CN117603990A - 一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 - Google Patents
一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 Download PDFInfo
- Publication number
- CN117603990A CN117603990A CN202311514132.1A CN202311514132A CN117603990A CN 117603990 A CN117603990 A CN 117603990A CN 202311514132 A CN202311514132 A CN 202311514132A CN 117603990 A CN117603990 A CN 117603990A
- Authority
- CN
- China
- Prior art keywords
- vvdreb2a
- grape
- gene
- drought
- anthocyanin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- 229930002877 anthocyanin Natural products 0.000 claims abstract description 43
- 235000010208 anthocyanin Nutrition 0.000 claims abstract description 43
- 239000004410 anthocyanin Substances 0.000 claims abstract description 43
- 150000004636 anthocyanins Chemical class 0.000 claims abstract description 43
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 42
- 241000219095 Vitis Species 0.000 claims abstract description 41
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 41
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 41
- 230000014509 gene expression Effects 0.000 claims abstract description 18
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 9
- 241000219094 Vitaceae Species 0.000 claims description 6
- 235000021021 grapes Nutrition 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 5
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 206010020649 Hyperkeratosis Diseases 0.000 abstract description 23
- 230000009261 transgenic effect Effects 0.000 abstract description 19
- 238000009825 accumulation Methods 0.000 abstract description 13
- 230000015572 biosynthetic process Effects 0.000 abstract description 13
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 241000196324 Embryophyta Species 0.000 abstract description 10
- 230000024346 drought recovery Effects 0.000 abstract description 10
- 230000002018 overexpression Effects 0.000 abstract description 10
- 230000001105 regulatory effect Effects 0.000 abstract description 8
- 239000013604 expression vector Substances 0.000 abstract description 7
- 230000009466 transformation Effects 0.000 abstract description 7
- 230000001052 transient effect Effects 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 235000013399 edible fruits Nutrition 0.000 abstract description 4
- 241000589158 Agrobacterium Species 0.000 abstract description 3
