄为栽培番茄。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
工业应用
本发明首先利用泛基因组挖掘出一个野生番茄基因，并将其命名为BFNE(branch and fruit number enhencer gene)基因，然后将BFNE基因超表达于野生番茄(Micro Tom小番茄)中，得到转BFNE基因番茄。通过实验证明：与野生番茄(Micro Tom小番茄)相比，转BFNE基因番茄表现为分枝数增多，果实数量与重量均增加。说明BFNE基因可改良番茄株型、提高番茄产量。本发明还发现BFNE基因还可用于鉴定或区分野生番茄和栽培番茄，可用于野生番茄和栽培番茄杂交育种与番茄品种改良。

Claims (17)

  1. 一种蛋白质，为如下a1)或a2)或a3)或a4)所示的蛋白质：
    a1)氨基酸序列是序列1所示的蛋白质；
    a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
    a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番茄株型或产量相关的蛋白质。
  2. 与蛋白质相关的生物材料，为下述A1)至A12)中的任一种：
    A1)编码所述蛋白质的核酸分子；
    A2)含有A1)所述核酸分子的表达盒；
    A3)含有A1)所述核酸分子的重组载体；
    A4)含有A2)所述表达盒的重组载体；
    A5)含有A1)所述核酸分子的重组微生物；
    A6)含有A2)所述表达盒的重组微生物；
    A7)含有A3)所述重组载体的重组微生物；
    A8)含有A4)所述重组载体的重组微生物；
    A9)含有A1)所述核酸分子的转基因植物细胞系；
    A10)含有A2)所述表达盒的转基因植物细胞系；
    A11)含有A3)所述重组载体的转基因植物细胞系；
    A12)含有A4)所述重组载体的转基因植物细胞系；
    所述蛋白质为如下a1)或a2)或a3)或a4)所示的蛋白质：
    a1)氨基酸序列是序列1所示的蛋白质；
    a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
    a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番 茄株型或产量相关的蛋白质。
  3. 根据权利要求2所述的生物材料，其特征在于：A1)所述核酸分子为如下1)或2)或3)所示的基因：
    1)其编码序列是序列2或序列3所示的DNA分子；
    2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码所述蛋白质的DNA分子；
    3)在严格条件下与1)或2)限定的核苷酸序列杂交，且编码所述蛋白质的DNA分子。
  4. 蛋白质或与所述蛋白质相关的生物材料在如下1)-5)任一种中的应用：
    1)调控番茄株型；
    2)调控番茄产量；
    3)培育株型改变和/或产量增加的转基因番茄；
    4)鉴定或区分野生番茄和栽培番茄；
    5)番茄育种；
    所述蛋白质为如下a1)或a2)或a3)或a4)所示的蛋白质：
    a1)氨基酸序列是序列1所示的蛋白质；
    a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
    a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番茄株型或产量相关的蛋白质；
    所述生物材料为下述A1)至A12)中的任一种：
    A1)编码所述蛋白质的核酸分子；
    A2)含有A1)所述核酸分子的表达盒；
    A3)含有A1)所述核酸分子的重组载体；
    A4)含有A2)所述表达盒的重组载体；
    A5)含有A1)所述核酸分子的重组微生物；
    A6)含有A2)所述表达盒的重组微生物；
    A7)含有A3)所述重组载体的重组微生物；
    A8)含有A4)所述重组载体的重组微生物；
    A9)含有A1)所述核酸分子的转基因植物细胞系；
    A10)含有A2)所述表达盒的转基因植物细胞系；
    A11)含有A3)所述重组载体的转基因植物细胞系；
    A12)含有A4)所述重组载体的转基因植物细胞系。
  5. 根据权利要求4所述的应用，其特征在于：所述调控番茄株型为调控番茄分枝。
  6. 根据权利要求4所述的应用，其特征在于：所述调控番茄产量为调控番茄果实数量和/或调控番茄果实重量。
  7. 根据权利要求4-6任一所述的应用，其特征在于：所述番茄为野生番茄。
  8. 一种培育株型改变和/或产量增加的转基因番茄的方法，包括如下步骤：提高受体番茄中蛋白质的含量和/或活性，得到转基因番茄；所述转基因番茄的分枝数和/或产量高于所述受体番茄；
    所述蛋白质为如下a1)或a2)或a3)或a4)所示的蛋白质：
    a1)氨基酸序列是序列1所示的蛋白质；
    a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
    a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番茄株型或产量相关的蛋白质。
  9. 根据权利要求8所述的方法，其特征在于：所述转基因番茄的产量高于所述受体番茄体现为所述转基因番茄的果实数量高于所述受体番茄和/或所述转基因番茄的果实重量高于所述受体番茄。
  10. 根据权利要求8所述的方法，其特征在于：所述提高受体植物中蛋白质的含量和/或活性的方法为在受体番茄中过表达所述蛋白质。
  11. 根据权利要求10所述的方法，其特征在于：所述过表达的方法为将所述蛋白质的编码基因导入受体植物中。
  12. 根据权利要求11所述的方法，其特征在于：所述蛋白质的编码基因如序列表中序列2所示。
  13. 根据权利要求8-12任一所述的方法，其特征在于：所述受体番茄为野生番茄。
  14. 由权利要求8-13任一所述方法制备得到的转基因番茄。
  15. 一种鉴定或区分野生番茄和栽培番茄的方法，包括如下步骤：检测待测番茄是否含有蛋白质或其编码基因：若待测番茄含有蛋白质或其编码基因，则待测番茄为野生番茄；若待测番茄不含有蛋白质或其编码基因，则待测番茄为栽培番茄；
    所述蛋白质为如下a1)或a2)或a3)或a4)所示的蛋白质：
    a1)氨基酸序列是序列1所示的蛋白质；
    a2)在序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与番茄株型或产量相关的蛋白质；
    a4)与序列1所示的氨基酸序列具有90％同一性、来源于番茄且与番茄株型或产量相关的蛋白质。
  16. 根据权利要求15所述的方法，其特征在于：所述蛋白质的编码基因为序列2或序列3所示的DNA分子。
  17. 根据权利要求15或16所述的方法，其特征在于：所述检测待测番茄是否含有蛋白质或其编码基因的方法包括如下步骤：提取待测番茄的基因组DNA，采用序列6所示的单链DNA和序列7所示的单链DNA进行PCR扩增，得到扩增产物；然后电泳检测所述扩增产物，若扩增得到大小为777bp的扩增产物条带，则待测番茄为野生番茄；若扩增得到大小为534bp的扩增产物条带，则待测番茄为栽培番茄。
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