技术领域technical field
本发明涉及一种棉花蛋白及其编码基因和应用。 The invention relates to a cotton protein, its coding gene and application. the
背景技术Background technique
短季棉的推广是解决我国粮棉争地、保证粮食安全的重要途径。早熟型是棉花品种的重要性状之一，而花期是衡量棉花熟性的重要指标之一。 The promotion of short-season cotton is an important way to solve the land competition for grain and cotton in my country and ensure food security. Early maturity is one of the important characters of cotton varieties, and flowering period is one of the important indicators to measure the maturity of cotton. the
花是植物的次生器官，高等植物的地上部分来自茎端分生组织(shoot apical meristem，SAM)，即位于茎顶端的一群未分化的小细胞，茎端分生组织进而发育成茎、叶、花序分生组织及花分生组织。开花决定过程是植物生殖生长启动的第一个阶段，是花发端和花器官形成的基础，所以开花决定过程直接控制作物生育期的早晚。因此克隆棉花开花相关基因，对其进行表达分析和转基因功能验证，为短季棉育种提供优质的基因资源。 Flowers are the secondary organs of plants. The aerial parts of higher plants come from the shoot apical meristem (SAM), which is a group of undifferentiated small cells at the top of the stem. The shoot apical meristem then develops into stems and leaves. , inflorescence meristem and flower meristem. The flowering decision process is the first stage of initiation of plant reproductive growth, and is the basis of flower initiation and flower organ formation, so the flowering decision process directly controls the crop growth period. Therefore, the genes related to flowering in cotton were cloned, and their expression analysis and transgenic function verification were performed to provide high-quality genetic resources for short-season cotton breeding. the
发明内容Contents of the invention
本发明的一个目的是提供一种蛋白，名称为Gh FPF1，来源于棉花(Gossypium spp)。 An object of the present invention is to provide a protein named Gh FPF1 derived from cotton (Gossypium spp). the
本发明所述蛋白是如下1)或2)的蛋白： The protein of the present invention is the protein of the following 1) or 2):
1)序列表中的SEQ ID №.2所示的氨基酸序列组成的蛋白质； 1) A protein composed of the amino acid sequence shown in SEQ ID №.2 in the sequence listing;
2)将序列表中的SEQ ID №.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物开花相关的由1)衍生的蛋白质。 2) The amino acid residue sequence of SEQ ID No. 2 in the sequence listing undergoes one or several amino acid residue substitutions and/or deletions and/or additions and is related to plant flowering. The protein derived from 1). the
序列表中SEQ ID №.2所示的氨基酸序列由109个氨基酸残基组成。 The amino acid sequence shown in SEQ ID №.2 in the sequence listing consists of 109 amino acid residues. the
上述1)和2)中的Gh FPF1蛋白可人工合成，也可先合成其编码基因，再进行生物表达得到。上述1)和2)中的Gh FPF1蛋白的编码基因可通过将序列表中SEQ ID №.1的第8-334位核苷酸所示的DNA序列缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变后得到。 The Gh FPF1 protein in the above 1) and 2) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The coding gene of the GhFPF1 protein in the above-mentioned 1) and 2) can delete the codon of one or several amino acid residues by the DNA sequence shown in the 8th-334th nucleotide of SEQ ID No. 1 in the sequence listing , and/or a missense mutation of one or several base pairs. the
编码所述Gh FPF1蛋白的核酸分子也属于本发明的保护范围。 Nucleic acid molecules encoding the Gh FPF1 protein also belong to the protection scope of the present invention. the
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、hnRNA或tRNA等。 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, hnRNA or tRNA. the
本发明的又一个目的是提供所述蛋白的编码基因。 Another object of the present invention is to provide the gene encoding the protein. the
所述编码基因具有下述核苷酸序列之一： The coding gene has one of the following nucleotide sequences:
1)序列表中SEQ ID №：1第8-334位的核苷酸序列； 1) The nucleotide sequence of the 8th-334th position of SEQ ID №: 1 in the sequence listing;
2)编码序列表中SEQ ID №：2蛋白质序列的多核苷酸序列； 2) The polynucleotide sequence of SEQ ID №: 2 protein sequence in the coding sequence list;