- 230000001276 controlling effect Effects 0.000 abstract description 2
- 230000001404 mediated effect Effects 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 238000013459 approach Methods 0.000 abstract 1
- 229930003935 flavonoid Natural products 0.000 abstract 1
- 150000002215 flavonoids Chemical class 0.000 abstract 1
- 235000017173 flavonoids Nutrition 0.000 abstract 1
- 230000008641 drought stress Effects 0.000 description 13
- 238000011282 treatment Methods 0.000 description 8
- 240000000560 Citrus x paradisi Species 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000011161 development Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 108091027981 Response element Proteins 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 229930000223 plant secondary metabolite Natural products 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 101150065143 DREB2A gene Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000003621 irrigation water Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
-
- 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
- C12N15/825—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 pigment 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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明中提供了一种赤霞珠葡萄耐旱基因VvDREB2A及其应用，该基因的核苷酸序列为SEQ ID NO:2。本发明构建了pRI101‑VvDREB2A过量表达与RNAi‑VvDREB2A抑制表达载体，通过农杆菌介导转化葡萄愈伤组织和瞬时转化果实验证功能。过量表达VvDREB2A明显提高了葡萄愈伤组织的耐旱性，并增加了葡萄愈伤与果实中花色苷积累，抑制其表达则影响的葡萄耐旱性与花色苷的积累。所公开的应用是将基因VvDREB2A应用于转基因育种或愈伤组织工厂，提高植株的抗旱能力以及通过调控类黄酮途径来提高葡萄花色苷的合成与积累。
Description
技术领域
本发明属于功能基因筛选应用技术领域，具体涉及一种赤霞珠葡萄抗旱基因VvDREB2A及其应用。
背景技术
干旱被视为对植物生长发育具有重要影响的关键非生物性胁迫因子之一，导致大多数农作物产量下降，并引发植株形态与生理方面的变化；在极端情况下，严重干旱胁迫会造成植株生长停滞及叶片枯萎，甚至导致植物死亡。为了应对干旱胁迫，植物已经进化出一系列复杂的生理生化机制，花青素作为重要的植物次生代谢产物，在各类环境胁迫下发挥着关键作用，其合成不仅增强了ROS清除和抗氧化能力，还能有效保护植物光合装置和细胞成分免受氧化损伤，从而显著提升植物的耐旱性，但严重干旱条件下，植物体内的花色苷也呈降低趋势。
葡萄是我国西北地区发展的重要经济果树，不仅可以鲜食还可以酿酒、制干，葡萄产业对于西北地区农业经济的发展起到了重要的支撑作用。但长期的灌溉水资源短缺，是限制葡萄产业发展的重要因素，培育耐旱性强、综合品质优的葡萄品种成为本地区葡萄产业发展的迫切需求。而转基因育种成为未来农业育种的重要方向，对于快速、定向培育目标品种意义重大。
发明内容
本发明的目的在于，提供一个赤霞珠葡萄抗旱基因VvDREB2A，并证明了该基因在增强耐旱性，提高花色苷积累方面的功能。
为了实现上述任务，本发明采用如下的技术解决方案：
一种赤霞珠葡萄抗旱基因VvDREB2A，所述的基因VvDREB2A的编码蛋白的序列如下：
MSSGVIERKRKSRSRRSGPNSVAETLARWKQYNDILDSVRKAPAKGSKK
GCMKGKGGPENSICGYRGVRQRTWGKWVAEIREPNRGSRLWLGTFPTAIEA
ALAYDEAARAMYGSSARLNLPNYTTSLKDSSSAPTTSVSDSTTTTSNYSEVCA
YEDSKKPVLPSIKHESGEGESGISGGMLSAVVKAEPATPVSLVTQGGGNDPVN
VGNEPVDAMKLQHEENGHSLDAMYFKNEDGGQDFLEGFPMDEMFDVDEFL
RAIDSDPLASYGTRQELGHDSGQVGSFETDNMQWEKPTDLSYQLQNPDAKL
