CN118373890A - Regulation method of plant economic traits based on IbbHLH49 protein - Google Patents
Regulation method of plant economic traits based on IbbHLH49 protein Download PDFInfo
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- CN118373890A CN118373890A CN202410441717.3A CN202410441717A CN118373890A CN 118373890 A CN118373890 A CN 118373890A CN 202410441717 A CN202410441717 A CN 202410441717A CN 118373890 A CN118373890 A CN 118373890A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
技术领域Technical Field
本发明涉及基因工程技术领域,尤其涉及一种基于IbbHLH49蛋白的植物经济性状的调控方法。The present invention relates to the technical field of genetic engineering, and in particular to a method for regulating plant economic traits based on IbbHLH49 protein.
背景技术Background technique
淀粉是植物进行光合作用的重要产物,是植物中含量最丰富、分布最广泛的碳水化合物,不仅是人类日常饮食中的主要能量来源,也是主要的饲料来源,同时作为工业生产的重要原材料,被广泛应用于酿酒、纺织、造纸以及化工等领域。淀粉主要存在于植物的种子、块根、块茎以及果实中,不同作物的淀粉含量各不相同。Starch is an important product of plant photosynthesis. It is the most abundant and widely distributed carbohydrate in plants. It is not only the main energy source in human daily diet, but also the main source of feed. At the same time, as an important raw material for industrial production, it is widely used in winemaking, textiles, papermaking, and chemical industries. Starch mainly exists in the seeds, roots, tubers, and fruits of plants. The starch content of different crops varies.
甘薯(Ipomoea batatas)块根中含有丰富的淀粉,其利用价值非常巨大。甘薯作为一种边际土地作物,在不与主粮争地的前提下,其种植面积会有进一步缩小的趋势。因此,利用基因工程培育高产高淀粉的甘薯品种是一种行之有效的手段。然而,甘薯块根中淀粉合成的转录调控机制目前仍然未知。Sweet potato (Ipomoea batatas) tubers are rich in starch, which has great utilization value. As a marginal land crop, the planting area of sweet potato will be further reduced if it does not compete with staple food. Therefore, it is an effective means to cultivate high-yield and high-starch sweet potato varieties using genetic engineering. However, the transcriptional regulatory mechanism of starch synthesis in sweet potato tubers is still unknown.
如何挖掘甘薯块根中与淀粉合成的相关基因,深入研究淀粉合成机制,结合植物基因工程对植物淀粉产量进行调控具有重要的意义。It is of great significance to explore the genes related to starch synthesis in sweet potato tubers, conduct in-depth research on the mechanism of starch synthesis, and regulate plant starch production in combination with plant genetic engineering.
发明内容Summary of the invention
本发明提供一种基于IbbHLH49蛋白的植物经济性状的调控方法,用以调控植物的经济性状,包括块根大小、块根产量、淀粉产量、支链淀粉比例中的至少一种。The present invention provides a method for regulating plant economic traits based on IbbHLH49 protein, which is used to regulate the economic traits of plants, including at least one of root tuber size, root tuber yield, starch yield, and amylopectin ratio.
第一方面,本发明提供一种植物经济性状的调控方法,包括:调控出发植株中IbbHLH49蛋白编码基因的表达,得到转基因植株,所述转基因植株的经济性状与出发植株不同,所述经济性状包括块根大小、块根产量、淀粉产量、支链淀粉比例中的至少一种;In a first aspect, the present invention provides a method for regulating plant economic traits, comprising: regulating the expression of a gene encoding an IbbHLH49 protein in a starting plant to obtain a transgenic plant, wherein the economic traits of the transgenic plant are different from those of the starting plant, and the economic traits include at least one of root size, root yield, starch yield, and amylopectin ratio;
所述IbbHLH49蛋白是如下A1)、A2)或A3)的蛋白质:The IbbHLH49 protein is the following protein A1), A2) or A3):
A1)氨基酸序列如SEQ ID NO:1所示的蛋白质;A1) a protein with an amino acid sequence as shown in SEQ ID NO: 1;
A2)将SEQ ID NO:1所示的氨基酸序列经过氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有80%以上的同一性且与植物淀粉合成相关的蛋白质;A2) a protein related to plant starch synthesis obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, which has more than 80% identity with the protein shown in A1);
A3)在A1)或A2)的N末端或/和C末端连接蛋白标签得到的融合蛋白。A3) A fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of A1) or A2).
上述方法中,所述IbbHLH49蛋白来源于甘薯(Ipomoea batatas)。In the above method, the IbbHLH49 protein is derived from sweet potato (Ipomoea batatas).
上述方法中,SEQ ID NO:1所示的蛋白质由521个残基组成,具体氨基酸序列如下所示:In the above method, the protein shown in SEQ ID NO: 1 consists of 521 residues, and the specific amino acid sequence is as follows:
MDKGGKDEAMAAKRGDDAMSFQSANVSSEWQMNGSNLANTPIGMIPNSNPMMVDAFCLNVWDQSASSASLGFCDANVHSNVTTSSPFGAGTSGFTTALRGGVDRGLGMAWHPANTMLKTGMLLPTAPAVVPPNLPQFPADSDFLQRAARFSCFSGGNLGDMMNPFESLSPYCRGITPTQRPQQVFVGNGLKPAPAGEISNGAADGSPLNNNSVIEYAVGSRNSAKEGGGAFGNEPNEPECSSRGGLDVSEGAGAESSASKKRKRSGQDAETDQNKGTPPPAEAATDQTDNQQKGDQNVTATPSKPGGKGGKQGSQASDNPKEDYIHIRARRGQATNSHSLAERVRREKISERMKFLQDLVPGCNKVTGKAVMLDEIINYVQSLQRQVEFLSMKLATVNPRLEFNIDSLLAKDILQSRAGSSSSLLSFPHDMTMPYPPVHHPPSTLIQAGLPSLGSSADAIRRTINPHLATASGSFKEPTPQVPSMWDDELHNVVQMGFNPSAPLDSQDIGSLPPGHMKSEPMDKGGKDEAMAAKRGDDAMSFQSANVSSEWQMNGSNLANTPIGMIPNSNPMMVDAFCLNVWDQSASSASLGFCDANVHSNVTTSSPFGAGTSGFTTALRGGVDRGLGMAWHPANTMLKTGMLLPTAPAVVPPNLPQFPADSDFLQRAARFSCFSGGNLGDMMNPFESLSPYCRGITPTQRPQQVFVGNGLKPAPAGEISNGAADGSPLNNNSVIEYAVGSRNSAKEGG GAFGNEPNEPECSSRGGLDVSEGAGAESSASK KRKRSGQDAETDQNKGTPPPAEAATDQTDNQQKGDQNVTATPSKPGGKGGKQGSQASDNPKEDYIHIRARRGQATNSHSLAERVRREKISERMKFLQDLVPGCNKVTGKAVMLDEIINYVQSLQRQVEFLSMKLATVNPRLEFNIDSLLAKDILQSRAGSSSSLLSFPHDMTMPYPPVHHPPSTLIQAGLPSLGSSADAIRRTINPHLATA SGSFKEPTPQVPSMWDDELHNVVQMGFNPSAPLDSQDIGSLPPGHMKSEP
上述方法中,同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。In the above method, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.
上述方法中,所述80%以上的同一性可以为至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的同一性。In the above method, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
上述方法中,蛋白标签(protein-tag)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。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。In the above method, protein tag refers to a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and/or a SUMO tag, etc.
上述方法中,所述调控可以为上调或增强或提高,也可为下调或抑制或降低。In the above method, the regulation may be up-regulation, enhancement or increase, or down-regulation, inhibition or reduction.
进一步地,上调或增强或提高IbbHLH49蛋白编码基因的表达具体包括方法M1):Furthermore, upregulating, enhancing or increasing the expression of the IbbHLH49 protein encoding gene specifically includes method M1):
M1)向出发植株中导入所述IbbHLH49蛋白的编码基因,上调或增强或提高所述IbbHLH49蛋白编码基因的表达,得到转基因植株;相比出发植株,所述转基因植株的块根大小、块根产量、淀粉产量、支链淀粉比例均高于出发植株。M1) introducing the coding gene of the IbbHLH49 protein into the starting plant, upregulating or enhancing or improving the expression of the coding gene of the IbbHLH49 protein, and obtaining a transgenic plant; compared with the starting plant, the tuber size, tuber yield, starch yield and amylopectin ratio of the transgenic plant are all higher than those of the starting plant.
上述方法M1)中,所述IbbHLH49蛋白的编码基因为B1)、B2)中的一种;In the above method M1), the coding gene of the IbbHLH49 protein is one of B1) and B2);
B1)核苷酸序列如SEQ ID NO:2所示的核酸分子;B1) a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO: 2;
B2)与SEQ ID NO:2所示的DNA分子具有80%以上的同一性,且编码IbbHLH49蛋白的DNA分子。B2) A DNA molecule that has more than 80% identity with the DNA molecule shown in SEQ ID NO: 2 and encodes the IbbHLH49 protein.