3)在高严谨条件下可与序列表中SEQ ID №：1限定的DNA序列杂交的核苷酸序列； 3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID №: 1 in the sequence listing under high stringency conditions;
4)与1)或2)或3)限定的DNA序列具有90％以上同源性，且编码相同功能蛋白质的DNA序列；具体的，所述同源性为95％以上；再具体的为96％以上；再具体的为97％以上；再具体的为98％以上；再具体的为99％以上。 4) A DNA sequence that has more than 90% homology with the DNA sequence defined in 1) or 2) or 3), and encodes the same functional protein; specifically, the homology is more than 95%; more specifically, it is 96% More specifically, more than 97%; more specifically, more than 98%; more specifically, more than 99%. the
上述高严谨条件可为用6×SSC，0.5％SDS的溶液，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。 The above-mentioned high stringency conditions can be 6×SSC, 0.5% SDS solution, hybridization at 65° C., and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively. the
其中，序列表中的SEQ ID №：1由401个核苷酸组成，其开放阅读框架(ORF)为自5′末端第8-334位核苷酸，编码序列表中SEQ ID №：2所示的蛋白质，即本发明所述的Gh FPF1蛋白。 Among them, SEQ ID №: 1 in the sequence listing consists of 401 nucleotides, its open reading frame (ORF) is nucleotides 8-334 from the 5′ end, and SEQ ID №: 2 in the coding sequence listing The protein shown is the GhFPF1 protein of the present invention. the
含有上述核酸分子的重组载体、表达盒、转基因细胞系或重组菌也属于本发明的保护范围。 Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above nucleic acid molecules also belong to the protection scope of the present invention. the
所述重组载体可为重组表达载体，也可为重组克隆载体。 The recombinant vector can be a recombinant expression vector or a recombinant cloning vector. the
所述重组表达载体可用现有的表达载体构建。所述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。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，它们可单独使用或与其它的启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子。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，如加入在植物中表达可产生颜色变化的酶或发光化合物的基因(GUS基因、GFP基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。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。 The recombinant expression vector can be constructed with existing expression vectors. The expression vector can 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 polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or with other promoters Used in combination; in addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in plants (GUS gene, GFP gene, luciferase Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene. the
扩增本发明所述编码基因全长或其任意片段的引物对也属于本发明保护的范围。 The pair of primers for amplifying the full-length coding gene of the present invention or any fragment thereof also falls within the protection scope of the present invention. the
本发明的另一个目的是提供本发明所述蛋白、编码基因和含有所述编码基因的重组载体、表达盒、转基因细胞系或重组菌在如下1)-3)至少一种中的应用： Another object of the present invention is to provide the protein of the present invention, encoding gene and recombinant vector containing said encoding gene, expression cassette, transgenic cell line or recombinant bacteria in at least one of the following 1)-3):
1)调节植物生育期； 1) Regulating plant growth period;
2)调节植物开花时间； 2) Regulating plant flowering time;
3)调节植物莲座叶数量； 3) Regulate the number of plant rosette leaves;
4)调节植物茎生叶数量。 4) Regulate the number of cauline leaves in plants. the
所述调节植物生育期为使植物的生育期提前； The regulation of plant growth period is to advance the growth period of plants;
所述调节植物开花为促进植物开花； The regulation plant flowering is to promote plant flowering;
所述调节植物莲座叶数量为减少植物莲座叶数量； Said regulation plant rosette leaf quantity is to reduce the plant rosette leaf quantity;
所述调节植物茎生叶数量为减少植物茎生叶数量； The number of stem leaves of the regulation plant is to reduce the number of stem leaves of plants;
所述植物为双子叶植物或单子叶植物；所述双子叶植物具体为拟南芥或棉花。 The plant is a dicotyledonous plant or a monocotyledonous plant; the specific cotyledonous plant is Arabidopsis thaliana or cotton. the
本发明的还一个目的是提供本发明所述的蛋白、编码基因和含有所述编码基因的重组载体、表达盒、转基因细胞系或宿主菌在培育在培育转基因植物中的应用。 Another object of the present invention is to provide the application of the protein, coding gene and recombinant vector containing the coding gene, expression cassette, transgenic cell line or host bacteria in the cultivation of transgenic plants according to the present invention. the
具体的，所述转基因植物具有如下至少一种性状：1)生育期提前；2)开花时间提前；3)莲座叶数量减少；4)茎生叶数量减少。 Specifically, the transgenic plant has at least one of the following traits: 1) the growth period is advanced; 2) the flowering time is advanced; 3) the number of rosette leaves is reduced; 4) the number of cauline leaves is reduced. the
具体的所述植物为双子叶植物或单子叶植物；所述双子叶植物具体为拟南芥或棉花。 Specifically, the plant is a dicotyledonous plant or a monocotyledonous plant; and the specific dicotyledonous plant is Arabidopsis thaliana or cotton. the
本发明再一个目的是提供一种培育转基因植物的方法，是将本发明所述的编码基因导入目的植物，得到转基因植物。 Another object of the present invention is to provide a method for cultivating transgenic plants, which is to introduce the coding gene described in the present invention into the target plant to obtain transgenic plants. the
所述转基因植物与所述目的植物相比，具有如下至少一种性状：1)生育期提前；2)开花时间提前；3)莲座叶数量减少；4)茎生叶数量减少。 Compared with the target plant, the transgenic plant has at least one of the following traits: 1) the growth period is advanced; 2) the flowering time is advanced; 3) the number of rosette leaves is reduced; 4) the number of cauline leaves is reduced. the
具体的，所述植物为双子叶植物或单子叶植物；所述双子叶植物具体为拟南芥或棉花。 Specifically, the plant is a dicotyledonous plant or a monocotyledonous plant; the specific cotyledonous plant is Arabidopsis thaliana or cotton. the
本研究从陆地棉中克隆出棉花Gh FPF1基因，成功构建植物过表达载体，采用农杆菌介导的花序浸染法转化模式植物拟南芥，与对照相比，转基因拟南芥比非转基因拟南芥提前开花5.1天，同时莲座叶与茎生叶的总数比野生型拟南芥少2.7片，因此利用该基因可用来培育生育期短的棉花，为转基因植物的培育奠定了基础。 In this study, the cotton Gh FPF1 gene was cloned from upland cotton, the plant overexpression vector was successfully constructed, and the model plant Arabidopsis thaliana was transformed by inflorescence dipping method mediated by Agrobacterium. Mustard flowered 5.1 days earlier, and the total number of rosette leaves and cauline leaves was 2.7 less than that of wild-type Arabidopsis. Therefore, the gene can be used to breed cotton with a short growth period, laying the foundation for the cultivation of transgenic plants. the
附图说明Description of drawings
图1为转Gh FPF1基因拟南芥在DNA分子水平上的鉴定图，其中编号1为空白对照、2为野生型拟南芥、3为阳性对照(重组质粒)、4-10分别为转基因拟南芥各个株系。 Fig. 1 is the identification diagram of the transgenic Arabidopsis thaliana at the DNA molecular level, wherein number 1 is the blank control, 2 is the wild type Arabidopsis, 3 is the positive control (recombinant plasmid), and 4-10 are the transgenic Arabidopsis respectively. Various strains of A. thaliana. the
图2为T3代转Gh FPF1基因拟南芥在RNA分子水平上的鉴定图，WT为野生型拟南芥对照，COL3、COL7、COL4分别为转基因拟南芥各个株系。 Figure 2 is the identification diagram of Arabidopsis thaliana transgenic with Gh FPF1 gene inthe third generation at the RNA molecular level, WT is the wild-type Arabidopsis control, and COL3, COL7, and COL4 are the transgenic Arabidopsis lines respectively.
图3为野生型和转Gh FPF1基因T3代拟南芥生长3周时的表型对比图，其中A代表野生型拟南芥，B代表转基因拟南芥。 Figure 3 is a phenotypic comparison of wild-type and GhFPF1-transferredT3 generation Arabidopsis thaliana grown for 3 weeks, where A represents wild-type Arabidopsis, and B represents transgenic Arabidopsis.
图4为野生型和转Gh FPF1基因T3代拟南芥生长4周时的表型对比图，其中A代表野生型拟南芥，B代表转基因拟南芥。 Figure 4 is a phenotypic comparison of wild-type and Gh FPF1 gene transgenicT3 generation Arabidopsis thaliana grown for 4 weeks, wherein A represents wild-type Arabidopsis, and B represents transgenic Arabidopsis.
具体实施方式Detailed ways
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。 The experimental methods used in the following examples are conventional methods unless otherwise specified. the
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。 The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. the
实施例1、棉花基因Gh FPF1的制备 Embodiment 1, the preparation of cotton gene Gh FPF1
1、RNA的提取 1. Extraction of RNA
取中棉所36种子(购自中国农业科学院棉花研究所)浸泡一天后种于小花盆中，置于光照培养室中生长一周后，取整个幼苗置于液氮中速冻后存放于-70°冰箱中。提取上述材料的RNA。 Take 36 seeds from China Cotton Research Institute (purchased from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences) and soak them for one day, plant them in small flower pots, and place them in a light culture room for one week. ° in the refrigerator. RNA was extracted from the above materials. the
2、cDNA的制备 2. Preparation of cDNA
将步骤1制备得到的RNA反转录为cDNA。 Reverse transcribe the RNA prepared in step 1 into cDNA. the
3、基因的扩增 3. Gene amplification
引物序列为： The primer sequence is:
上游引物P1 5’-AGAGAAAATGAGCGGTCCTTG-3’ Upstream primer P1 5'-AGAGAAAATGAGCGGTCCTTG-3'
下游引物P2 5’-GCCCGAACATGGTGATTAAG-3’ Downstream primer P2 5'-GCCCGAACATGGTGATTAAG-3'
以上述设计的引物和步骤2所制备得到的中棉所36cDNA为模板，进行PCR扩增。 Using the primers designed above and the Zhongmian Institute 36 cDNA prepared in step 2 as a template, perform PCR amplification. the
将PCR扩增产物进行测序。测序结果表明，上述PCR扩增得到具有序列表中SEQ ID№：1的核苷酸序列，共401bp，其中编码区长327bp，该编码区序列如序列表中SEQID №：1中第8-334位核苷酸所示，编码序列表中SEQ ID №：2所示的氨基酸序列，共109个氨基酸残基。将该具有序列表中SEQ ID №：1中第8-334位的核苷酸序列的片段命名为Gh FPF1。 The PCR amplification products were sequenced. Sequencing results show that the above PCR amplification has a nucleotide sequence of SEQ ID №: 1 in the sequence listing, with a total of 401 bp, of which the coding region is 327 bp long. The amino acid sequence shown in SEQ ID №: 2 in the coded sequence list has a total of 109 amino acid residues. The fragment having the nucleotide sequence of positions 8-334 in SEQ ID No. 1 in the sequence listing is named Gh FPF1. the
实施例2、棉花基因Gh FPF1的功能验证 Example 2, Functional Verification of Cotton Gene Gh FPF1
(一)、表达载体的构建 (1) Construction of expression vector
(1)带有特定酶切位点的目的基因片段的获得 (1) Obtaining target gene fragments with specific restriction sites
上游引物：5’-CTAGTCTAGAATGAGCGGTCCTTGGTGTTT-3’含酶切位点XbaI(T/CTAGA) Upstream primer: 5'-CTAGTCTAGAATGAGCGGTCCTTGGTGTTT-3' containing enzyme cutting site XbaI(T/CTAGA)
下游引物：5’-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3’含酶切位点SmaI(CCC/GGG) Downstream primer: 5'-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3' with enzyme cutting site SmaI(CCC/GGG)
以上述设计的含有酶切位点的引物，以中棉所36的幼苗的cDNA为模板，进行PCR扩增。将扩增获得的带有酶切位点的目的片段连接至pGEM-T Easy克隆载体，转化DH5α感受态细胞，通过PCR及酶切验证和序列测定筛选出序列正确的重组载体。 PCR amplification was carried out with the above designed primers containing restriction sites and the cDNA of the seedlings of Zhongmian Institute 36 as a template. The amplified target fragment with restriction sites was connected to the pGEM-T Easy cloning vector, transformed into DH5α competent cells, and the recombinant vector with the correct sequence was screened out by PCR, restriction restriction verification and sequence determination. the
(2)pBI121-Gh FPF1植物表达载体的构建 (2) Construction of pBI121-Gh FPF1 plant expression vector
将步骤(1)制备的重组载体和pBI121质粒分别用SmaI和XbaI双酶切，电泳回收目的基因片段和pBI121载体的大片段产物；将目的基因片段和pBI121的酶切大片段产物用T4连接酶连接过夜；连接产物转化大肠杆菌DH5α，37℃培养过夜；挑取单克隆摇菌，测序验证序列的正确性。所得质粒中插入的外源基因的序列为SEQ ID №：1第8-337位核苷酸，将该质粒命名为pBI121-Gh FPF1。 The recombinant vector prepared in step (1) and the pBI121 plasmid were double-digested with SmaI and XbaI respectively, and the large fragment product of the target gene fragment and pBI121 vector was recovered by electrophoresis; the large fragment product of the target gene fragment and pBI121 was digested with T4 ligase Ligated overnight; the ligated product was transformed into Escherichia coli DH5α, and cultured overnight at 37°C; a single clone was picked and sequenced to verify the correctness of the sequence. The sequence of the exogenous gene inserted in the obtained plasmid is SEQ ID No.: 1 nucleotides 8-337, and the plasmid is named pBI121-Gh FPF1. the
(二)、转Gh FPF1基因拟南芥的获得 (2), the acquisition of transgenic Arabidopsis thaliana
1、重组农杆菌的获得 1. Acquisition of recombinant Agrobacterium
将质粒pBI121-Gh FPF1转入农杆菌LBA4404感受态细胞中，得到重组菌。提取重组菌的质粒送去测序，将测序正确的含有质粒pBI121-Gh FPF1的重组菌命名为LBA4404/pBI121-Gh FPF1。 The plasmid pBI121-Gh FPF1 was transformed into Agrobacterium LBA4404 competent cells to obtain recombinant bacteria. The plasmids of the recombinant bacteria were extracted and sent for sequencing, and the recombinant bacteria containing the plasmid pBI121-Gh FPF1 sequenced correctly were named LBA4404/pBI121-Gh FPF1. the
2、采用花序浸染法转化拟南芥 2. Transformation of Arabidopsis thaliana by inflorescence dipping method
(1)将-20℃保存的重组农杆菌菌液20μl接种到1ml LB液体培养基中，28℃、180rpm振荡培养过夜活化，取活化菌液200μl加入到20ml LB液体培养基28℃、180rpm振荡培养 (1) Inoculate 20 μl of the recombinant Agrobacterium solution stored at -20°C into 1ml LB liquid medium, and activate overnight at 28°C and 180rpm with shaking culture. Take 200μl of the activated bacteria solution and add it to 20ml LB liquid medium at 28°C and shake at 180rpm nourish
(2)待菌液OD值约为1.2时，3000转/每分离心菌液收集菌体 (2) When the OD value of the bacterial liquid is about 1.2, collect the bacterial cells by centrifuging the bacterial liquid at 3000 rpm
(3)转化介质配方为：1/2MS(大量元素减半，其他不变)、5％蔗糖，0.01μg/ml苄氨基嘌呤(BAP)，0.03％silwetL-77，20mg/L乙酰丁香酮，KOH调pH值至5.7(Steven et al.1998)。 (3) Transformation medium formula is: 1/2MS (major elements are halved, others remain unchanged), 5% sucrose, 0.01 μ g/ml benzylaminopurine (BAP), 0.03% silwetL-77, 20 mg/L acetosyringone, KOH adjusted the pH value to 5.7 (Steven et al.1998). the
(4)用上述转化介质悬浮菌体，调OD至0.8开始浸染 (4) Use the above-mentioned transformation medium to suspend the bacteria, adjust the OD to 0.8 and start to impregnate
(5)将拟南芥花序置于转化介质中30-50s，浸染后用保鲜膜将拟南芥包起来，暗培养一天后置于正常条件下培养。 (5) Put the Arabidopsis inflorescence in the transformation medium for 30-50 seconds, wrap the Arabidopsis with plastic wrap after dipping, culture in the dark for one day, and culture under normal conditions. the
3、转Gh FPF1基因拟南芥植株的鉴定 3. Identification of transgenic Arabidopsis plants with Gh FPF1 gene
(1)转Gh FPF1基因拟南芥植株中Gh FPF1基因的检测 (1) Detection of Gh FPF1 gene in transgenic Arabidopsis plants
将收获的种子消毒后种植在含卡那霉素的1/2MS上(琼脂浓度0.6％)，后进行4℃春化3天，转移到培养室中培养10天左右，二阴性植株叶片变黄，不再生长，将生长正常的植株移栽至小花盆中种植，待生长一个月后，检测植株中是否转入棉花基因GhFPF1。待测植株中Gh FPF1基因的制备方法与实施例1相同，以ddH2O为空白对照，非转基因拟南芥DNA为阴性对照，重组子质粒为阳性对照，PCR扩增时所用引物序列为： The harvested seeds were sterilized and planted on 1/2MS containing kanamycin (agar concentration 0.6%), then vernalized at 4°C for 3 days, transferred to the culture room for about 10 days, and the leaves of double-negative plants turned yellow , no longer grow, transplant the normal growing plants into small flowerpots for planting, and after one month of growth, detect whether the cotton gene GhFPF1 is transferred into the plants. The preparation method of the Gh FPF1 gene in the plant to be tested is the same as that in Example 1, ddH2 O is used as the blank control, non-transgenic Arabidopsis DNA is used as the negative control, and the recombinant plasmid is used as the positive control. The primer sequences used in PCR amplification are:
上游引物：5’-GATGTGATATCTCCACTGACGT-3’ Upstream primer: 5'-GATGTGATATCTCCACTGACGT-3'
下游引物：5’-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3’ Downstream primer: 5'-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3'
其中，上游引物与表达载体相匹配，下游引物与基因相匹配。 Wherein, the upstream primer matches the expression vector, and the downstream primer matches the gene. the
将扩增产物在1.5％琼脂糖凝胶上进行电泳检测，检测结果见图1，上述选取的生长正常的植株和阳性对照组中均可以检测到约450bp的条带(基因序列加上表达载体上的一小段序列共约450bp)，而空白对照和野生型拟南芥阴性对照组中均未检测到相应的DNA分子片段，说明Gh FPF1基因已整合到上述拟南芥基因组中。 The amplified product is detected by electrophoresis on a 1.5% agarose gel, and the test results are shown in Fig. 1. A band of about 450bp (gene sequence plus expression vector) can be detected in the above-mentioned selected normal plants and the positive control group. A small segment of the sequence above is about 450bp in total), while the corresponding DNA molecular fragments were not detected in the blank control and the wild-type Arabidopsis negative control group, indicating that the Gh FPF1 gene has been integrated into the above-mentioned Arabidopsis genome. the
(2)qRT-PCR检测 (2) qRT-PCR detection
收获步骤(1)筛选出的7株阳性株的T0代种子，将种子繁殖至T3代，将T3代种子和野生型拟南芥种子同等条件下种植，两周分别整株取样至-70℃保存。随机选取三 个转基因株系COL3、COL4、COL7进行qRT-PCR检测。按照实施例1的方法分别提取转基因和野生型拟南芥RNA，并反转录为cDNA，以该cDNA为模板、以拟南芥基因UBQ5为内参，，进行qRT-PCR检测，扩增Gh FPF1基因和UBQ5内参基因使用的引物序列为： Harvest the T0 generation seeds of the 7 positive strains screened in step (1), propagate the seeds to the T3 generation, plant the T3 generation seeds and the wild-type Arabidopsis seeds under the same conditions, and sample the whole plant in two weeks. Store at -70°C. Three transgenic lines COL3, COL4, and COL7 were randomly selected for qRT-PCR detection. According to the method of Example 1, transgenic and wild-type Arabidopsis RNA were extracted respectively, and reverse-transcribed into cDNA. Using the cDNA as a template and the Arabidopsis gene UBQ5 as an internal reference, qRT-PCR detection was performed to amplify GhFPF1 The primer sequences used for the gene and UBQ5 internal reference gene are:
Gh FPF1上游引物：5’-AAACTCAGGTTCGGACCAAAGG-3’ Gh FPF1 upstream primer: 5'-AAACTCAGGTTCGGACCAAAGG-3'
下游引物：5’-CCGTCGTATCTTTCCCAACCAA-3’ Downstream primer: 5'-CCGTCGTATCTTTTCCCAACCAA-3'
UBQ5上游引物：5’-TAACCCTTGAGGTTGAATCATC-3’ UBQ5 upstream primer: 5'-TAACCCTTGAGGTTGAATCATC-3'
下游引物：5’-GTCGATTCCTTCTGGATGTTGT-3’ Downstream primer: 5'-GTCGATTCCTTCTGGATGTTGT-3'
qRT-PCR检测结果见图2，结果表明，Gh FPF1基因在不同的转基因T3代植株中均有不同(高)水平的转录，但在野生型拟南芥中没有检测到Gh FPF1基因的转录产物。qRT-PCR检测结果进一步证明了Gh FPF1基因已整合到转基因T3代拟南芥的基因组中并且成功转录为mRNA。 The results of qRT-PCR detection are shown in Figure 2. The results show that the Gh FPF1 gene has different (high) levels of transcription in different transgenic T3 generation plants, but no transcription of the Gh FPF1 gene was detected in wild-type Arabidopsis product. The results of qRT-PCR further proved that the GhFPF1 gene had been integrated into the genome of the transgenic T3 Arabidopsis thaliana and was successfully transcribed into mRNA.
(三)、转Gh FPF1基因拟南芥植株的表型 (3), Phenotype of transgenic Arabidopsis plants with Gh FPF1 gene
1、将转基因T3代拟南芥与野生型拟南芥同等条件下种植栽培，分别观察生长3周和4周时的表型，结果见图3、4。从图3可以看出，转基因拟南芥抽薹早于野生型拟南芥；从图4可以看出，转基因拟南芥的开花时间早于野生型拟南芥。该实验结果表明，转基因拟南芥的生育期比非转基因拟南芥明显提前。 1. Plant and cultivate the transgenicT3 Arabidopsis and wild-type Arabidopsis under the same conditions, and observe the phenotypes at 3 and 4 weeks of growth respectively. The results are shown in Figures 3 and 4. It can be seen from Figure 3 that the bolting of transgenic Arabidopsis is earlier than that of wild-type Arabidopsis; it can be seen from Figure 4 that the flowering time of transgenic Arabidopsis is earlier than that of wild-type Arabidopsis. The experimental results showed that the growth period of the transgenic Arabidopsis was significantly earlier than that of the non-transgenic Arabidopsis.
2、选取7个转基因T3代株系与野生型和转空载体拟南芥同等条件下种植栽培，每个株系30棵，统计开花时间，并将莲座叶与茎生叶一并进行计数，结果见表1。表1中，WT为野生型拟南芥，COL1-COL7为选取的7个T3代转基因拟南芥株系。 2. Select 7 transgenic T3 generation lines and plant them under the same conditions as the wild type and empty vector Arabidopsis thaliana, 30 plants for each line, count the flowering time, and count the rosette leaves and cauline leaves together, The results are shown in Table 1. In Table 1, WT refers to wild-type Arabidopsis thaliana, and COL1-COL7 refers to selected 7 transgenic Arabidopsis lines of T3 generation. the
表1 Table 1
结果显示，转基因拟南芥比野生型拟南芥提前开花5.1天，莲座叶与茎生叶的总 数比野生型拟南芥少2.7片；转基因拟南芥比转空载体拟南芥提前开花5.0天，莲座叶与茎生叶的总数比转空载体拟南芥少2.6片。表1的统计结果进一步说明棉花Gh FPF1基因在拟南芥中具有调控生育期的作用，具体体现为开花时间早、莲座叶和茎生叶数量减少。 The results showed that the transgenic Arabidopsis flowered 5.1 days earlier than the wild-type Arabidopsis, and the total number of rosette leaves and cauline leaves was 2.7 less than that of the wild-type Arabidopsis; the transgenic Arabidopsis flowered earlier than the empty vector Arabidopsis On day 5.0, the total number of rosette leaves and cauline leaves was 2.6 less than that of the empty vector Arabidopsis. The statistical results in Table 1 further indicated that the cotton Gh FPF1 gene played a role in regulating the growth period in Arabidopsis, which was embodied in the early flowering time and the reduction in the number of rosette leaves and cauline leaves. the
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310136123.3ACN103183732B (en) | 2013-04-18 | 2013-04-18 | Cotton Gh FPP1 protein as well as coding gene and application thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310136123.3ACN103183732B (en) | 2013-04-18 | 2013-04-18 | Cotton Gh FPP1 protein as well as coding gene and application thereof |
| Publication Number | Publication Date |
|---|---|
| CN103183732A CN103183732A (en) | 2013-07-03 |
| CN103183732Btrue CN103183732B (en) | 2014-08-20 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310136123.3AExpired - Fee RelatedCN103183732B (en) | 2013-04-18 | 2013-04-18 | Cotton Gh FPP1 protein as well as coding gene and application thereof |
| Country | Link |
|---|---|
| CN (1) | CN103183732B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924511B (en)* | 2016-07-11 | 2019-07-09 | 中国农业科学院棉花研究所 | A kind of Leaf shape, plant type GAP-associated protein GAP GhBRH1-L and its encoding gene and application |
| CN108753796A (en)* | 2018-07-02 | 2018-11-06 | 中国农业科学院棉花研究所 | A kind of the DB genes and its coding albumen and application of the control limited fruit branch character of cotton |
| CN108949781A (en)* | 2018-08-15 | 2018-12-07 | 中国农业科学院棉花研究所 | A kind of limited fruit branch DB gene of cotton, coding albumen and its application |
| CN113046459A (en)* | 2019-12-27 | 2021-06-29 | 中国农业科学院棉花研究所 | Flanking sequence of GhFPF1 gene-transferred cotton insertion site and specificity identification method thereof |
| CN111607604B (en)* | 2020-03-27 | 2021-12-14 | 中国农业科学院棉花研究所 | Application of cotton GHPSAT2 gene in promoting plant flowering |
| CN111218460B (en)* | 2020-03-27 | 2021-09-21 | 中国农业科学院棉花研究所 | Application of cotton GhACO gene in promoting plant flowering |
| CN112794890B (en)* | 2021-03-25 | 2022-05-27 | 南京农业大学 | Flowering regulatory protein ELF 3-beta and coding gene and application thereof |
| CN115851821B (en)* | 2023-02-14 | 2023-04-25 | 河南大学三亚研究院 | Application of BBX16 gene in improving plant salt tolerance |
| CN117904139B (en)* | 2024-03-05 | 2025-02-11 | 中国科学院遗传与发育生物学研究所 | Application of TaFPFL1-2B gene in improving plant drought resistance |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997025433A1 (en)* | 1996-01-09 | 1997-07-17 | Eidg. Technische Hochschule Zürich Ethz | Regulation of flowering in plants |
| WO2006067219A1 (en)* | 2004-12-22 | 2006-06-29 | Vib Vzw | Methods and means to increase the amounts of carbohydrates in plants |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8299318B2 (en)* | 2007-07-05 | 2012-10-30 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997025433A1 (en)* | 1996-01-09 | 1997-07-17 | Eidg. Technische Hochschule Zürich Ethz | Regulation of flowering in plants |
| WO2006067219A1 (en)* | 2004-12-22 | 2006-06-29 | Vib Vzw | Methods and means to increase the amounts of carbohydrates in plants |
| Title |
|---|
| FPF1 modulates the competence to flowering in Arabidopsis;Melzer等;《The Plant Journal》;19991231;第18卷(第4期);395－405* |
| FPF1 Promotes Flowering in Arabidopsis;Kania等;《The Plant Cell》;19971231;第9卷;1327－1338* |
| Kania等.FPF1 Promotes Flowering in Arabidopsis.《The Plant Cell》.1997,第9卷1327-1338. |
| Melzer等.FPF1 modulates the competence to flowering in Arabidopsis.《The Plant Journal》.1999,第18卷(第4期),395-405. |
| Publication number | Publication date |
|---|---|
| CN103183732A (en) | 2013-07-03 |
| Publication | Publication Date | Title |
|---|---|---|
| CN103183732B (en) | Cotton Gh FPP1 protein as well as coding gene and application thereof | |
| CN104988158B (en) | Cabbage type rape nuclear factor NF-YA genes BnNF-YA12 and its application | |
| CN104829700A (en) | Corn CCCH-type zinc finger protein, and encoding gene ZmC3H54 and application thereof | |
| CN103288944B (en) | bHLH17 protein, its coding gene and its application | |
| CN115305252B (en) | Receptor kinase gene OsIFBR for regulating and controlling rice resistance | |
| CN110628808A (en) | Arabidopsis AtTCP5 Gene and Its Application in Regulating Plant Height | |
| CN112226455A (en) | A rice grain length and grain weight related protein and its encoding gene and application | |
| WO2020221029A1 (en) | Zea mays receptor-like kinase gene zmrlk7 and use thereof | |
| CN113234729B (en) | Gene GauRev2 capable of obviously improving verticillium wilt resistance of cotton and application thereof | |
| CN103360484B (en) | Upland cotton protein GhMADS22, and coding gene and application thereof | |
| CN108795944A (en) | Cotton long-chain non-coding RNA-lnc973 and its application in plant salt endurance | |
| CN113584047B (en) | Barley HvNAT2 gene and application thereof | |
| CN104610438B (en) | Cotton stress response related protein GhGeBP and coding gene and application thereof | |
| CN103288943B (en) | bHLH13 protein, its coding gene and its application | |
| CN112341532B (en) | Application of OsDSK2a protein or its encoding gene in regulating rice blast resistance | |
| CN104177482B (en) | A kind of plant stress tolerance is correlated with SbSNAC1 albumen and encoding gene thereof and application | |
| CN119899860B (en) | Application of ZmPHR1 protein and encoding gene thereof in regulation and control of drought resistance of plants | |
| NL2030997B1 (en) | Zea mays receptor-like kinase 7 (zmrlk7) gene related to kernel and plant type development of maize and use thereof | |
| CN114644701B (en) | Use of proteins derived from corn and related biomaterials | |
| CN117844829B (en) | Soybean heat-resistant protein GmBSK1 and application of encoding gene thereof in regulation and control of plant stress resistance | |
| CN110229801B (en) | Gene for controlling rice leaf senescence and protein coded by same | |
| CN113136388B (en) | Application of rice OsMAPKKK5 gene in improving plant height and grain shape of rice | |
| CN108659109A (en) | A kind of wheat powdery mildew resistance-associated protein TaSTKR1 and its encoding gene and application | |
| CN108424438A (en) | A kind of wheat powdery mildew resistance-associated protein TaWRKY49 and its encoding gene and application | |
| CN110194791B (en) | Application of SPL3 protein in regulation and control of plant inflorescence or carpopodium development |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20140820 Termination date:20210418 | |
| CF01 | Termination of patent right due to non-payment of annual fee |