LGSLNHMEQVPSDFDYCYGFLQPGKQLDPCIGLNDQGLLDLELSDMGF(SEQ ID NO:1)；
所述的基因，其一种具体的核苷酸序列如下：
ATGTCGTCCGGAGTCATTGAAAGGAAGAGAAAGTCTCGAAGCCGGCG
AAGTGGACCTAATTCCGTGGCCGAGACTCTTGCAAGATGGAAACAATACA
ACGATATACTTGATTCTGTCCGCAAAGCTCCAGCAAAAGGTTCAAAGAAG
GGGTGTATGAAAGGTAAAGGGGGGCCCGAGAATTCAATATGTGGTTACAG
GGGTGTGAGGCAGAGGACATGGGGTAAATGGGTTGCTGAGATTCGGGAGC
CAAACAGAGGGAGTAGGCTATGGTTAGGTACCTTCCCAACTGCCATTGAA
GCTGCTCTTGCATATGACGAAGCTGCAAGGGCCATGTATGGTTCTTCTGCC
CGTCTTAATCTTCCAAATTACACCACGTCTTTGAAGGATTCTTCTTCGGCTC
CAACTACATCGGTTTCTGATTCCACCACGACAACATCCAACTACTCTGAAG
TGTGTGCATATGAGGATTCAAAGAAGCCTGTTTTACCGAGTATCAAACATG
AAAGTGGCGAAGGTGAATCAGGAATAAGTGGTGGTATGCTTTCTGCAGTG
GTTAAAGCTGAACCTGCCACACCAGTTAGTTTGGTAACACAGGGAGGAGG
TAATGATCCTGTCAATGTAGGTAATGAACCTGTTGATGCAATGAAGCTCCA
GCATGAAGAAAACGGTCATTCTTTGGATGCCATGTACTTCAAGAATGAAGA
CGGAGGACAGGATTTCTTGGAAGGATTTCCTATGGATGAAATGTTTGATGT
GGATGAATTTTTGAGGGCCATAGACTCTGACCCCCTTGCTAGCTATGGTAC
AAGGCAGGAATTGGGTCATGATTCTGGGCAAGTAGGGAGCTTTGAAACTG
ATAATATGCAGTGGGAAAAGCCAACGGATTTATCTTACCAACTGCAAAATC
CAGATGCTAAGCTTCTTGGGAGTTTGAATCATATGGAGCAAGTGCCTTCTG
ACTTTGATTATTGCTATGGCTTCTTGCAGCCAGGCAAGCAGTTAGATCCCTG
TATAGGGTTGAATGATCAGGGGTTACTTGATTTGGAATTATCAGATATGGGG
TTCTAA(SEQ ID NO:2)；
本发明还提供所述的VvDREB2A基因的一种用途，是在增强葡萄抗旱中的应用；
本发明再一个方面还提供所述的VvDREB2A基因的另一种用途，是在增加葡萄中花色苷含量的应用。
本发明再一个方面还提供一种筛选抗旱葡萄个体的方法，所述的方法是通过检测所述基因的表达量来筛选个体。
本发明发现过量表达VvDREB2A转基因愈伤的生长速度明显快于野生对照，相对电导率则低于对照；同时，VvDREB2A过表达转化愈伤组织中、瞬时转化葡萄果实中花色苷的含量显著高于对照，RT-qPCR分析表明，与花色苷合成相关基因VvMYBA2、VvUFGT、VvCHI、VvCHS、Vv4CL和VvF3'5'H的表达水平均被显著上调。以上结果表明赤霞珠葡萄抗逆基因VvDREB2A在过量表达增强了愈伤组织抗旱性，同时也能够促进葡萄愈伤与果实中的花色苷积累。通过调控VvDREB2A基因表达可以起到调控植物耐旱性，并改变植物花色苷水平的目的。
附图说明
图1为干旱胁迫对VvDREB2A转基因愈伤的影响图，其中a图所示为经过干旱胁迫处理的葡萄愈伤组织的鲜重测定，b图所示为干旱胁迫处理后葡萄愈伤的相对电导率测定。每个实验重复3次，不同字母表示处理间有显著性差异(p＜0.05)。
图2为VvDREB2A转基因愈伤在干旱处理后花色苷积累的差异图，其中a图所示为转基因葡萄愈伤组织的花色苷含量，b图所示为转基因愈伤花色苷相关基因表达量的测定。qRT-PCR中以PRI101的值为参考并设置为1，每个实验重复3次，不同字母表示处理间有显著性差异(p＜0.05)。
图3为VvDREB2A瞬时转化葡萄果实对花色苷积累的影响图，其中a图所示为VvDREB2A瞬时转化葡萄果实中花色苷含量，b图所示为瞬时转化葡萄果实后花色苷相关基因的表达。qRT-PCR中以空载体pRI101/TRV的值为参考并设置为1，每个实验重复3次，*表示与对照相比有显著差异(P<0.05)。
图4为验证了VvDREB2A与VvMYBA2、VvCHI和VvF3'5'H的启动子结合图，其中a图展示了相关基因启动子上与VvDREB2A的结合位点；b图酵母单杂交试验证明了VvDREB2A能与VvMYBA2、VvCHI和VvF3'5'H启动子相互作用。酵母菌株在SD/-Leu和SD/-Leu+ABA培养基上生长3天。实验重复了3次，代表性图片如图所示；c图通过EMSA(凝胶迁移实验)验证了VvDREB2A与proVvMYBA2的标记DNA探针之间的相互作用；d图双荧光素酶试验表明，在本生烟草叶片中，VvDREB2A激活了proVvMYBA2::Luc的表达。
具体实施方式
本发明发现葡萄耐旱相关基因VvDREB2A，不仅能够增强葡萄的耐旱能力，而且对花色苷的积累具有重要的正向调控作用，可在未来葡萄转基因育种、愈伤组织工厂中应用。