SEQ ID NO:2所示的核酸分子的核苷酸序列如下所示:The nucleotide sequence of the nucleic acid molecule shown in SEQ ID NO:2 is as follows:
ATGGATAAGGGTGGCAAGGATGAGGCCATGGCAGCAAAGAGAGGTGATGATGCTATGAGCTTTCAGTCAGCAAATGTGTCATCTGAGTGGCAAATGAATGGTTCCAATCTCGCGAATACGCCTATTGGAATGATTCCCAATAGCAATCCGATGATGGTGGATGCGTTTTGCCTGAATGTTTGGGACCAATCTGCAAGTTCAGCAAGCTTAGGCTTTTGTGATGCTAATGTTCATAGCAATGTTACCACTTCTAGCCCATTTGGAGCTGGAACAAGTGGGTTCACCACCGCCTTAAGAGGCGGTGTCGATAGAGGTCTTGGTATGGCGTGGCATCCAGCTAACACGATGTTGAAAACGGGGATGCTTCTGCCTACTGCTCCTGCAGTGGTTCCTCCGAACTTGCCTCAGTTCCCAGCTGATTCCGATTTTCTTCAAAGGGCAGCGAGGTTTTCGTGCTTCAGTGGAGGAAACTTGGGCGATATGATGAACCCCTTTGAGTCTCTGAGTCCTTATTGTAGAGGCATAACGCCAACTCAAAGGCCTCAACAGGTGTTTGTAGGTAATGGACTAAAACCTGCACCCGCGGGTGAAATCTCAAACGGGGCTGCTGATGGTAGCCCGCTAAATAACAATAGCGTGATTGAATATGCCGTTGGATCTAGAAATAGTGCAAAAGAAGGCGGAGGAGCCTTTGGAAATGAACCTAACGAGCCCGAATGTAGTAGCCGTGGTGGCCTTGATGTATCAGAGGGTGCAGGGGCAGAGTCTTCTGCCTCAAAGAAAAGGAAAAGAAGTGGGCAGGACGCTGAAACCGATCAAAACAAGGGAACTCCACCACCAGCTGAAGCAGCAACAGATCAGACTGATAACCAGCAGAAAGGAGATCAAAACGTGACCGCAACTCCCAGCAAGCCAGGTGGTAAAGGCGGTAAGCAGGGGTCCCAAGCTTCAGATAATCCTAAAGAAGATTACATCCACATTCGGGCTAGGAGAGGCCAGGCCACGAATAGCCACAGTCTTGCAGAAAGAGTTAGAAGGGAGAAAATCAGTGAAAGAATGAAGTTTCTTCAGGATCTCGTGCCCGGTTGTAACAAGGTCACTGGCAAAGCAGTAATGCTTGATGAAATCATTAATTATGTACAGTCACTCCAACGACAGGTTGAGTTCTTGTCGATGAAGCTTGCAACAGTAAACCCACGGCTCGAGTTCAACATTGATAGTCTCCTAGCAAAAGATATCCTCCAGTCCAGGGCTGGCTCTTCGTCTTCTCTGTTGTCTTTTCCACATGATATGACTATGCCTTATCCACCAGTACACCATCCACCATCAACGCTGATTCAAGCAGGTCTTCCTAGCCTGGGAAGTTCTGCAGATGCAATACGAAGAACCATCAACCCGCACTTGGCAACTGCGAGTGGGAGCTTCAAGGAGCCTACACCTCAGGTACCTAGTATGTGGGACGATGAGCTCCATAACGTTGTCCAAATGGGCTTCAATCCAAGCGCTCCCCTCGACAGCCAAGATATAGGTTCTCTACCACCAGGCCATATGAAATCTGAGCCCTGAATGGATAAGGGTGGCAAGGATGAGGCCATGGCAGCAAAGAGAGGTGATGATGCTATGAGCTTTCAGTCAGCAAATGTGTCATCTGAGTGGCAAATGAATGGTTCCAATCTCGCGAATACGCCTATTGGAATGATTCCCAATAGCAATCCGATGATGGTGGATGCGTTTTGCCTGAATGTTTGGGACCAATCTGCAAGTTCAGCAAGCTTAGGCTTTTGTGATGCTAATGTTCATAGCAATGTTACCACTTCTAGCCCATT TGGAGCTGGAACAAGTGGGTTCACCACCGCCTTAAGAGGCGGTGTCGATAGAGGTCTTGGTATGGCGTGGCATCCAGCTAACACGATGTTGAAAACGGGGATGCTTCTGCCTACTGCTCCTGCAGTGGTTC CTCCGAACTTGCCTCAGTTCCCAGCTGATTCCGATTTTCTTCAAAGGGCAGCGAGGTTTTCGTGCTTCAGTGGAGGAAACTTGGGCGATATGATGAACCCCTTTGAGTCTCTGAGTCCTTATTGTAGAGGCATAACGCCAACTCAAAGGCCTCAACAGGTTTGTAGGTAATGGACTAAAACCTGCACCCGCGGGTGAAATCTCAAACGGGGCTGCTGATGGTAGCCCGCTAAATAACAATAGCGTGATTGAATATGCCGT TGGATCTAGAAATAGTGCAAAAGAAGGCGGAGGAGCCTTTGGAAATGAACCTAACGAGCCCGAATGTAGTAGCCGTGGTGGCCTTGATGTATCAGAGGGTGCAGGGGCAGAGTCTTCTGCCTCAAAGAAA AGGAAAAGAAGTGGGCAGGACGCTGAAACCGATCAAAACAAGGGAACTCCACCACCAGCTGAAGCAGCAACAGATCAGACTGATAACCAGCAGAAAGGAGATCAAAACGTGACCGCAACTCCCAGCAAGCCAGGTGGTAAAGGCGGTAAGCAGGGGTCCCAAGCTTCAGATAATCCTAAAGAAGATTACATCCACATTCGGGCTAGGAGAGGCCAGGCCACGAATAGCCACAGTCTTGCAGAAAGAGTTAGAAGGGAAAAAAA TCAGTGAAAGAATGAAGTTTCTTCAGGATCTCGTGCCCGGTTGTAACAAGGTCACTGGCAAAGCAGTAATGCTTGATGAAATCATTAATTATGTACAGTCACTCCAACGACAGGTTGAGTTCTTGTCGA TGAAGCTTGCAACAGTAAACCCACGGCTCGAGTTCAACATTGATAGTCTCCTAGCAAAAGATATCCTCCAGTCCAGGGCTGGCTCTTCGTCTTCTCTGTTGTCTTTTCCACATGATATGACTATGCCTTATCCACCAGTACACCATCCACCATCAACGCTGATTCAAGCAGGTCTTCCTAGCCTGGGAAGTTCTGCAGATGCAATACGAAGAACCATCAACCCGCACTTGGCAACTGCGAGTGGGAGCTTCAAGGAGCCTAC ACCTCAGGTACCTAGTATGTGGGACGATGAGCTCCATAACGTTGTCCAAATGGGCTTCAATCCAAGCGCTCCCCTCGACAGCCAAGATATAGGTTCTCTACCACCAGGCCATTGAAATCTGAGCCCTGA
上述方法M1)中,所述80%以上的同一性可以为至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%的同一性。In the above method M1), the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
上述方法M1)中,通过构建含有IbbHLH49蛋白的编码基因的重组表达载体,并导入出发植株中,以上调或增强或提高IbbHLH49蛋白编码基因的表达。进一步地,所述含有IbbHLH49蛋白的编码基因的重组表达载体可以为pCAMBIA1300-IbbHLH49-GFP。具体地,pCAMBIA1300-IbbHLH49-GFP为利用限制性内切酶KpnI和BamHI将IbbHLH49蛋白的编码基因插入至pCAMBIA1300-GFP载体得到的。重组表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织,并将转化的植物细胞或组织培育成植株。In the above method M1), a recombinant expression vector containing the coding gene of the IbbHLH49 protein is constructed and introduced into the starting plant to up-regulate, enhance or improve the expression of the IbbHLH49 protein coding gene. Further, the recombinant expression vector containing the coding gene of the IbbHLH49 protein can be pCAMBIA1300-IbbHLH49-GFP. Specifically, pCAMBIA1300-IbbHLH49-GFP is obtained by inserting the coding gene of the IbbHLH49 protein into the pCAMBIA1300-GFP vector using restriction endonucleases KpnI and BamHI. The recombinant expression vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant cells or tissues are cultivated into plants.
上述方法中,下调/抑制/降低IbbHLH49蛋白编码基因的表达具体包括方法M2):In the above method, down-regulating/inhibiting/reducing the expression of the IbbHLH49 protein encoding gene specifically includes method M2):
M2)通过基因敲除或基因沉默的方法,下调或抑制或降低所述IbbHLH49蛋白编码基因表达,得到所述转基因植株。M2) down-regulating, inhibiting or reducing the expression of the IbbHLH49 protein encoding gene by gene knockout or gene silencing to obtain the transgenic plant.
上述方法M2)中,所述基因敲除(gene knockout)是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。In the above method M2), the gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.
所述基因沉默(gene silencing)是指在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默以不改变DNA序列为前提,使基因不表达或低表达。基因沉默可发生在两种水平上,一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默,另一种是转录后基因沉默,即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活,包括反义RNA、共抑制(co-suppression)、基因压抑(quelling)、RNA干扰(RNAi)和微小RNA(miRNA)介导的翻译抑制等。The gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.
进一步地,本发明通过构建shRNA载体发挥RNAi功能,以降低IbbHLH49蛋白编码基因的表达水平,具体地,所述shRNA的一条链的序列为核苷酸序列如SEQ IDNo:2的第1-200位的DNA片段转录得到的序列。进一步地,通过构建含有式(I)所示的DNA分子的重组表达载体,表达上述shRNA后下调或抑制或降低所述IbbHLH49蛋白编码基因表达,得到所述转基因植株:Furthermore, the present invention constructs an shRNA vector to exert RNAi function to reduce the expression level of the IbbHLH49 protein encoding gene. Specifically, the sequence of one strand of the shRNA is a sequence obtained by transcribing the DNA fragment of positions 1-200 of the nucleotide sequence such as SEQ ID No: 2. Further, by constructing a recombinant expression vector containing a DNA molecule represented by formula (I), the expression of the shRNA down-regulates or inhibits or reduces the expression of the IbbHLH49 protein encoding gene, and the transgenic plant is obtained:
SEQ反向-X-SEQ正向(I);SEQ reverse-X-SEQ forward (I);
所述SEQ正向的序列是序列表中SEQ ID NO:2所示序列的第1位-第200位;所述SEQ反向的序列与所述SEQ正向的序列反向互补;所述X是所述SEQ正向与所述SEQ反向之间的间隔序列,所述X与所述SEQ正向及所述SEQ反向均不互补。The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.
下调或抑制或降低所述IbbHLH49蛋白编码基因表达所获得的转基因植株,其块根大小、块根产量、淀粉产量、支链淀粉比例均低于出发植株。The transgenic plants obtained by down-regulating, inhibiting or reducing the expression of the IbbHLH49 protein encoding gene have lower root size, root yield, starch yield and amylopectin ratio than the starting plants.
上述方法中,所述植物为如下任一种:In the above method, the plant is any one of the following:
D1)双子叶植物;D1) Dicotyledons;
D2)管状花目植物;D2) Tubulariaceae;
D3)旋花科植物;D3) Convolvulaceae;
D4)番薯属植物;D4) Ipomoea batatas;
D5)甘薯。D5) Sweet potato.
可以理解的是,扩增上述IbbHLH49蛋白编码基因全长或其任一片段的引物对也属于本发明的保护范围。It is understandable that primer pairs for amplifying the full length of the above-mentioned IbbHLH49 protein encoding gene or any fragment thereof also fall within the scope of protection of the present invention.
第二方面,本发明提供上述任一所述的IbbHLH49蛋白和/或调控IbbHLH49蛋白编码基因的表达的物质在如下任一方面的应用:In a second aspect, the present invention provides the use of any of the above-mentioned IbbHLH49 proteins and/or substances for regulating the expression of genes encoding IbbHLH49 proteins in any of the following aspects:
D1)调控植物块根大小中的应用;D1) Application in regulating the size of plant root tubers;
D2)调控植物块根产量中的应用;D2) Application in regulating plant root yield;
D3)调控植物淀粉含量中的应用;D3) Application in regulating starch content of plants;
D4)调控植物支链淀粉比例中的应用;D4) Application in regulating the proportion of plant amylopectin;
D5)调控植物中淀粉合成相关酶活性中的应用;D5) Application in regulating the activity of enzymes related to starch synthesis in plants;
D6)在植物育种中的应用;D6) Application in plant breeding;
D7)制备调控植物淀粉含量的产品中的应用。D7) Application in the preparation of products for regulating the starch content of plants.
上述应用中,所述植物育种的目的可以为培育经济性状改变的植物,例如,培育淀粉含量提高或者淀粉含量降低的植物、培育块根大小和/或块根产量改变的植物、支链淀粉比例改变的植物。植物育种中的应用具体可为将含有IbbHLH49蛋白或蛋白质的编码基因IbbHLH49的植物与其它植物进行杂交以进行植物育种。In the above application, the purpose of plant breeding can be to cultivate plants with changed economic traits, for example, to cultivate plants with increased starch content or reduced starch content, to cultivate plants with changed root size and/or root yield, or to cultivate plants with changed amylopectin ratio. The application in plant breeding can specifically be to hybridize plants containing IbbHLH49 protein or protein encoding gene IbbHLH49 with other plants for plant breeding.
上述应用中,调控所述IbbHLH49蛋白含量或活性的物质为与所述IbbHLH49蛋白相关的生物材料;In the above application, the substance that regulates the content or activity of the IbbHLH49 protein is a biological material related to the IbbHLH49 protein;
所述生物材料为下述b1)-b10)中的任一种:The biological material is any one of the following b1) to b10):
b1)编码所述IbbHLH49蛋白的核酸分子;b1) a nucleic acid molecule encoding the IbbHLH49 protein;
b2)含有b1)所述核酸分子的表达盒、重组载体、重组微生物、转基因植物细胞系、转基因植物组织或转基因植物器官;b2) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in b1);
b3)下调或抑制或降低所述IbbZIP11蛋白的编码基因的表达的RNA分子;b3) an RNA molecule that downregulates, inhibits or reduces the expression of the gene encoding the IbbZIP11 protein;
b4)转录b3)所述RNA分子的编码基因;b4) transcribing the gene encoding the RNA molecule described in b3);
b5)含有b4)所述编码基因的表达盒;b5) an expression cassette containing the coding gene described in b4);
b6)含有b4)所述编码基因的重组载体、或含有b5)所述表达盒的重组载体;b6) a recombinant vector containing the coding gene described in b4) or a recombinant vector containing the expression cassette described in b5);
b7)含有b4)所述编码基因的重组微生物、或含有b5)所述表达盒的重组微生物、或含有b6)所述重组载体的重组微生物;b7) a recombinant microorganism containing the coding gene described in b4), or a recombinant microorganism containing the expression cassette described in b5), or a recombinant microorganism containing the recombinant vector described in b6);
b8)含有b4)所述编码基因的转基因植物细胞系、或含有b5)所述表达盒的转基因植物细胞系、或含有b6)所述重组载体的转基因植物细胞系;b8) a transgenic plant cell line containing the coding gene described in b4), or a transgenic plant cell line containing the expression cassette described in b5), or a transgenic plant cell line containing the recombinant vector described in b6);
b9)含有b4)所述编码基因的转基因植物组织、或含有b5)所述表达盒的转基因植物组织、或含有b6)所述重组载体的转基因植物组织;b9) transgenic plant tissue containing the coding gene described in b4), or transgenic plant tissue containing the expression cassette described in b5), or transgenic plant tissue containing the recombinant vector described in b6);
b10)含有b4)所述编码基因的转基因植物器官、或含有b5)所述表达盒的转基因植物器官、或含有b6)所述重组载体的转基因植物器官。b10) A transgenic plant organ containing the coding gene described in b4), or a transgenic plant organ containing the expression cassette described in b5), or a transgenic plant organ containing the recombinant vector described in b6).
上述应用中,b1)所述的编码所述IbbHLH49蛋白的核酸分子可以为B1)核苷酸序列如SEQ ID NO:2所示的核酸分子,或者B2)与SEQ ID NO:2所示的DNA分子具有80%以上的同一性,且编码IbbHLH49蛋白的DNA分子。In the above application, the nucleic acid molecule encoding the IbbHLH49 protein described in b1) can be B1) a nucleic acid molecule whose nucleotide sequence is as shown in SEQ ID NO: 2, or B2) a DNA molecule that has more than 80% identity with the DNA molecule shown in SEQ ID NO: 2 and encodes the IbbHLH49 protein.
上述应用中,b3)中抑制或降低所述IbbZIP11蛋白的编码基因的表达的RNA分子为由如式(I)所示的DNA分子转录得到的RNA:In the above application, the RNA molecule that inhibits or reduces the expression of the gene encoding the IbbZIP11 protein in b3) is an RNA transcribed from a DNA molecule as shown in formula (I):
SEQ反向-X-SEQ正向(I);SEQ reverse-X-SEQ forward (I);
所述SEQ正向的序列是序列表中SEQ ID NO:2所示序列的第1位-第200位;所述SEQ反向的序列与所述SEQ正向的序列反向互补;所述X是所述SEQ正向与所述SEQ反向之间的间隔序列,所述X与所述SEQ正向及所述SEQ反向均不互补。The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.
上述应用中,b4)所述的核酸分子为式(I)所示的DNA分子;In the above application, the nucleic acid molecule described in b4) is a DNA molecule represented by formula (I);
SEQ反向-X-SEQ正向(I);SEQ reverse-X-SEQ forward (I);
所述SEQ正向的序列是序列表中SEQ ID NO:2所示序列的第1位-第200位;所述SEQ反向的序列与所述SEQ正向的序列反向互补;所述X是所述SEQ正向与所述SEQ反向之间的间隔序列,所述X与所述SEQ正向及所述SEQ反向均不互补。The sequence of the SEQ forward direction is the 1st to 200th position of the sequence shown in SEQ ID NO: 2 in the sequence list; the sequence of the SEQ reverse direction is reverse complementary to the sequence of the SEQ forward direction; the X is an interval sequence between the SEQ forward direction and the SEQ reverse direction, and the X is not complementary to either the SEQ forward direction or the SEQ reverse direction.
上述应用中,b2)或b5)所述的表达盒是指能够在宿主细胞中表达IbbHLH49蛋白的DNA分子或者能够转录b3)所述的RNA的DNA分子,该DNA分子不但可包括启动目的基因转录的启动子,还可包括终止目的基因转录的终止子。进一步地,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和诱导型启动子。启动子的例子包括但不限于:花椰菜花叶病毒的组成型启动子35S;来自西红柿的创伤诱导型启动子,亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol120:979-992);来自烟草的化学诱导型启动子,发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导);西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸曱酯诱导);热休克启动子(美国专利5,187,267);四环素诱导型启动子(美国专利5,057,422);种子特异性启动子,如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7)),种子贮存蛋白质特异的启动子(例如,菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4:3047-3053)。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。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(参见,例如:Odell等人(I985)Nature 313:810;Rosenberg等人(1987)Gene,56:125;Guerineau等人(1991)Mol.Gen.Genet,262:141;Proudfoot(1991)Cell,64:671;Sanfacon等人GenesDev.,5:141;Mogen等人(1990)Plant Cell,2:1261;Munroe等人(1990)Gene,91:151;Ballad等人(1989)Nucleic Acids Res.17:7891;Joshi等人(1987)Nucleic Acid Res.,15:9627)。In the above application, the expression cassette described in b2) or b5) refers to a DNA molecule capable of expressing the IbbHLH49 protein in a host cell or a DNA molecule capable of transcribing the RNA described in b3), and the DNA molecule may include not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, 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. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent No. 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J.4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Genet, 262:141; Proudfoot (1991) Cell, 262:14 ... Cell, 2: 1261; Munroe et al. (1990) Gene, 91: 151; Ballad et al. (1989) Nucleic Acids Res. 17: 7891; Joshi et al. (1987) Nucleic Acid Res., 15: 9627).
上述应用中,b2)或b6)所述的重组载体中包括b1)或b3)所述编码基因表达盒。进一步地,可用植物表达载体构建含有所述编码基因表达盒的重组载体。所述植物表达载体可为Gateway系统载体或双元农杆菌载体等,如pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1300、pCAMBIA1300-GFP、pCAMBIA1302、pCAMBIA2300、pCAMBIA2301、pCAMBIA1301、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb。使用IbbHLH49构建重组载体时,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,如花椰菜花叶病毒(CAMV)35S启动子、泛生素基因Ubiqutin启动子(pUbi)等,它们可单独使用或与其它的植物启动子结合使用;此外,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。In the above application, the recombinant vector described in b2) or b6) includes the encoding gene expression cassette described in b1) or b3). Further, a recombinant vector containing the encoding gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-GFP, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using IbbHLH49 to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; In addition, when using the gene of the present invention to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation start region can come from a transcription start region or a structural gene.
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)、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。In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.
进一步地,b2)或b6)所述的重组载体可以为pCAMBIA1300-IbbHLH49-GFP和pCAMBIA1300-IbbHLH49-35SI-X。重组载体pCAMBIA1300-IbbHLH49-GFP的构建过程如下:(1)用限制性内切酶KpnI和BamHI双酶切载体pCAMBIA1300-GFP得到载体骨架;(2)用序列表中如SEQ ID NO:2所示的DNA分子替换pCAMBIA1300-GFP的限制性内切酶KpnI和BamHI识别序列间的片段得到的重组质粒pCAMBIA1300-IbbHLH49-GFP,重组载体pCAMBIA1300-IbbHLH49-GFP表达序列表中SEQ ID NO:1所示的IbbHLH49蛋白。重组载体pCAMBIA1300-IbbHLH49-35SI-X的构建过程如下:(1)用限制性内切酶BamH I和SalI双酶切载体pCAMBIA1300-35SI-X得到载体骨架;(2)用序列表中SEQ ID NO:2中的第1-200bp所示的DNA分子替换pCAMBIA1300-35SI-X的限制性内切酶BamHI和SalI识别序列间的片段;(3)用序列表中SEQ ID NO:2中的第1-200bp所示的DNA分子的反向互补序列替换pCAMBIA1300-35SI-X的限制性内切酶KpnI和SacI识别序列间的片段得到的重组载体pCAMBIA1300-IbbHLH49-35SI-X,重组载体pCAMBIA1300-IbbHLH49-35SI-X含有式(I)所示的DNA分子。Further, the recombinant vector described in b2) or b6) can be pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X. The construction process of the recombinant vector pCAMBIA1300-IbbHLH49-GFP is as follows: (1) the vector pCAMBIA1300-GFP is double-digested with restriction endonucleases KpnI and BamHI to obtain the vector backbone; (2) the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP is obtained by replacing the fragment between the restriction endonuclease KpnI and BamHI recognition sequences of pCAMBIA1300-GFP with the DNA molecule shown in SEQ ID NO:2 in the sequence list, and the recombinant vector pCAMBIA1300-IbbHLH49-GFP expresses the IbbHLH49 protein shown in SEQ ID NO:1 in the sequence list. The construction process of the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X is as follows: (1) the vector pCAMBIA1300-35SI-X is double-digested with restriction endonucleases BamHI and SalI to obtain a vector backbone; (2) the DNA molecule shown in the 1st to 200bp in SEQ ID NO:2 in the sequence list is used to replace the fragment between the restriction endonuclease BamHI and SalI recognition sequences of pCAMBIA1300-35SI-X; (3) the fragment between the restriction endonuclease KpnI and SacI recognition sequences of pCAMBIA1300-35SI-X is replaced with the reverse complementary sequence of the DNA molecule shown in the 1st to 200bp in SEQ ID NO:2 in the sequence list to obtain the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X, and the recombinant vector pCAMBIA1300-IbbHLH49-35SI-X contains the DNA molecule shown in formula (I).
上述应用中,b2)或b7)所述的重组微生物可以为酵母、细菌、藻类或真菌。所述细菌可为革兰氏阳性细菌或革兰氏阴性细菌。所述革兰氏阴性细菌可为根癌农杆菌(Agrobacterium tumefaciens)。所述根癌农杆菌(Agrobacterium tumefaciens)具体可为根癌农杆菌EHA105。In the above application, the recombinant microorganism described in b2) or b7) can be yeast, bacteria, algae or fungi. The bacteria can be gram-positive bacteria or gram-negative bacteria. The gram-negative bacteria can be Agrobacterium tumefaciens. The Agrobacterium tumefaciens can specifically be Agrobacterium tumefaciens EHA105.
上述应用中,所述重组微生物具体可以为EHA105/pCAMBIA1300-IbbHLH49-GFP、EHA105/pCAMBIA1300-IbbHLH49-35SI-X。EHA105/pCAMBIA1300-IbbHLH49-GFP是将重组质粒pCAMBIA1300-IbbHLH49-GFP转化根癌农杆菌EHA105得到的重组农杆菌;EHA105/pCAMBIA1300-IbbHLH49-35SI-X是将重组质粒pCAMBIA1300-IbbHLH49-35SI-X转化根癌农杆菌EHA105得到的重组农杆菌。In the above application, the recombinant microorganism may specifically be EHA105/pCAMBIA1300-IbbHLH49-GFP or EHA105/pCAMBIA1300-IbbHLH49-35SI-X. EHA105/pCAMBIA1300-IbbHLH49-GFP is a recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP into Agrobacterium tumefaciens EHA105; EHA105/pCAMBIA1300-IbbHLH49-35SI-X is a recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X into Agrobacterium tumefaciens EHA105.
上述应用中,b2)或b9)所述的转基因植物组织可来源于根、茎、叶、花、果实、种子、花粉、胚和花药。In the above applications, the transgenic plant tissue described in b2) or b9) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.
上述应用中,b2)或b10)所述的转基因植物器官可为转基因植物的根、茎、叶、花、果实和种子。In the above application, the transgenic plant organ described in b2) or b10) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.
第三方面,本发明提供上述任一所述的IbbHLH49蛋白相关的生物材料。In a third aspect, the present invention provides any of the above-mentioned IbbHLH49 protein-related biological materials.
第四方面,本发明提供一种培育转基因植株的方法,包括:调控出发植株中IbbHLH49蛋白编码基因的表达,得到转基因植株,所述转基因植株的块根大小、块根产量、淀粉产量、支链淀粉比例中的至少一项与出发植株不同。In a fourth aspect, the present invention provides a method for cultivating transgenic plants, comprising: regulating the expression of the IbbHLH49 protein encoding gene in a starting plant to obtain a transgenic plant, wherein at least one of the root tuber size, root tuber yield, starch yield, and amylopectin ratio of the transgenic plant is different from that of the starting plant.
本发明提供一种IbbHLH49蛋白、编码基因的目前未知的功能及其用途,通过调控该IbbHLH49蛋白在甘薯中的含量或表达得到转IbbHLH49基因的甘薯植株,经实验表明,相比野生型甘薯植株,过表达IbbHLH49基因的甘薯植株具有更大的块根大小和更高的块根产量,且有关淀粉合成的相关酶的表达量增高,淀粉含量以及支链淀粉比例提高;而干扰表达IbbHLH49基因的甘薯植株具有更小的块根大小和更低的块根产量,且有关淀粉合成的相关酶的表达量降低,淀粉含量以及支链淀粉比例降低;说明IbbHLH49蛋白及编码基因对调控植物经济性状起着重要的作用。本发明所提供的IbbHLH49蛋白及其编码基因在植物经济性状中具有重要的应用价值,在农业领域具有一定的应用空间和市场前景。The present invention provides an IbbHLH49 protein, a currently unknown function of a coding gene and its use. By regulating the content or expression of the IbbHLH49 protein in sweet potatoes, a sweet potato plant transfected with the IbbHLH49 gene is obtained. Experiments show that, compared with wild-type sweet potato plants, sweet potato plants overexpressing the IbbHLH49 gene have larger root size and higher root yield, and the expression of related enzymes related to starch synthesis increases, and the starch content and the proportion of amylopectin increase; while the sweet potato plants with interference expression of the IbbHLH49 gene have smaller root size and lower root yield, and the expression of related enzymes related to starch synthesis decreases, and the starch content and the proportion of amylopectin decrease; indicating that the IbbHLH49 protein and the coding gene play an important role in regulating plant economic traits. The IbbHLH49 protein and the coding gene provided by the present invention have important application value in plant economic traits, and have certain application space and market prospects in the agricultural field.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为转基因甘薯植株的PCR鉴定结果;其中,A为过表达IbbHLH49基因甘薯阳性植株的PCR鉴定结果,B为干扰表达IbbHLH49基因甘薯阳性植株的PCR鉴定结果;M为DNA分子Marker,W为阴性对照水,P为阳性对照质粒(pCAMBIA1300-IbbHLH49-GFP和pCAMBIA1300-IbbHLH49-35SI-X),WT为野生型甘薯植株的基因组DNA,OE49-1~OE49-13为过表达IbbHLH49基因甘薯阳性植株的基因组DNA,Ri49-1~Ri49-14为干扰表达IbbHLH49基因甘薯阳性植株的基因组DNA;Figure 1 is the PCR identification result of transgenic sweet potato plants; wherein A is the PCR identification result of sweet potato positive plants overexpressing the IbbHLH49 gene, and B is the PCR identification result of sweet potato positive plants interfering with the expression of the IbbHLH49 gene; M is a DNA molecule marker, W is a negative control water, P is a positive control plasmid (pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X), WT is the genomic DNA of wild-type sweet potato plants, OE49-1 to OE49-13 are the genomic DNA of sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-1 to Ri49-14 are the genomic DNA of sweet potato positive plants interfering with the expression of the IbbHLH49 gene;
图2为IbbHLH49基因在转IbbHLH49基因甘薯阳性植株和野生型甘薯植株中的表达量分析;其中,A为过表达IbbHLH49基因甘薯阳性植株中IbbHLH49基因的相对表达量,B为干扰表达IbbHLH49基因甘薯阳性植株中IbbHLH49基因的相对表达量,WT为野生型甘薯植株的cDNA,OE49-1~OE49-13和Ri49-1~Ri49-14为转IbbHLH49基因甘薯阳性植株的cDNA;Figure 2 is an expression analysis of the IbbHLH49 gene in sweet potato positive plants transgenic for IbbHLH49 and wild-type sweet potato plants; wherein A is the relative expression of the IbbHLH49 gene in sweet potato positive plants overexpressing the IbbHLH49 gene, B is the relative expression of the IbbHLH49 gene in sweet potato positive plants interfering with the expression of the IbbHLH49 gene, WT is the cDNA of the wild-type sweet potato plant, OE49-1 to OE49-13 and Ri49-1 to Ri49-14 are the cDNA of sweet potato positive plants transgenic for IbbHLH49;
图3为转IbbHLH49基因甘薯植株和野生型甘薯植株的块根照片;其中,WT为野生型甘薯植株;OE49-9和OE49-12为过表达IbbHLH49基因甘薯阳性植株,Ri49-7和Ri49-14为干扰表达IbbHLH49基因甘薯阳性植株;图中比例尺为10cm;Figure 3 is a photo of the root tubers of the IbbHLH49 transgenic sweet potato plants and the wild-type sweet potato plants; wherein WT is the wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene; the scale bar in the figure is 10 cm;
图4为转IbbHLH49基因甘薯植株和野生型甘薯植株块根的石蜡切片;其中,WT为野生型甘薯植株;OE49-9和OE49-12为过表达IbbHLH49基因甘薯阳性植株,Ri49-7和Ri49-14为干扰表达IbbHLH49基因甘薯阳性植株;图中细胞内颗粒为淀粉粒,比例尺为100μm;Figure 4 is a paraffin section of the root tubers of the IbbHLH49 transgenic sweet potato plants and the wild-type sweet potato plants; wherein WT is the wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants interfering with the expression of the IbbHLH49 gene; the intracellular granules in the figure are starch granules, and the scale bar is 100 μm;
图5为转IbbHLH49基因甘薯植株和野生型甘薯植株块根的产量、淀粉含量及支链淀粉比例测定结果;其中,A为转IbbHLH49基因甘薯植株和野生型甘薯植株块根的产量测定结果,B为转IbbHLH49基因甘薯植株和野生型甘薯植株淀粉含量测定结果,C为转IbbHLH49基因甘薯植株和野生型甘薯植株支链淀粉比例测定结果;WT为野生型甘薯植株;OE49-9和OE49-12为过表达IbbHLH49基因甘薯阳性植株,Ri49-7和Ri49-14为干扰表达IbbHLH49基因甘薯阳性植株;Figure 5 shows the yield, starch content and amylopectin ratio of the tuberous roots of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants; wherein A is the yield of the tuberous roots of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants, B is the starch content of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants, and C is the amylopectin ratio of the IbbHLH49-transgenic sweet potato plants and the wild-type sweet potato plants; WT is a wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene;
图6为转IbbHLH49基因甘薯植株和野生型甘薯植株块根中AGPase与SBE活性测定结果;其中,A为转IbbHLH49基因甘薯植株和野生型甘薯植株块根中AGPase活性测定结果,B为转IbbHLH49基因甘薯植株和野生型甘薯植株块根中SBE活性测定结果;WT为野生型甘薯植株;OE49-9和OE49-12为过表达IbbHLH49基因甘薯阳性植株,Ri49-7和Ri49-14为干扰表达IbbHLH49基因甘薯阳性植株。Figure 6 shows the results of AGPase and SBE activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants; wherein, A is the result of AGPase activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants, and B is the result of SBE activity determination in the tuberous roots of sweet potato plants transgenic for IbbHLH49 gene and wild-type sweet potato plants; WT is a wild-type sweet potato plant; OE49-9 and OE49-12 are sweet potato positive plants overexpressing the IbbHLH49 gene, and Ri49-7 and Ri49-14 are sweet potato positive plants with interference expression of the IbbHLH49 gene.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明中的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例,它们不应该理解成对本发明的限制。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。在本发明的描述中,需要理解的是,所用到的术语仅仅是用于描述的目的,而不能理解为指示或暗示相对重要性。In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.
以下实施例中,甘薯品系‘H283’为本实验室保存。公众可从中国农业大学甘薯生物学与生物技术重点实验室获得,以重复本实验,不可作为其它用途使用。In the following examples, the sweet potato line 'H283' is stored in this laboratory. The public can obtain it from the Key Laboratory of Sweet Potato Biology and Biotechnology of China Agricultural University to repeat this experiment, but it cannot be used for other purposes.
甘薯品种“栗子香”为本实验室保存,记载于如下文献中:“薛璐瑶.甘薯IbbHLH118、IbNF-YA1和IbNF-YA10基因的功能及分子调控机制研究,博士学位论文,中国农业大学,2022.”。公众可从中国农业大学甘薯生物学与生物技术重点实验室获得,以重复本实验,不可作为其它用途使用。The sweet potato variety “Lizixiang” is preserved in this laboratory and is recorded in the following document: “Xue Luyao. Function and molecular regulation mechanism of sweet potato IbbHLH118, IbNF-YA1 and IbNF-YA10 genes, PhD dissertation, China Agricultural University, 2022.” The public can obtain it from the Key Laboratory of Sweet Potato Biology and Biotechnology of China Agricultural University to repeat this experiment, but it cannot be used for other purposes.
克隆载体pMD19-T为宝生物工程(大连)公司产品,产品目录号为6013。The cloning vector pMD19-T is a product of Takara Biotechnology (Dalian) Co., Ltd., with the product catalog number being 6013.
载体pCAMBIA1300-GFP记载于如下文献中:Luo HS,Meng DX,Liu HB,Xie MJ,YinCF,Liu F,Dong ZB,Jin WW.Ectopic Expression of the Transcriptional Regulatorsilky3Causes Pleiotropic Meristem and Sex Determination Defects in MaizeInflorescences.Plant Cell.2020,32:3750-3773;公众可从申请人处获得上述生物材料,以重复本实验,不可作为其它用途使用。The vector pCAMBIA1300-GFP is recorded in the following literature: Luo HS, Meng DX, Liu HB, Xie MJ, Yin CF, Liu F, Dong ZB, Jin WW. Ectopic Expression of the Transcriptional Regulatorsilky3Causes Pleiotropic Meristem and Sex Determination Defects in MaizeInflorescences. Plant Cell.2020,32:3750-3773; the public can obtain the above biological materials from the applicant to repeat this experiment, but they cannot be used for other purposes.
pCAMBIA1300-35SI-X购于武汉转导实验室有限公司,在pCAMBIA1300的基础上改造,在MCS处加35S启动子+Intro+NOS终止子,正反向序列分别插入,形成发卡结构。pCAMBIA1300-35SI-X was purchased from Wuhan Transduction Laboratory Co., Ltd. and was modified based on pCAMBIA1300. 35S promoter + Intro + NOS terminator were added to the MCS, and the forward and reverse sequences were inserted respectively to form a hairpin structure.
植物总RNA提取试剂盒为全式金(TransGen Biotech,北京)的Transzol植物总RNA提取试剂盒(目录号:ET111)。The plant total RNA extraction kit was the Transzol Plant Total RNA Extraction Kit (Catalog No.: ET111) from TransGen Biotech, Beijing.
HiFiScript gDNA Removal RT MasterMix反转录试剂盒(目录号:CW2020M)为康为世纪生物科技有限公司产品。HiFiScript gDNA Removal RT MasterMix Reverse Transcription Kit (Catalog Number: CW2020M) is a product of Kangwei Century Biotechnology Co., Ltd.
无缝克隆酶来自于翌圣生物科技(上海)股份有限公司的HieffPlus OneStep Cloning Kit试剂盒(10911ES)。Seamless cloning enzyme comes from Hieff of Yisheng Biotechnology (Shanghai) Co., Ltd. Plus OneStep Cloning Kit (10911ES).
下述实施例采用SPSS26.0统计软件对数据进行处理,实验结果以平均值±标准偏差表示,采用Student’s t-test检验,*表示具有显著性差异(P<0.05),**表示具有极显著性差异(P<0.01)。下述实施例中,如无特殊说明,处理组均设置三个重复。The following examples were processed using SPSS 26.0 statistical software, and the experimental results were expressed as mean ± standard deviation, and Student's t-test was used. * indicates significant difference (P < 0.05), and ** indicates extremely significant difference (P < 0.01). In the following examples, unless otherwise specified, three replicates were set for each treatment group.
实施例1、IbbHLH49基因的获得Example 1. Acquisition of IbbHLH49 gene
1、cDNA模板的获得1. Obtaining cDNA template
将离体培养4周的H283植株液氮冷冻研磨,用植物总RNA提取试剂盒提取RNA,并用HiFiScript gDNA Removal RT MasterMix反转录试剂盒反转录出第一链cDNA。H283 plants cultured in vitro for 4 weeks were frozen in liquid nitrogen and ground, RNA was extracted using a plant total RNA extraction kit, and the first-strand cDNA was reverse transcribed using a HiFiScript gDNA Removal RT MasterMix reverse transcription kit.
2、通过Sweetpotato Garden数据库(http://sweetpotato-garden.kazusa.or.jp/index.html)对IbbHLH49基因进行Blastx分析,找出完整且与EST序列相似性高的基因ORF。2. Perform Blastx analysis on the IbbHLH49 gene using the Sweetpotato Garden database (http://sweetpotato-garden.kazusa.or.jp/index.html) to find the complete gene ORF with high similarity to the EST sequence.
3、设计并人工合成引物O49-F和O49-R,以步骤1获得的cDNA为模板,进行PCR扩增,获得约1566bp的PCR扩增产物,和克隆载体pMD19-T连接后得到重组载体并测序。引物序列如下:3. Design and synthesize primers O49-F and O49-R, use the cDNA obtained in step 1 as a template, perform PCR amplification, obtain a PCR amplification product of about 1566 bp, connect it with the cloning vector pMD19-T to obtain a recombinant vector and sequence it. The primer sequences are as follows:
O49-F:5′-ATGGATAAGGGTGGCAAGGAT-3′O49-F:5′-ATGGATAAGGGTGGCAAGGAT-3′
O49-R:5′-TCAGGGCTCAGATTTCATATGG-3′O49-R:5′-TCAGGGCTCAGATTTCATATGG-3′
结果表明,PCR扩增产物的核苷酸序列如SEQ ID NO:2所示,将该序列所示的基因命名为IbbHLH49基因,其编码的蛋白命名为IbbHLH49蛋白或蛋白质IbbHLH49,该蛋白的氨基酸序列如SEQ ID NO:1所示。The results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO:2, the gene shown in the sequence was named IbbHLH49 gene, the protein encoded by it was named IbbHLH49 protein or protein IbbHLH49, and the amino acid sequence of the protein was as shown in SEQ ID NO:1.
实施例2、IbbHLH49蛋白在提高植物淀粉含量中的应用Example 2: Application of IbbHLH49 protein in increasing starch content in plants
一、重组质粒pCAMBIA1300-IbbHLH49-GFP的构建1. Construction of recombinant plasmid pCAMBIA1300-IbbHLH49-GFP
1、人工合成SEQ ID NO:2自5′末端起第1至1563位所示的双链DNA分子。以该双链DNA分子为模板,以1300-F-KpnI和1300-R-BamHI为引物进行PCR扩增,得到N端含有限制性内切酶KpnI和C端含有限制性内切酶BamHI的双链DNA分子片段1。1. Artificially synthesize the double-stranded DNA molecule shown in positions 1 to 1563 from the 5′ end of SEQ ID NO: 2. Use the double-stranded DNA molecule as a template and 1300-F-KpnI and 1300-R-BamHI as primers to perform PCR amplification to obtain a double-stranded DNA molecule fragment 1 containing restriction endonuclease KpnI at the N-terminus and restriction endonuclease BamHI at the C-terminus.
1300-F-KpnI:5′-ACGGGGGACGAGCTCGGTACCATGGATAAGGGTGGCAAGGAT-3'(下划线为限制性内切酶KpnI的识别序列);1300-F-KpnI: 5′-ACGGGGGACGAGCTC GGTACC ATGGATAAGGGTGGCAAGGAT-3′ (the underline is the recognition sequence of restriction endonuclease KpnI);
1300-R-BamHI:5′-CATGTCGACTCTAGAGGATCCGGGCTCAGATTTCATATGG-3'(下划线为限制性内切酶BamHI的识别序列)。1300-R-BamHI: 5′-CATGTCGACTCTAGA GGATCC GGGCTCAGATTTCATATGG-3′ (the underline is the recognition sequence of the restriction endonuclease BamHI).
2、用限制性内切酶KpnI和BamHI双酶切载体pCAMBIA1300-GFP,回收约10444bp的载体骨架2。2. Double-digest the vector pCAMBIA1300-GFP with restriction endonucleases KpnI and BamHI to recover the vector backbone 2 of about 10444 bp.
3、将片段1与载体骨架2使用无缝克隆酶进行同源重组连接,得到重组质粒pCAMBIA 1300-IbbHLH49-GFP。3. Fragment 1 and vector backbone 2 were homologously recombined using seamless cloning enzyme to obtain the recombinant plasmid pCAMBIA 1300-IbbHLH49-GFP.
根据测序结果,对重组质粒pCAMBIA1300-IbbHLH49-GFP进行结构描述如下:用核苷酸序列是SEQ ID No:1的DNA分子替换质粒pCAMBIA1300-GFP的限制性内切酶KpnI和BamHI识别序列间的小片段,保持pCAMBIA1300-GFP的其他核苷酸序列不变,得到重组表达载体pCAMBIA1300-IbbHLH49-GFP,重组表达载体pCAMBIA1300-IbbHLH49-GFP表达SEQ IDNO:1所示的IbbHLH49蛋白。According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-IbbHLH49-GFP was described as follows: a small fragment between the restriction endonuclease KpnI and BamHI recognition sequences of the plasmid pCAMBIA1300-GFP was replaced with a DNA molecule whose nucleotide sequence was SEQ ID No: 1, while keeping the other nucleotide sequences of pCAMBIA1300-GFP unchanged, to obtain the recombinant expression vector pCAMBIA1300-IbbHLH49-GFP, and the recombinant expression vector pCAMBIA1300-IbbHLH49-GFP expressed the IbbHLH49 protein shown in SEQ ID NO: 1.
二、重组质粒pCAMBIA1300-IbbHLH49-35SI-X的构建2. Construction of recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X
1、人工合成核苷酸序列如SEQ ID NO:2所示的双链DNA分子。以该双链DNA分子为模板,选取基因的1-200bp的序列作为干扰序列,以RNAi-F1-BamHI和RNAi-R1-SalI为引物进行PCR扩增,得到正义链,以RNAi-F2-SacI和RNAi-R2-KpnI为引物进行PCR扩增,得到反义链。1. Artificially synthesize a double-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2. Using the double-stranded DNA molecule as a template, select a 1-200 bp sequence of the gene as an interference sequence, use RNAi-F1-BamHI and RNAi-R1-SalI as primers to perform PCR amplification to obtain the sense strand, and use RNAi-F2-SacI and RNAi-R2-KpnI as primers to perform PCR amplification to obtain the antisense strand.
RNAi-F1-BamHI:5'-ACGGGGGACTCTAGTGGATCCATGGATAAGGGTGGCAAGGATGAG-3'(下划线为限制性内切酶BamHI的识别序列);RNAi-F1-BamHI: 5′-ACGGGGGACTCTAGT GGATCC ATGGATAAGGGTGGCAAGGATGAG-3′ (underlined is the recognition sequence of restriction endonuclease BamHI);
RNAi-R1-SalI:5'-AATTACCCTCTACTAGTCGACGAACTTGCAGATTGGTCCCAAACAT-3'(下划线为限制性内切酶SalI的识别序列);RNAi-R1-SalI: 5′-AATTACCCTCTACTA GTCGAC GAACTTGCAGATTGGTCCCAAACAT-3′ (underlined is the recognition sequence of restriction endonuclease SalI);
RNAi-F2-SacI:5'-CGATCGGGGAAATTCGAGCTCATGGATAAGGGTGGCAAGGATGAG-3'(下划线为限制性内切酶SacI的识别序列);RNAi-F2-SacI: 5′-CGATCGGGGAAATTC GAGCTC ATGGATAAGGGTGGCAAGGATGAG-3′ (the underline is the recognition sequence of restriction endonuclease SacI);
RNAi-R2-KpnI:5'-GTTAGGATTTCTAGAGGTACCGAACTTGCAGATTGGTCCCAAACAT-3'(下划线为限制性内切酶KpnI的识别序列)。RNAi-R2-KpnI: 5′-GTTAGGATTTCTAGA GGTACC GAACTTGCAGATTGGTCCCAAACAT-3′ (the underline is the recognition sequence of the restriction endonuclease KpnI).
2、将上述正义链序列按5'-3′方向克隆进载体pCAMBIA1300-35SI-X的多克隆位点1(5'-BamHI-SalI-3')处;2. Clone the above positive chain sequence into the multiple cloning site 1 (5'-BamHI-SalI-3') of the vector pCAMBIA1300-35SI-X in the 5'-3' direction;
3、测序验证成功后,以上述构建成功的克隆为目标载体,再次将上述反义链序列按5'-3'方向克隆进载多克隆位点2(5'-KpnI-SacI-3')处,测序验证第二次插入成功,即构建完成pCAMBIA1300-IbbHLH49-35SI-X质粒。3. After successful sequencing verification, the above successfully constructed clone was used as the target vector, and the antisense chain sequence was cloned again into the multiple cloning site 2 (5'-KpnI-SacI-3') in the 5'-3' direction. Sequencing verified that the second insertion was successful, and the pCAMBIA1300-IbbHLH49-35SI-X plasmid was constructed.
测序结果表明:重组质粒pCAMBIA1300-IbbHLH49-35SI-X含有式(I)所示的DNA分子:The sequencing results showed that the recombinant plasmid pCAMBIA1300-IbbHLH49-35SI-X contained the DNA molecule represented by formula (I):
SEQ反向-X-SEQ正向(I);SEQ reverse-X-SEQ forward (I);
所述SEQ正向的序列是SEQ ID NO:2的第1-200位;所述SEQ反向的序列与所述SEQ正向的序列反向互补;所述X是所述SEQ正向与所述SEQ反向之间的间隔序列,所述X与所述SEQ正向及所述SEQ反向均不互补。The SEQ forward sequence is positions 1-200 of SEQ ID NO: 2; the SEQ reverse sequence is reverse complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to either the SEQ forward or the SEQ reverse.
三、转基因甘薯植株的获得3. Obtaining transgenic sweet potato plants
1、将重组质粒pCAMBIA1300-IbbHLH49-GFP和pCAMBIA1300-IbbHLH49-35SI-X分别转化根癌农杆菌EHA105,得到重组农杆菌,将重组农杆菌分别命名为EHA105/pCAMBIA1300-IbbHLH49-GFP和EHA105/pCAMBIA1300-IbbHLH49-35SI-X。1. The recombinant plasmids pCAMBIA1300-IbbHLH49-GFP and pCAMBIA1300-IbbHLH49-35SI-X were transformed into Agrobacterium tumefaciens EHA105, respectively, to obtain recombinant Agrobacterium, and the recombinant Agrobacterium were named EHA105/pCAMBIA1300-IbbHLH49-GFP and EHA105/pCAMBIA1300-IbbHLH49-35SI-X, respectively.
2、剥取长约0.5mm的栗子香的茎尖分生组织,置于含有2.0mg L-1 2,4-D的MS固体培养基上,27±1℃黑暗诱导培养4~6周,得到胚性愈伤组织。然后将胚性愈伤组织转移至含有2.0mg L-1 2,4-D的MS液体培养基中振荡培养扩繁3~4周后,得到直径为0.7~1.3mm的可以用于转化的胚性细胞悬浮培养系。2. Peel off the stem tip meristem of Lizixiang about 0.5 mm long, place it on MS solid medium containing 2.0 mg L -1 2,4-D, and induce culture in the dark at 27±1℃ for 4 to 6 weeks to obtain embryonic callus. Then transfer the embryonic callus to MS liquid medium containing 2.0 mg L -1 2,4-D and culture for 3 to 4 weeks with shaking to obtain embryonic cell suspension culture lines with a diameter of 0.7 to 1.3 mm that can be used for transformation.
3、农杆菌的培养:在抗性平板上活化农杆菌菌液,挑取单菌落接种于20mL的已加入对应抗生素的LB液体培养基中,28℃下200rpm振荡过夜培养,直到OD600值在0.5~0.7范围内。5000rpm离心并去上清液,用等体积的LB培养基清洗菌体两遍,等体积的含有2.0mgL-1 2,4-D的MS液体培养基清洗菌体一遍后,用等体积的含有2.0mg L-1 2,4-D的MS液体培养基重悬菌体。3. Cultivation of Agrobacterium: Activate Agrobacterium culture on a resistant plate, pick a single colony and inoculate it into 20 mL of LB liquid medium to which the corresponding antibiotic has been added, and culture it overnight at 28°C with shaking at 200 rpm until the OD 600 value is within the range of 0.5 to 0.7. Centrifuge at 5000 rpm and remove the supernatant, wash the cells twice with an equal volume of LB medium, wash the cells once with an equal volume of MS liquid medium containing 2.0 mg L -1 2,4-D, and resuspend the cells with an equal volume of MS liquid medium containing 2.0 mg L -1 2,4-D.
4、侵染与共培养:将栗子香胚性细胞团悬浮于制备好的农杆菌菌液中,摇动片刻使其充分散开,以使胚性细胞团充分接触菌液,静置5min,然后用吸管将菌液吸出,将侵染过的胚性细胞团移至含有30mg L-1AS和2.0mg L-1 2,4-D的MS固体培养基上进行共培养,固体培养基上铺1层普通滤纸,27±1℃,暗培养3天。4. Infection and co-cultivation: Suspend the chestnut-fragrant embryonic cell mass in the prepared Agrobacterium bacterial solution, shake it for a while to fully disperse it so that the embryonic cell mass can fully contact the bacterial solution, let it stand for 5 minutes, then use a pipette to suck out the bacterial solution, and move the infected embryonic cell mass to MS solid culture medium containing 30 mg L -1 AS and 2.0 mg L -1 2,4-D for co-cultivation. Cover the solid culture medium with a layer of ordinary filter paper and culture it in the dark at 27±1℃ for 3 days.
5、选择培养与拟转基因植株的再生:将共培养3天后的胚性细胞团用刀片轻轻刮下,用含有500mg L-1头孢霉素和2.0mg L-1 2,4-D的MS液体培养基洗涤3次,再转入含有100mg L-1头孢霉素和2.0mg L-1 2,4-D的MS液体培养基中,延迟培养1周。然后将液体培养基尽量吸干,摆在铺有1~2层滤纸并含有5mg L-1潮霉素、100mg L-1头孢霉素和2.0mg L-12,4-D的固体MS培养基上进行选择培养,培养条件为27±1℃,暗培养。2周后将生长状态良好的愈伤组织转移至铺有1层滤纸并含有11mg L-1潮霉素、100mg L-1头孢霉素和2.0mg L-12,4-D的固体MS培养基上进行选择培养。其后每2周继代1次。将在含有11mg L-1潮霉素、100mg L-1头孢霉素和2.0mg L-1 2,4-D的固体MS培养基上选择培养8周后形成的抗性愈伤组织转移至含有100mg L-1头孢霉素和1mg L-1ABA的MS固体培养基上,培养条件为27±1℃、每日13h、3000lux的光照,诱导形成体细胞胚。诱导2~4周,将成熟的体细胞胚转移到MS固体培养基上,培养条件为27±1℃、每日13h、3000lux的光照,培养4~8周后,再生出完整的拟转基因植株。将再生的小植株切下,继代培养在MS固体培养基上,培养条件为27±1℃、每日13h、3000lux的光照,每6周继代1次5. Selection culture and regeneration of transgenic plants: After 3 days of co-culture, the embryonic cell mass was gently scraped off with a blade, washed 3 times with MS liquid medium containing 500 mg L -1 cephalosporin and 2.0 mg L -1 2,4-D, and then transferred to MS liquid medium containing 100 mg L -1 cephalosporin and 2.0 mg L -1 2,4-D, and cultured for 1 week. Then the liquid medium was dried as much as possible and placed on a solid MS medium covered with 1 to 2 layers of filter paper and containing 5 mg L -1 hygromycin, 100 mg L -1 cephalosporin and 2.0 mg L -1 2,4-D for selection culture. The culture conditions were 27±1℃ and dark culture. After 2 weeks, the callus with good growth status was transferred to a solid MS medium covered with 1 layer of filter paper and containing 11 mg L -1 hygromycin, 100 mg L -1 cephalosporin and 2.0 mg L -1 2,4-D for selection culture. Subculture once every 2 weeks thereafter. The resistant callus formed after 8 weeks of selective culture on solid MS medium containing 11 mg L -1 hygromycin, 100 mg L -1 cephalosporin and 2.0 mg L -1 2,4-D was transferred to MS solid medium containing 100 mg L -1 cephalosporin and 1 mg L -1 ABA. The culture conditions were 27±1℃, 13h per day, and 3000lux of light to induce the formation of somatic embryos. After 2 to 4 weeks of induction, the mature somatic embryos were transferred to MS solid medium. The culture conditions were 27±1℃, 13h per day, and 3000lux of light. After 4 to 8 weeks of culture, complete transgenic plants were regenerated. The regenerated plantlets were cut and subcultured on MS solid medium. The culture conditions were 27±1℃, 13h per day, and 3000lux of light. Subculture once every 6 weeks.
6、转基因植株的鉴定:使用PCR检测和qRT-PCR检测相结合的方法。6. Identification of transgenic plants: using a combination of PCR and qRT-PCR detection.
1)PCR检测方法如下:1) The PCR detection method is as follows:
提取野生型甘薯和拟转IbbHLH49基因甘薯株系的DNA,进行PCR鉴定。使用pCAMBIA1300-IbbHLH66-GFP和pCAMBIA1300-IbbHLH49-35SI-X重组质粒为阳性对照,水和野生型WT为阴性对照,引物如下:DNA of wild-type sweet potato and the sweet potato lines to be transformed with IbbHLH49 gene were extracted for PCR identification. pCAMBIA1300-IbbHLH66-GFP and pCAMBIA1300-IbbHLH49-35SI-X recombinant plasmids were used as positive controls, water and wild-type WT were used as negative controls, and the primers were as follows:
OE49-F:5'-AGGAAGTTCATTTCATTTGGAGA-3'OE49-F: 5'-AGGAAGTTTCATTTCATTTGGAGA-3'
OE49-R:5'-TCAGGGCTCAGATTTCATATGG-3'OE49-R:5'-TCAGGGCTCAGATTTCATATGG-3'
Ri49-F:5'-CTTCGCAAGACCCTTCCTCT-3'Ri49-F: 5'-CTTCGCAAGACCCTTCCTCT-3'
Ri49-R:5'-TCGGGGAAATTCGAGCTC-3'Ri49-R: 5'-TCGGGGAAATTCGAGCTC-3'
将扩增得到的PCR产物在1%(w/v)的琼脂糖凝胶中进行电泳分离,PCR阳性植株应该具有一条特异的1612bp和623bp的电泳条带,记录下PCR阳性植株的株系号。The amplified PCR products were separated by electrophoresis in 1% (w/v) agarose gel. The PCR-positive plants should have a specific electrophoresis band of 1612 bp and 623 bp. The strain number of the PCR-positive plants was recorded.
结果如图1所示,结果显示只有阳性对照和拟转基因甘薯植株OE49-1~OE49-13在1612bp附近出现了电泳条带,Ri49-1~Ri49-14在623bp附近出现了电泳条带,而野生型甘薯和阴性对照没有出现条带,初步确定了本发明获得了甘薯转基因阳性植株OE49-1~OE49-13和Ri49-1~Ri49-14。The results are shown in Figure 1, which show that only the positive control and the pseudo-transgenic sweet potato plants OE49-1 to OE49-13 showed electrophoresis bands near 1612 bp, and Ri49-1 to Ri49-14 showed electrophoresis bands near 623 bp, while the wild-type sweet potato and the negative control did not show any bands. It was preliminarily determined that the present invention obtained the sweet potato transgenic positive plants OE49-1 to OE49-13 and Ri49-1 to Ri49-14.
2)qRT-PCR2) qRT-PCR
提取阳性甘薯植株的RNA,反转录得到cDNA,进行qRT-PCR,以野生型为对照。RNA was extracted from positive sweet potato plants, reverse transcribed to obtain cDNA, and qRT-PCR was performed, with the wild type as the control.
Ibactin基因为内参:Ibactin gene as internal reference:
Ibactin-F:5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′Ibactin-F: 5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′
Ibactin-R:5′-GGAAAATTAGAAGCACTTCCTGTGAAC-3′Ibactin-R: 5′-GGAAAATTAGAAGCACTTCCTGTGAAC-3′
IbbHLH49引物序列为:The IbbHLH49 primer sequence is:
qRT49-F:5′-ATCCACATTCGGGCTAGGAG-3′qRT49-F: 5′-ATCCACATTCGGGCTAGGAG-3′
qRT49-R:5′-CGGGTTTACTGTTGCAAGCT-3′qRT49-R: 5′-CGGGTTTACTGTTGCAAGCT-3′
结果如图2所示,结果表明,IbbHLH49基因的表达量在过表达转基因甘薯植株中显著上调,在干扰表达转基因甘薯植株中显著下调。选取2个过表达转基因甘薯植株(OE49-9和OE49-12)和2个干扰表达转基因甘薯植株(Ri49-7和Ri49-14)扩繁,获得IbbHLH49 T1代转基因甘薯株系OE49-9、OE49-12、Ri49-7和Ri49-14,进行后续试验。The results are shown in Figure 2. The results showed that the expression level of the IbbHLH49 gene was significantly upregulated in the overexpression transgenic sweet potato plants and significantly downregulated in the interference expression transgenic sweet potato plants. Two overexpression transgenic sweet potato plants (OE49-9 and OE49-12) and two interference expression transgenic sweet potato plants (Ri49-7 and Ri49-14) were selected for propagation to obtain the IbbHLH49 T1 generation transgenic sweet potato lines OE49-9, OE49-12, Ri49-7 and Ri49-14 for subsequent experiments.
四、过表达IbbHLH49转基因甘薯植株淀粉含量鉴定4. Identification of starch content in transgenic sweet potato plants overexpressing IbbHLH49
WT为野生型栗子香,转基因甘薯为IbbHLH49 T1代转基因甘薯株系OE49-9、OE49-12、Ri49-7和Ri49-14的植株。WT is the wild type Li Zixiang, and the transgenic sweet potato is the plants of the IbbHLH49 T 1 generation transgenic sweet potato lines OE49-9, OE49-12, Ri49-7 and Ri49-14.
1、IbbHLH49转基因甘薯块根的观察1. Observation of IbbHLH49 transgenic sweet potato root tubers
取种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根,清洗干净后观察。Sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in an isolated field for 4 months were taken, cleaned and observed.
结果如图3所示,过表达转基因植株甘薯块根明显大于野生型植株的甘薯块根,而干扰表达转基因植株甘薯块根则明显小于野生型植株的甘薯块根。The results are shown in Figure 3. The sweet potato tubers of the overexpressing transgenic plants were significantly larger than those of the wild-type plants, while the sweet potato tubers of the interference-expressing transgenic plants were significantly smaller than those of the wild-type plants.
2、IbbHLH49转基因甘薯块根的石蜡切片观察2. Paraffin section observation of IbbHLH49 transgenic sweet potato root tubers
取种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根,清洗干净后,取块根中间部位制作石蜡切片。方法如下:The sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months were taken, cleaned, and the middle part of the tubers was taken to make paraffin sections. The method is as follows:
1)将材料浸泡在FAA固定液固定48小时以上。1) Immerse the material in FAA fixative for more than 48 hours.
2)将固定好的材料进行脱水处理,脱水时依次用50%、70%、80%、95%、100%的梯度浓度酒精,每个浓度处理30分钟。2) Dehydrate the fixed material using gradient concentrations of alcohol: 50%, 70%, 80%, 95%, and 100%, with each concentration treated for 30 minutes.
3)用二甲苯与100%酒精1:1混合后处理2次样品,每次30分钟,使植物细胞脱色变得透明。3) Treat the sample twice with a mixture of xylene and 100% alcohol in a ratio of 1:1, each time for 30 minutes, to decolorize the plant cells and make them transparent.
4)将处理好的材料放入石蜡中30分钟,进行2次处理,每次40℃烘箱敞口过夜。4) Place the treated material in paraffin for 30 minutes, and perform two treatments, each time in an oven at 40°C uncovered overnight.
5)将熔化的石蜡倒入包埋器中,用热镊子把材料迅速移入包埋器中,并摆好位置,观察面向下摆放。5) Pour the melted paraffin into the embedding container, use hot tweezers to quickly move the material into the embedding container, and position it with the observation side facing downward.
6)随后将包埋器移入水面,缓慢浸入水中,等石蜡完全凝结以后,从水中取出,并注明包埋块的名称和日期等。6) Then move the embedding device to the water surface and slowly immerse it in the water. After the paraffin is completely solidified, take it out of the water and mark the name and date of the embedded block.
7)对包埋的材料进行修块、粘块、整修后,用少许石蜡将蜡块固定在木块上,然后将木块固定在切片机上,并调节切片刀固定器或材料固定器,使切片刀与蜡块靠近,调节切片厚度为2mm,进行切片,并将切片用毛笔移在铺有黑纸的盘中。7) After trimming, gluing and finishing the embedded material, fix the wax block on the wood block with a little paraffin wax, then fix the wood block on the microtome, and adjust the slicer holder or material holder to bring the slicer close to the wax block, adjust the slice thickness to 2 mm, slice, and move the slices with a brush to a plate covered with black paper.
8)将切好的蜡带,置于烘片台上,使切片展开烫平,后取一小滴甘油涂在载玻片上,用小拇指外侧把它涂成薄层,涂抹面积要超过蜡带所占面积,涂抹得越薄、越均匀越好37℃烘干。8) Place the cut wax strips on the drying table to flatten the slices. Then take a small drop of glycerin and apply it on the slide. Use the outside of your little finger to apply it into a thin layer. The area applied should be larger than the area occupied by the wax strips. The thinner and more evenly applied, the better. Dry at 37°C.
9)把玻片放入盛有二甲苯的染缸中以去除石蜡(10~20分钟),反复操作2次。9) Place the slide in a dyeing jar containing xylene to remove the paraffin (10 to 20 minutes), and repeat the operation twice.
10)最后进行染色,将脱蜡完全的材料,通过以下程序进行染色:1/2二甲苯+1/2纯酒精→100%酒精→95%酒精→85%酒精→70%酒精→50%酒精→1%番红染色(4小时以上)→50%酒精→70%酒精→85%酒精→95%酒精→0.5%固绿(1分钟)→95%酒精→100%酒精→100%酒精(未注明时间的步骤反应时间均为3分钟)。10) Finally, dyeing is performed. The completely dewaxed material is dyed through the following procedure: 1/2 xylene + 1/2 pure alcohol → 100% alcohol → 95% alcohol → 85% alcohol → 70% alcohol → 50% alcohol → 1% safranin dye (more than 4 hours) → 50% alcohol → 70% alcohol → 85% alcohol → 95% alcohol → 0.5% Fast Green (1 minute) → 95% alcohol → 100% alcohol → 100% alcohol (the reaction time for steps without time indication is 3 minutes).
11)置入二甲苯中3分钟至透明。11) Place in xylene for 3 minutes until transparent.
12)滴加中性树胶对所制标本进行封藏。12) Add neutral gum to seal the prepared specimen.
切片制作完成后,使用正倒置一体显微镜(Revolve;Echo)观察并拍照记录。After the slices were prepared, they were observed and photographed using an upright and inverted microscope (Revolve; Echo).
结果如图4所示,过表达转基因植株甘薯块根细胞中淀粉粒的数目要明显多于野生型植株,而干扰表达转基因植株块根细胞中淀粉粒的数目则明显少于野生型植株。The results are shown in Figure 4. The number of starch granules in the root cells of sweet potato overexpressing transgenic plants is significantly greater than that of wild-type plants, while the number of starch granules in the root cells of transgenic interference expression plants is significantly less than that of wild-type plants.
2、IbbHLH49转基因甘薯块根的产量测定2. Yield determination of IbbHLH49 transgenic sweet potato root tubers
对种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根进行产量测定。The sweet potato root yield of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants grown in an isolated field for 4 months was measured.
结果如图5中A所示,过表达转基因植株甘薯单株块根产量与野生型植株相比增加了9.4~17.2%,而干扰表达转基因植株甘薯单株块根产量与野生型植株相比降低了11.0~13.3%。The results are shown in Figure 5A. The root yield per plant of sweet potato in the overexpression transgenic plants increased by 9.4-17.2% compared with the wild-type plants, while the root yield per plant of sweet potato in the interference expression transgenic plants decreased by 11.0-13.3% compared with the wild-type plants.
3、IbbHLH49转基因甘薯块根的总淀粉含量测定3. Determination of total starch content in IbbHLH49 transgenic sweet potato roots
使用淀粉含量测试盒(苏州科铭生物技术有限公司,DF-2-Y)测量甘薯植株块根中的淀粉含量。甘薯植株的块根选择种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根。The starch content in the tubers of sweet potato plants was measured using a starch content test kit (Suzhou Keming Biotechnology Co., Ltd., DF-2-Y). The tubers of sweet potato plants were selected from the tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.
结果如图5中B所示,过表达转基因植株甘薯淀粉含量与野生型植株相比增加了5.1~16.7%,而干扰表达转基因植株甘薯淀粉含量与野生型植株相比减少了4.0~8.4%。The results are shown in FIG. 5B . The starch content of sweet potatoes in the overexpression transgenic plants increased by 5.1 to 16.7% compared with the wild-type plants, while the starch content of sweet potatoes in the interference expression transgenic plants decreased by 4.0 to 8.4% compared with the wild-type plants.
4、IbbHLH49转基因甘薯块根的支链淀粉比例测定4. Determination of the proportion of amylopectin in IbbHLH49 transgenic sweet potato roots
取种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根,清洗干净后烘干研磨破碎,使用去离子水溶解后将混合物过100目网筛。将过滤得到的悬浊液静置沉淀后去除上清,所得到的沉淀物置于烘箱中40℃烘干至恒重,得到甘薯块根淀粉。使用支链淀粉含量测试盒(ZHDF-2-Y)对所得甘薯块根淀粉的支链淀粉比例进行测定。The sweet potato tubers of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months were taken, cleaned, dried, ground and crushed, dissolved in deionized water and the mixture was passed through a 100-mesh sieve. The filtered suspension was allowed to settle and the supernatant was removed. The obtained precipitate was placed in an oven at 40°C and dried to constant weight to obtain sweet potato tuber starch. The amylopectin content test kit (ZHDF-2-Y) was used to determine the amylopectin ratio of the obtained sweet potato tuber starch.
结果如图5中C所示,过表达转基因植株甘薯支链淀粉比例与野生型植株相比增加了2.3~3.6%,而干扰表达转基因植株甘薯支链淀粉比例与野生型植株相比减少了2.9~6.5%。The results are shown in Figure 5C. The amylopectin ratio of the overexpression transgenic plants increased by 2.3-3.6% compared with the wild-type plants, while the amylopectin ratio of the interference expression transgenic plants decreased by 2.9-6.5% compared with the wild-type plants.
5、IbbHLH49转基因甘薯块根中AGPase的活性测定5. Determination of AGPase Activity in IbbHLH49 Transgenic Sweet Potato Roots
使用AGPase活性测定试剂盒(苏州科铭生物技术有限公司,AGP-2A-Y)测量甘薯植株块根中AGPase的活性。甘薯植株的块根选择种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根。The activity of AGPase in the tuberous roots of sweet potato plants was measured using an AGPase activity assay kit (Suzhou Keming Biotechnology Co., Ltd., AGP-2A-Y). The tuberous roots of sweet potato plants were selected from the tuberous roots of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.
结果如图6中A所示,过表达转基因植株甘薯块根中AGPase的活性要显著高于野生型植株,而干扰表达转基因植株甘薯块根中AGPase的活性要显著低于野生型植株。The results are shown in Figure 6A. The activity of AGPase in the sweet potato tubers of the overexpressing transgenic plants was significantly higher than that of the wild-type plants, while the activity of AGPase in the sweet potato tubers of the interference-expressing transgenic plants was significantly lower than that of the wild-type plants.
6、IbbHLH49转基因甘薯块根中SBE的活性测定6. Determination of SBE activity in IbbHLH49 transgenic sweet potato roots
使用SBE活性测定试剂盒(苏州科铭生物技术有限公司,SBE-2-Y)测量甘薯植株块根中SBE的活性。甘薯植株的块根选择种植于隔离大田4个月的IbbHLH49 T1代转基因植株OE49-9、OE49-12、Ri49-7和Ri49-14与野生型植株的甘薯块根。The activity of SBE in the tuberous roots of sweet potato plants was measured using an SBE activity assay kit (Suzhou Keming Biotechnology Co., Ltd., SBE-2-Y). The tuberous roots of sweet potato plants were selected from the sweet potato tuberous roots of IbbHLH49 T1 transgenic plants OE49-9, OE49-12, Ri49-7 and Ri49-14 and wild-type plants planted in the isolated field for 4 months.
结果如图6中B所示,过表达转基因植株甘薯块根中SBE的活性要显著高于野生型植株,而干扰表达转基因植株甘薯块根中SBE的活性要显著低于野生型植株。The results are shown in Figure 6B. The activity of SBE in the sweet potato tubers of overexpressing transgenic plants was significantly higher than that of wild-type plants, while the activity of SBE in the sweet potato tubers of interference-expressing transgenic plants was significantly lower than that of wild-type plants.
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
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