本发明利用同源克隆技术，根据赤霞珠葡萄基因组序列采用反转录PCR(ReverseTranscription-Polymerase Chain Reaction，RT-PCR)，以赤霞珠葡萄总RNA反转录合成cDNA第一链为模板，首次扩增了赤霞珠葡萄中VvDREB2A，该基因完整开放阅读框序列全长1068bp(SEQ ID NO:2)，编码335个氨基酸(SEQ ID NO:1)。
为进一步研究赤霞珠葡萄基因VvDREB2A的具体功能，发明人构建了pRI-VvDREB2A过量表达载体与RNAi-VvDREB2A抑制表达载体，并将其在葡萄愈伤组织中表达。发现VvDREB2A能正向调节葡萄愈伤组织的耐旱性，pRI-VvDREB2A的转基因愈伤组织在干旱胁迫下的生长快于对照，而RNAi-VvDREB2A转基因愈伤组织生长慢于对照。经过干旱胁迫处理后，与野生对照相比，过表达VvDREB2A的愈伤组织中相对电导率较低，表明VvDREB2A提高了葡萄愈伤的耐旱性
在研究中，发明人测定了转基因愈伤组织在干旱胁迫下的花色苷含量以及转基因愈伤中花色苷相关基因表达情况，结果表明，VvDREB2A能够促进葡萄愈伤组织中花色苷的积累。
因此，本发明具体包含如下的内容：
1、利用同源克隆技术，以欧洲葡萄赤霞珠叶片总RNA反转录合成cDNA第一链为模板，扩增得到了赤霞珠抗旱基因VvDREB2A序列，该赤霞珠抗旱基因VvDREB2A的编码区序列为SEQ ID NO:2。
2、构建pRI-VvDREB2A过量表达载体与RNAi-VvDREB2A抑制表达载体，并通过农杆菌介导将其导入野生型‘佳美’葡萄愈伤组织。筛选获得了表现型良好的VvDREB2A转基因株系。
3、VvDREB2A正向调节葡萄愈伤组织的耐旱性，过表达转基因愈伤组织相较野生对照在干旱胁迫下的生长速度快，且相对电导率低。此外，过表达转基因愈伤与葡萄果实中花色苷的积累也明显多于野生对照,且与花色苷合成相关的基因VvMYBA2、VvUFGT、VvCHI、VvCHS、Vv4CL和VvF3'5'H表达水平均显著上调。以上结果都表明赤霞珠葡萄抗逆基因VvDREB2A在上调花色苷积累的过程中起重要作用。
下面结合实施例和附图对本发明进行详细的描述。
实施例1：VvDREB2A葡萄转基因愈伤组织对干旱胁迫的响应
DREB2A是一种APETALA2/乙烯响应元件结合因子型转录因子，受到干旱和盐胁迫的诱导，为了确定其在葡萄中的作用，本发明扩增了赤霞珠VvDREB2A的CDS序列(SEQ IDNO:2)，使用下表1所示引物构建pRI101-VvDREB2A过表达载体pCAMBIA2300-RNAi-VvDREB2A抑制表达载体，转化佳美葡萄愈伤组织。将转基因愈伤与野生型愈伤组织分别置于含有20％ PEG6000的培养基中，暗培养20天。
与野生型愈伤相比，VvDREB2A过表达愈伤在干旱胁迫下的生长更快，相对电导率更低，而VvDREB2A抑制表达的转基因愈伤在干旱胁迫下的生长速度受到了明显的抑制，并且其相对电导率显著高于野生对照(图1)。这些结果表明VvDREB2A正调节葡萄愈伤组织的耐旱性。
表1：试验所用引物的序列表
实施例2：VvDREB2A参与干旱胁迫下葡萄愈伤花色苷的积累
花色苷是一类重要的植物次生代谢物，在转化VvDREB2A的过程中发现其对花色苷的合成也具有调控作用。对转基因愈伤组织进行PEG6000模拟干旱处理20天后，测定不同葡萄愈伤组织花色苷含量。结果表明，VvDREB2A的过表达处理下葡萄中花色苷的积累量最高，而抑制葡萄愈伤组织中VvDREB2A的表达则表现出相反的表型(图2A)。同时，定量分析显示，VvDREB2A过表达通过促进包括VvMYBA2、VvUFGT、VvCHI、VvCHS、Vv4CL和VvF3'5'H在内的花色苷生物合成基因的表达，增加了花色苷的生物合成。
实施例3VvDREB2A直接调控花色苷合成相关基因的表达
为了确定VvDREB2A在植物花色苷合成中的作用，在葡萄果实转色初期进行葡萄瞬时转化验证基因功能，利用农杆菌介导将过表达载体pRI-VvDREB2A与病毒干扰载体TRV-VvDREB2A转化离体葡萄果实。如图3A所示，VvDREB2A瞬时过表达的葡萄中花色苷的含量显著高于对照，而抑制VvDREB2A功能的葡萄中花色苷含量则低于对照。VvDREB2A过表达显著提高了花色苷合成途径相关基因VvMYBA2、VvUFGT、VvCHI、VvCHS、Vv4CL和VvF3'5'H的表达水平(图3B)，VvDREB2A可能通过调控花色苷合成相关基因来影响葡萄花色苷含量，
为进一步明确其机制，使用酵母单杂交(Y1H)试验验证VvDREB2A与花色苷合成相关基因启动子的结合。如图4B所示，在细菌溶液中稀1000倍后，共表达pGADT7-VvDREB2A和pVvCHI-pABAi、pGADT7-VvDREB2A和pVvF3'5'H-pABAi以及pGADT7-VvDREB2A和pVvMYBA2-pABAi的酵母细胞能够在SD/-Leu+ABA的培养基上生长，而空载体对照则无法正常生长。接着通过EMSA试验以及双荧光素酶实验验证了VvDREB2A与proVvMYBA2互作。以上结果表明VvDREB2A能与花色苷合成相关基因VvCHI、VvF3'5'H和VvMYBA2的启动子结合而发挥调控作用。
Claims (7)
1.一种基因，其特征在于，所述的基因，其编码蛋白的氨基酸序列为SEQ ID NO:1。
2.如权利要求1所述的基因，其特征在于，所述的基因的核苷酸序列为SEQ ID NO:2。
3.权利要求1所述的基因在增强葡萄抗旱中的应用。
4.一种增加葡萄抗旱的方法，其特征在于，所述的方法是在葡萄中增加权利要求1所述的方法的表达量。
5.权利要求1所述的基因在增加葡萄中花色苷含量的应用。
6.一种增加葡萄中花色苷含量的方法，其特征在于，所述的方法是在葡萄中增加权利要求1所述的基因的表达量。
7.一种筛选抗旱葡萄个体的方法，其特征在于，所述的方法是通过检测利要求1所述的基因的表达量来筛选个体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311514132.1A CN117603990A (zh) | 2023-11-14 | 2023-11-14 | 一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311514132.1A CN117603990A (zh) | 2023-11-14 | 2023-11-14 | 一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117603990A true CN117603990A (zh) | 2024-02-27 |
Family
ID=89948780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311514132.1A Pending CN117603990A (zh) | 2023-11-14 | 2023-11-14 | 一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117603990A (zh) |
-
2023
- 2023-11-14 CN CN202311514132.1A patent/CN117603990A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | De novo transcriptome sequencing and comparative analysis of differentially expressed genes in kiwifruit under waterlogging stress | |
| CN111778265B (zh) | 玉米赤霉素氧化酶的突变基因、突变体、表达载体和应用 | |
| Zou et al. | Comparison of transcriptomes undergoing waterlogging at the seedling stage between tolerant and sensitive varieties of Brassica napus L. | |
| CN116284300B (zh) | 一种增强马铃薯抗旱性的扩展蛋白基因StEXLB1及其应用 | |
| CN113337526A (zh) | 玉米甲硫氨酸亚砜还原酶基因ZmMSRB3及应用 | |
| CN105063085A (zh) | 甘蓝型油菜基因BnMPK3及其抗油菜菌核病的应用 | |
| Zhang et al. | Advances in genetic modification of cassava | |
| CN107418972A (zh) | 一种获得生长特性改良水稻品种的方法 | |
| CN112266922B (zh) | OsMAPKK4基因在改良水稻抗病性中的应用 | |
| CN110964740B (zh) | 一种高黄酮醇烟草的制备方法及其应用 | |
| CN109943579B (zh) | 一种岷江百合LrCCoAOMT基因及其应用 | |
| CN108251435B (zh) | 野生毛葡萄商-24抗病基因VqJAZ4及其应用 | |
| CN114958906B (zh) | 与烟草低钾胁迫相关的基因、启动子及其应用 | |
| CN114369616B (zh) | 番茄sisps基因在提高植物耐高温性中的应用 | |
| CN105294846A (zh) | Cic1蛋白在调控植物低温耐性中的应用 | |
| CN117603990A (zh) | 一种赤霞珠葡萄抗旱基因VvDREB2A及其应用 | |
| CN112760334B (zh) | 调控番茄果实糖含量的基因及其应用 | |
| Zhu et al. | MdbZIP74 negatively regulates osmotic tolerance and adaptability to moderate drought conditions of apple plants | |
| CN108795942B (zh) | 一种水稻外因胁迫诱导表达启动子Psubs3及其应用 | |
| CN113584047A (zh) | 大麦HvNAT2基因及其用途 | |
| CN114085848B (zh) | 马铃薯StSUMO1和StSCE1的应用 | |
| CN116732048B (zh) | 水稻转录因子基因OsbZIP48在获得高锌水稻籽粒和/或调节氮素吸收中的应用 | |
| US20230123814A1 (en) | Use of alr1 gene or alr1 protein of aluminum ion receptor in regulating plant aluminum resistance | |
| CN117210487A (zh) | BnaMATE43b基因在提高植物抗氧化性和/或提高植物耐铝毒性中的应用 | |
| CN118307653A (zh) | 小麦转录因子TaHRP1在小麦抗病育种中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |