技术领域technical field
本发明涉及野葡萄VyGOLS基因及其编码蛋白在干旱胁迫中的应用，属于植物基因工程技术领域。The invention relates to the application of wild grapeVyGOLS gene and its encoded protein in drought stress, and belongs to the technical field of plant genetic engineering.
背景技术Background technique
葡萄种类繁多，具有重要的食用价值和经济价值。干旱（Drought）对葡萄生长发育过程和产量品质有严重的影响，已经成为制约葡萄生长和提高果品质量的主要因素之一，尤其是近年来全球气候的变化和我国南方干旱的频频出现，使葡萄产业受到很大的威胁。在世界范围内都面临缺水问题的大背景之下，发掘抗旱葡萄资源、研究葡萄抗旱基因对提高葡萄抗旱性、培育抗旱新品种及节水栽培等都具有重要的科学价值和意义。There are many kinds of grapes, which have important edible value and economic value. Drought has a serious impact on the growth and development process and yield quality of grapes, and has become one of the main factors restricting grape growth and improving fruit quality. Especially in recent years, global climate changes and frequent droughts in southern my country have made grape The industry is under great threat. Under the background of water shortage problem all over the world, exploring drought-resistant grape resources and studying grape drought-resistant genes have important scientific value and significance for improving the drought resistance of grapes, cultivating new drought-resistant varieties and water-saving cultivation.
葡萄的抗旱性（Drought resistance）是由多基因控制的数量性状，对干旱等逆境应答和适应需要多个基因的参与，且受到多种途径的调控。在轻度干旱胁迫时，渗透调节是植物体适应水分亏缺的主要途径，植物一方面提高合成渗透调节物质相关酶基因的转录水平，促进基因的表达，增加表达产物的积累；另一方面植物增强抗氧化、解毒相关酶基因（如SOD、CAT、GST 等）的表达水平。当胁迫强度超出渗透调节的能力时，LEA 蛋白及糖类的脱水保护物质积累，保护生物大分子和生物膜系统免受损害。由此，葡萄的抗旱能力取决于自身的组织结构和生理特性，归根结底在于分子水平的差异。随着葡萄全基因组序列的公布，许多与干旱胁迫相关的基因及其调控因子已经得到分离与克隆，并转化到葡萄或模式植物中并对其功能进行分析和验证。Drought resistance of grapes is a quantitative trait controlled by multiple genes. The response and adaptation to drought and other adversities require the participation of multiple genes and are regulated by multiple pathways. Under mild drought stress, osmotic regulation is the main way for plants to adapt to water deficit. On the one hand, plants increase the transcription level of genes related to the synthesis of osmotic regulatory substances, promote gene expression, and increase the accumulation of expression products; on the other hand, plants Enhance the expression level of antioxidant and detoxification related enzyme genes (such as SOD, CAT, GST, etc.). When the stress intensity exceeds the capacity of osmotic regulation, LEA proteins and carbohydrates accumulate dehydration protection substances to protect biomacromolecules and biofilm systems from damage. As a result, the drought resistance of grapes depends on their own tissue structure and physiological characteristics, and ultimately lies in the differences at the molecular level. With the publication of the complete genome sequence of grape, many genes and their regulators related to drought stress have been isolated and cloned, and transformed into grape or model plants, and their functions have been analyzed and verified.
GOLS 是棉子糖合成的关键限速酶。目前，在拟南芥中已经鉴定出7个GOLS基因和三个可能的GOLS基因，AtGOLS1被干旱，高盐和高温胁迫诱导表达；AtGOLS2被干旱和高盐胁迫诱导表达；而AtGOLS3基因仅被低温胁迫诱导表达。在拟南芥中过量表达AtGOLS2基因使转基因植物叶片中大量积累肌醇半乳糖苷和棉子糖，并显著提高了植株抗旱能力和强氧化剂-甲基紫精（MV）的耐受性。在水稻中超表达AtGOLS2，使转基因水稻耐旱能力显著提高，同时在干旱条件下转基因水稻株系产量显著高于对照。将一个盐芥中的肌醇半乳糖苷合成酶（TsGOLS2）导入拟南芥中，提高了转基因拟南芥的耐盐和耐渗透胁迫的能力（Selvaraj, M.G., et al., (2017). Overexpression of an Arabidopsis thaliana galactinolsynthase gene improves drought tolerance in transgenic rice and increasedgrain yield in the field. Plant biotechnology journal, 15(11), 1465-1477.）。用强光，高温同时处理或者过氧化氢处理拟南芥植株会使AtGOLS1和AtGOLS2基因mRNA表达量上升。拟南芥AtGOLS1基因的突变体atgols1受到高温胁迫后叶片中检测不到肌醇半乳糖苷和棉子糖积累，这个结果表明AtGOLS1基因的功能可能是负责在热激胁迫下合成肌醇半乳糖苷。在烟草中超表达苜蓿肌醇半乳糖苷合成酶基因（MfGOLS1）提高了转基因烟草耐寒和耐渗透能力，同时转基因植株体内RFOs含量显著提高。GOLS is a key rate-limiting enzyme in raffinose synthesis. At present, sevenGOLS genes and three possibleGOLS genes have been identified in Arabidopsis.AtGOLS1 is induced by drought, high salt and high temperature stress;AtGOLS2 is induced by drought and high salt stress; andAtGOLS3 is only induced by low temperature. Stress-induced expression. Overexpression ofAtGOLS2 gene in Arabidopsis thaliana resulted in massive accumulation of inositol galactoside and raffinose in leaves of transgenic plants, and significantly improved plant drought resistance and tolerance to the strong oxidant methyl viologen (MV). Overexpression ofAtGOLS2 in rice significantly improved the drought tolerance of transgenic rice, and the yield of transgenic rice lines was significantly higher than that of the control under drought conditions. The introduction of inositol galactoside synthase (TsGOLS2 ) from a salt mustard into Arabidopsis thaliana improved the salt and osmotic stress tolerance of transgenic Arabidopsis (Selvaraj, MG, et al., (2017). Overexpression of an Arabidopsis thaliana galactinolsynthase gene improves drought tolerance in transgenic rice and increased grain yield in the field. Plant biotechnology journal, 15(11), 1465-1477.). The mRNA expression ofAtGOLS1 andAtGOLS2 genes increased when Arabidopsis plants were treated with strong light, high temperature or hydrogen peroxide. The ArabidopsisAtGOLS1 mutant atgols1 could not detect the accumulation of galacto-inositol and raffinose in leaves after high temperature stress. This result suggests that the function ofAtGOLS1 gene may be responsible for the synthesis of galacto-inositol under heat stress. . Overexpression of alfalfa inositol galactoside synthase gene (MfGOLS1 ) in tobacco improved the cold tolerance and penetration tolerance of transgenic tobacco, and the RFOs content in transgenic plants was significantly increased.
虽然，现有技术中已经有了一些物种中GOLS基因的报道，但是野葡萄中的GOLS基因尚未有人报道，人们对于野葡萄GOLS基因的具体功能也缺乏认识。Although there have been reports ofGOLS genes in some species in the prior art, theGOLS gene in wild grape has not been reported yet, and people lack knowledge about the specific function of the wild grapeGOLS gene.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种野葡萄VyGOLS基因，其能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达，促进转基因植株抗旱性增强。The purpose of the present invention is to provide a wild grapeVyGOLS gene, which can increase the accumulation of stress-resistance-related substances and the expression of drought-resistance-related genes in transgenic plants, and promote the enhanced drought resistance of the transgenic plants.
本发明还提供了野葡萄VyGOLS蛋白，该蛋白能够促进转基因植株中积累抗逆相关物质导致转基因植株抗旱性增强。The invention also provides the wild grape VyGOLS protein, which can promote the accumulation of stress-resistance-related substances in the transgenic plants, resulting in enhanced drought resistance of the transgenic plants.
本发明还提供了包含野葡萄VyGOLS基因的重组表达载体，该载体携带野葡萄VyGOLS基因，因此能够超表达VyGOLS基因，进而增强植物抗旱性。The present invention also provides a recombinant expression vector comprising the wild grapeVyGOLS gene. The vector carries the wild grapeVyGOLS gene, so that theVyGOLS gene can be overexpressed, thereby enhancing the drought resistance of the plant.
本发明还提供了上述的包含野葡萄VyGOLS基因的重组表达载体的制备方法，能够制得该载体。The present invention also provides a method for preparing the above-mentioned recombinant expression vector comprising theVyGOLS gene of Vitis vinifera, which can be prepared.
本发明还提供了上述的野葡萄VyGOLS基因和重组表达载体在植物品种育种中的应用，能够获得抗旱植物品种。The present invention also provides the application of the above-mentioned wild grapeVyGOLS gene and the recombinant expression vector in plant variety breeding, so that drought-resistant plant varieties can be obtained.
为了实现上述目的，本发明所采用的技术方案是：In order to achieve the above object, the technical scheme adopted in the present invention is:
野葡萄VyGOLS基因，其编码的氨基酸序列如SEQ ID NO.2所示。Wild grapeVyGOLS gene, the encoded amino acid sequence is shown in SEQ ID NO.2.
本发明中利用强启动子（花椰菜花叶病毒35S启动子）驱动原理的转基因技术，将VyGOLS基因的超量表达载体转入拟南芥中，从而获得转基因拟南芥植株；实验证明，相对于转化空载体的拟南芥植株，超量表达VyGOLS基因导致转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达，转基因植株抗旱性增强。In the present invention, the transgenic technology driven by a strong promoter (cauliflower mosaic virus 35S promoter) is used to transfer the overexpression vector of theVyGOLS gene into Arabidopsis thaliana, thereby obtaining transgenic Arabidopsis thaliana plants; Transformed Arabidopsis plants with empty vector, overexpressingVyGOLS gene leads to the accumulation of stress resistance-related substances and the expression of drought resistance-related genes in transgenic Arabidopsis thaliana, and the drought resistance of transgenic plants is enhanced.
野葡萄VyGOLS基因，其核苷酸序列如SEQ ID NO.1中的186-1196位所示。The nucleotide sequence of the wild grapeVyGOLS gene is shown in positions 186-1196 in SEQ ID NO.1.
上述的核苷酸序列为野葡萄中天然存在的序列，也可以根据该序列进行密码子优化，得到的优化序列也具有同样的效果。The above-mentioned nucleotide sequence is a naturally occurring sequence in wild grape, and codon optimization can also be performed according to the sequence, and the obtained optimized sequence also has the same effect.
野葡萄VyGOLS蛋白，其氨基酸序列如SEQ ID NO.2所示。Wild grape VyGOLS protein, its amino acid sequence is shown in SEQ ID NO.2.
野葡萄VyGOLS蛋白是一个含336个氨基酸的蛋白，该蛋白能够促进转基因植株中积累抗逆相关物质导致转基因植株抗旱性增强。The wild grape VyGOLS protein is a protein containing 336 amino acids, which can promote the accumulation of stress resistance-related substances in transgenic plants, resulting in enhanced drought resistance of transgenic plants.
重组表达载体，所述重组表达载体包含野葡萄VyGOLS基因，所述野葡萄VyGOLS基因的核苷酸序列如SEQ ID NO.1中的186-1196位所示。A recombinant expression vector comprising the wild grapeVyGOLS gene, and the nucleotide sequence of the wild grapeVyGOLS gene is shown in positions 186-1196 in SEQ ID NO.1.
本发明中的重组表达载体为植物过量表达载体，能够在植物中超表达目的基因。The recombinant expression vector in the present invention is a plant overexpression vector, which can overexpress the target gene in plants.
重组表达载体的制备方法，包括：根据如SEQ ID NO.1中的186-1196位所示的序列设计引物，克隆所述野葡萄VyGOLS基因，然后将所述野葡萄VyGOLS基因连接到pCAMBIA2300植物表达载体上，即得。A method for preparing a recombinant expression vector, comprising: designing primers according to the sequence shown in positions 186-1196 of SEQ ID NO.1, cloning the wild grapeVyGOLS gene, and then connecting the wild grapeVyGOLS gene to pCAMBIA2300 plant expression on the carrier, that is.
本发明中将野葡萄VyGOLS基因开放阅读框连接至植物过量表达载体pCAMBIA2300上，能够形成重组表达载体pCAMBIA2300-VyGOLS。In the present invention, the open reading frame of the wild grapeVyGOLS gene is connected to the plant overexpression vector pCAMBIA2300, and the recombinant expression vector pCAMBIA2300-VyGOLS can be formed.
上述的野葡萄VyGOLS基因在植物品种育种中的应用；具体的，在植物抗旱品种育种中的应用；更为具体的，在拟南芥抗旱品种育种中的应用。上述的的重组表达载体在植物品种育种中的应用；具体的，在植物抗旱品种育种中的应用；更为具体的，在拟南芥抗旱品种育种中的应用。Application of the above-mentioned wild grapeVyGOLS gene in plant variety breeding; specifically, application in plant drought-resistant variety breeding; more specifically, application in Arabidopsis drought-resistant variety breeding. Application of the above-mentioned recombinant expression vector in plant variety breeding; specifically, application in plant drought-resistant variety breeding; more specifically, in Arabidopsis drought-resistant variety breeding.
本发明通过植物基因工程技术，从燕山葡萄中分离克隆出与抗旱相关基因完整编码区段的DNA片段，并验证了该基因的功能，发现超量表达之后转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达，转基因植株抗旱性增强。因此，野葡萄VyGOLS基因及其重组表达载体能够用于植物抗旱品种育种。The invention uses plant genetic engineering technology to isolate and clone the DNA fragment of the complete coding segment of the drought-resistance-related gene from Yanshan grapes, and verify the function of the gene. Accumulation and expression of drought resistance-related genes, enhanced drought resistance of transgenic plants. Therefore, the wild grapeVyGOLS gene and its recombinant expression vector can be used for plant drought-resistant variety breeding.
附图说明Description of drawings
图1为本发明中野葡萄VyGOLS基因表达特性分析图；Fig. 1 is theVyGOLS gene expression characteristic analysis figure of the wild grape in the present invention;
图2为本发明中野葡萄VyGOLS基因过量表达载体的鉴定图；Fig. 2 is the identification diagram of wild grapeVyGOLS gene overexpression vector in the present invention;
图3为本发明中转VyGOLS基因拟南芥植株的PCR鉴定图；Fig. 3 is the PCR identification diagram ofVyGOLS gene Arabidopsis plant in the present invention;
图4为本发明中转VyGOLS基因拟南芥植株的抗旱性鉴定图；Fig. 4 is the identification diagram of drought resistance of Arabidopsis thaliana plants transfected withVyGOLS gene of the present invention;
图5为本发明中转VyGOLS基因拟南芥植株的生理特性分析图；Fig. 5 is the physiological characteristic analysis diagram ofVyGOLS gene Arabidopsis plant in the present invention;
图6为本发明中转基因拟南芥植株中抗旱相关基因的表达分析图。Figure 6 is a graph showing the expression analysis of drought resistance-related genes in the transgenic Arabidopsis plants of the present invention.
具体实施方式Detailed ways
下面结合具体实施例对本发明做进一步的详细说明。除特殊说明的之外，各实施例及试验例中所用的设备和试剂均可从商业途径得到。The present invention will be further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources.
野葡萄VyGOLS基因的实施例1Example 1 of the wild grapeVyGOLS gene
本实施例中野葡萄VyGOLS基因，其核苷酸序列如SEQ ID NO.1中的186-1196位所示。In this example, the nucleotide sequence of theVyGOLS gene of Vitis vinifera is shown in positions 186-1196 in SEQ ID NO.1.
野葡萄VyGOLS蛋白的实施例1Example 1 of wild grape VyGOLS protein
本实施例中野葡萄VyGOLS蛋白，其氨基酸序列如SEQ ID NO.2所示。In this example, the VyGOLS protein of wild grape, its amino acid sequence is shown in SEQ ID NO.2.
重组表达载体的实施例1Example 1 of recombinant expression vector
本实施例中重组表达载体包含野葡萄VyGOLS基因，所述野葡萄VyGOLS基因的核苷酸序列如SEQ ID NO.1中的186-1196位所示。In this example, the recombinant expression vector comprises the wild grapeVyGOLS gene, and the nucleotide sequence of the wild grapeVyGOLS gene is shown in positions 186-1196 in SEQ ID NO.1.
重组表达载体的制备方法的实施例1Example 1 of the preparation method of the recombinant expression vector
本实施例中重组表达载体的制备方法，包括：根据如SEQ ID NO.1中的186-1196位所示的序列设计引物，克隆所述野葡萄VyGOLS基因，然后将所述野葡萄VyGOLS基因连接到pCAMBIA2300植物表达载体上，即得。The preparation method of the recombinant expression vector in this embodiment includes: designing primers according to the sequence shown in positions 186-1196 in SEQ ID NO.1, cloning the wild grapeVyGOLS gene, and then connecting the wild grapeVyGOLS gene To pCAMBIA2300 plant expression vector, that is obtained.
野葡萄VyGOLS基因在植物品种育种中的应用的实施例1Example 1 of the application of wild grapeVyGOLS gene in plant variety breeding
本实施例中野葡萄VyGOLS基因能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达，促进转基因植株抗旱性增强，因此可以用于植物抗旱品种育种中的应用，具体的可以用于拟南芥抗旱品种的育种。In this example, the wild grapeVyGOLS gene can increase the accumulation of stress-resistance-related substances and the expression of drought-resistance-related genes in the transgenic plants, and promote the drought resistance of the transgenic plants. Breeding of drought-tolerant varieties of Arabidopsis.
重组表达载体在植物品种育种中的应用的实施例1Example 1 of the application of recombinant expression vector in plant variety breeding
本实施例中重组表达载体包含野葡萄VyGOLS基因，因此可以将其转化入植物中，以得到抗旱的植物品种。In this example, the recombinant expression vector contains the wild grapeVyGOLS gene, so it can be transformed into plants to obtain drought-resistant plant varieties.
试验例1 葡萄VyGOLS基因的表达特性分析Test Example 1 Analysis of the expression characteristics of grapeVyGOLS gene
燕山葡萄组培苗继代培养16 d后，选择生长健壮表现一致的幼苗用于各种逆境处理。After subculture of Yanshan grape tissue culture seedlings for 16 days, the seedlings with consistent growth, robustness and performance were selected for various adversity treatments.
干旱处理：将葡萄幼苗从培养基中拔出，置于滤纸上暴露在室温为（32±1）℃、相对湿度为55%、光周期为光照14 h/黑暗10 h 的条件下处理，在0、2、6、12、24 h取样。Drought treatment: The grape seedlings were pulled out from the medium, placed on filter paper, and exposed to the conditions of room temperature (32±1) °C, relative humidity of 55%, and photoperiod of light for 14 h/dark for 10 h. 0, 2, 6, 12, 24 h sampling.
低温处理：将组培苗置于温度为（4±1）℃、相对湿度为75%、光周期为光照14 h/黑暗10 h的条件下培养，在0、2、6、12、24 h取样。Low temperature treatment: The tissue culture seedlings were cultured under the conditions of temperature of (4±1) °C, relative humidity of 75%, and photoperiod of 14 h light/10 h dark. sampling.
盐胁迫：在三角瓶中加入20 mL 100 mmol·L-1的NaCl溶液，在温度为（25±1）℃、相对湿度为75%、光周期为光照14 h/黑暗10 h的条件下培养，在0、2、6、12、24 h取样。Salt stress: 20 mL of 100 mmol·L-1 NaCl solution was added to the Erlenmeyer flask, and the temperature was (25±1) °C, the relative humidity was 75%, and the photoperiod was 14 h light/10 h dark. , sampling at 0, 2, 6, 12, and 24 h.
在三角瓶中加入等体积的蒸馏水作为盐胁迫处理的对照。正常培养的组培苗作为干旱和低温处理的对照。An equal volume of distilled water was added to the flask as a control for salt stress treatment. The normal cultured tissue culture seedlings served as the control of drought and low temperature treatment.
在大田生长8~10 a的燕山葡萄，在转色期取葡萄果实，于盛花期取根系（第一新生侧根）、茎（新展开叶下第4~5片叶的茎段）、叶（新展开叶下第4~5片）、花序和卷须（新生枝条的第1个枝）等样品。用plus植物总RNA提取试剂盒（天根）提取葡萄叶片总RNA。普通反转录用PrimeScriptII1st Strand cDNA Synthesis Kit（TaKaRa）合成cDNA第一链。Yanshan grapes grown in the field for 8-10 years, grape fruits are taken at the veraison stage, and roots (the first new lateral roots), stems (stem segments of the 4th to 5th leaves under the newly expanded leaves), leaves ( Samples such as the 4th to 5th pieces under the newly expanded leaves), inflorescences and tendrils (the first branch of the new branches). Total RNA from grape leaves was extracted with plus plant total RNA extraction kit (Tiangen). General reverse transcription uses PrimeScriptII 1st Strand cDNA Synthesis Kit (TaKaRa) to synthesize the first strand of cDNA.
具体操作步骤如下：在PCR管中加入：Random 6 mers（50 μM） 1μL，dNTP Mixture（10 mM each）1 μL，Total RNA 2 μg，RNase free dH2O补齐至10 μL，充分混匀，瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5 min，冰上急冷。The specific operation steps are as follows: Add: Random 6 mers (50 μM) 1 μL, dNTP Mixture (10 mM each) 1 μL, Total RNA 2 μg, RNase free dH2 O to 10 μL into the PCR tube, mix well, Briefly centrifuge the solution to the bottom of the PCR tube. React at 65°C for 5 min on a PCR machine, and quench on ice.
根据VyGOLS基因序列设计实时荧光定量PCR引物，Real-time quantitative PCR primers were designed according to theVyGOLS gene sequence,
正向引物序列为qRT-VyGOLS-F：5'-GGGGACTATGTGAAAGGGGTT-3'（如SEQ ID NO.3所示）；The forward primer sequence is qRT-VyGOLS-F: 5'-GGGGACTATGTGAAAGGGGTT-3' (as shown in SEQ ID NO.3);
反向引物序列为qRT-VyGOLS-R：5'-GGATTTGGTTCTCAGGAGGG-3'（如SEQ ID NO.4所示）。The reverse primer sequence is qRT-VyGOLS-R: 5'-GGATTTGGTTCTCAGGAGGG-3' (as shown in SEQ ID NO. 4).
以VyGAPDH基因为内参，UsingVyGAPDH gene as internal reference,
正向引物序列为qRT-VyGAPDH-F：5'-CCCTTGTCCTCCCAACTCT-3'（如SEQ ID NO.5所示）；The forward primer sequence is qRT-VyGAPDH-F: 5'-CCCTTGTCCTCCCAACTCT-3' (as shown in SEQ ID NO.5);
反向引物序列为qRT-VyGAPDH-R：5'-CCTTCTCAGCACTGTCCCT-3'（如SEQ ID NO.6所示）。The reverse primer sequence is qRT-VyGAPDH-R:5'-CCTTCTCAGCACTGTCCCT-3' (shown in SEQ ID NO. 6).
实时荧光定量PCR按照TaKaRa SYBR® Premix Ex Taq™ II （Perfect RealTime）说明在Bio-Rad IQ5 Real-Time PCR Detection System（Bio-Rad Laboratories,HercμLes, CA）上进行。25 μL的反应体系：1 μL的反转录模板；正反向引物各1 μL； 12.5 μL 的2×SYBR® Premix Ex Taq™（2×）；9 μL的nuclease-free water。反应程序为：95℃，30 s；40 cycles of 95℃ for 5 s；57℃ for 30 s；72℃ for 30 s。结果采用2-ΔΔC(t)法进行分析。Real-time PCR was performed on a Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Herc μLes, CA) according to TaKaRa SYBR® Premix Ex Taq™ II (Perfect RealTime) instructions. 25 μL reaction system: 1 μL of reverse transcription template; 1 μL of forward and reverse primers; 12.5 μL of 2×SYBR® Premix Ex Taq™ (2×); 9 μL of nucleus-free water. The reaction program was: 95°C for 30 s; 40 cycles of 95°C for 5 s; 57°C for 30 s; 72°C for 30 s. The results were analyzed using the 2-ΔΔC(t) method.
结果如图1所示，结果表明VyGOLS基因主要在根系中表达，其次在叶里表达量较高，在茎、花、果、卷须中的表达量较低；在低温、干旱、高盐处理后2h，VyGOLS转录本快速积累，在6h大道峰值，随后逐渐降低。The results are shown in Figure 1. The results show thatthe VyGOLS gene is mainly expressed in the root system, followed by a higher expression in leaves, and a lower expression in stems, flowers, fruits, and tendrils; after low temperature, drought, and high salt treatments At 2h,VyGOLS transcripts accumulated rapidly, peaked at avenue 6h, and then gradually decreased.
试验例2 野葡萄VyGOLS基因过量表达载体的构建Test example 2 Construction of wild grapeVyGOLS gene overexpression vector
为研究葡萄VyGOLS基因的功能，将包含有VyGOLS基因编码区在内的共1011bp的ORF片段正确插入植物过量表达载体pCAMBIA2300- GFP上。In order to study the function of grapeVyGOLS gene, a total of 1011 bp ORF fragment including theVyGOLS gene coding region was correctly inserted into the plant overexpression vector pCAMBIA2300-GFP.
根据前期克隆到的VyGOLS基因ORF序列，设计可以扩增VyGOLS基因ORF的上下游引物VyGOLS-ORF-F和VyGOLS-ORF-R；根据pCAMBIA2300-GFP载体上的酶切位点，在引物VyGOLS-ORF-F的5’端加上酶切位点XbaI，具体序列为：According to the ORF sequence ofVyGOLS gene cloned earlier, the upstream and downstream primersVyGOLS -ORF-F andVyGOLS -ORF-R were designed to amplify the ORF ofVyGOLS gene; Add the enzyme cleavage siteXba I to the 5' end of -F, and the specific sequence is:
VyGOLS-ORF-XbaI-F：5’-GGGTCTAGAATGGCCCCAGGAGTGCCCGCAGA-3’（如SEQ ID NO.7所示）；VyGOLS -ORF-Xba I-F: 5'-GGGTCTAGA ATGGCCCCAGGAGTGCCCGCAGA-3' (as shown in SEQ ID NO. 7);
在引物VyGOLS-ORF-R的5’端加上酶切位点KpnI，具体序列为：Add an enzyme cleavage siteKpn I to the 5' end of the primerVyGOLS -ORF-R, and the specific sequence is:
VyGOLS-ORF-KpnI-R：5’-GGGGGTACCTCAAGCAGCAGAGGGTGCGGGAA-3’（如SEQ ID NO.8所示）。VyGOLS -ORF-Kpn I-R: 5'-GGGGGTACC TCAAGCAGCAGAGGGTGCGGGAA-3' (as shown in SEQ ID NO. 8).
以pMD18-T-VyGOLS质粒为模板，用VyGOLS-ORF-XbaI-F与VyGOLS-ORF-KpnI-R进行扩增，回收目的条带后连接到pMD19-T克隆载体，转化TOP10感受态细胞，在附加Amp的LB培养基上进行蓝白斑筛选，分别经过菌液PCR与质粒酶切检测，pMD19-T-VyGOLS阳性克隆送公司测序。用XbaI、KpnI双酶切重组克隆载体pMD19-T-VyGOLS与植物表达载体pCAMBIA2300-GFP，回收线性化载体与目标片段，连接并转化TOP10，经Kan抗生素筛选，挑取单克隆摇菌，菌液检测后提质粒酶切检测。检测结果如图2所示，M：DNA分子质量标准；泳道1：VyGOLS基因过量表达载体的双酶切鉴定；泳道2：VyGOLS基因过量表达载体；结果表明本试验例中构建成功植物表达载体pCAMBIA2300-VyGOLS。将其转化入农杆菌中，用于转染植物。Using pMD18-T-VyGOLS plasmid as a template, amplify withVyGOLS -ORF-Xba I-F andVyGOLS -ORF-Kpn I-R, recover the target band, connect it to pMD19-T cloning vector, transform TOP10 competent cells, add Amp The blue and white spots were screened on the LB medium of the pMD19-T-VyGOLS, and the positive clones of pMD19-T-VyGOLS were sent to the company for sequencing after bacterial liquid PCR and plasmid digestion respectively. The recombinant cloning vector pMD19-T-VyGOLS and the plant expression vector pCAMBIA2300-GFP were digested withXbaI andKpn I double enzymes, the linearized vector and the target fragment were recovered, ligated and transformed into TOP10, and screened by Kan antibiotics. After the liquid detection, the plasmid was digested and detected. The test results are shown in Figure 2, M: DNA molecular quality standard; Lane 1: Double-enzyme digestion identification ofVyGOLS gene overexpression vector; Lane 2:VyGOLS gene overexpression vector; The results show that the plant expression vector pCAMBIA2300 was successfully constructed in this test example -VyGOLS . It is transformed into Agrobacterium for transfection of plants.
试验例3葡萄VyGOLS基因在拟南芥中的过量表达Test Example 3 Overexpression of grapeVyGOLS gene in Arabidopsis thaliana
将含有重组植物表达载体的农杆菌划线培养在LB平板（含60 mg/L的Gent，100 mg/L的Kan）上划线，置于28℃条件下培养24 h；挑取单克隆在10 mL LB液体培养基（附加相应的抗生素）中，在28℃条件下培养24 h；取5 mL菌液转移至50 mL新鲜的LB液体培养基中，在28℃条件下继续培养，至菌液OD600达到0.6左右；转移至离心瓶或离心管中，室温条件下，转速为4000 rpm离心10 min，去除上清液收集菌体；重悬于渗透缓冲液（0.5×MS, 5 %蔗糖，0.03% Silwet L-77（GE Health）），调OD600至0.8；将拟南芥花序上已有的果荚去掉，花序完全浸入渗透液中10-30 s（或用移液器直接将渗透液滴在花序上），立即去掉拟南芥叶或茎秆上的渗透液，将植株平放在托盘中，用塑料薄膜覆盖托盘，24 h后取下薄膜，于温室中继续培养；为提高转化效率，7天之后用同样方法再次侵染；经过转化的拟南芥植株进行正常管理，待果荚现白色时进行收种子。The Agrobacterium containing the recombinant plant expression vector was streaked on an LB plate (containing 60 mg/L Gent, 100 mg/L Kan), and cultured at 28 °C for 24 h; In 10 mL of LB liquid medium (with corresponding antibiotics), culture at 28 °C for 24 h; transfer 5 mL of bacterial liquid to 50 mL of fresh LB liquid medium, and continue to cultivate at 28 °C until the bacteria The OD600 of the solution reaches about 0.6; transfer to a centrifuge bottle or centrifuge tube, centrifuge at 4000 rpm for 10 min at room temperature, remove the supernatant and collect the bacteria; resuspend in permeation buffer (0.5×MS, 5% sucrose, 0.03% Silwet L-77 (GE Health)), adjust the OD600 to 0.8; remove the existing fruit pods on the Arabidopsis inflorescence, and completely immerse the inflorescence in the infiltrate for 10-30 s (or directly transfer the infiltrate with a pipette) drop on the inflorescence), immediately remove the permeate on the leaves or stems of Arabidopsis thaliana, place the plants flat in the tray, cover the tray with plastic film, remove the film after 24 hours, and continue to cultivate in the greenhouse; in order to improve the transformation Efficiency, the same method was used to infect again after 7 days; the transformed Arabidopsis thaliana plants were managed normally, and seeds were harvested when the pods turned white.
对上述通过卡那霉素初步筛选得到的VyGOLS转基因植株及转化空载体植株，进一步在DNA水平进行鉴定，采用改进的SDS 微量提取法提取总DNA。分别以上述所提取的VyGOLS转基因植株及转化空载体植株的DNA为模板，在35S启动子上设计上游引物，与基因特异的下游引物组成引物对，进行 PCR检测。引物如下所示：The above-mentionedVyGOLS transgenic plants and transformed empty vector plants obtained by preliminary screening with kanamycin were further identified at the DNA level, and the total DNA was extracted by an improved SDS microextraction method. Using the DNAs of theVyGOLS transgenic plants and transformed empty vector plants extracted above as templates, upstream primers were designed on the 35S promoter to form primer pairs with gene-specific downstream primers for PCR detection. The primers are as follows:
检测引物-F：5’- GAAGATGCCTCTGCCGACAGTG-3’（如SEQ ID NO.9所示）；Detection primer-F: 5'-GAAGATGCCTCTGCCGACAGTG-3' (as shown in SEQ ID NO. 9);
检测引物-R：5’-AGTACTCCGTCGGCTACTGCCA-3’（如SEQ ID NO.10所示）。反应体系（25 µL）为：10×buffer 2.5 µL；d NTPs 0.5 µL；Taq 酶 0.3 µL；ddH2O 16.2 µL；Primer F 1.5µL；Primer R 1.5 µL；DNA 2.5 µL。反应程序为：94℃预变性 5 min；35 个循环，94℃变性30 S，58℃退火 30 S，72℃延伸 1 min；72℃延伸10min，4℃保存。PCR 产物在 1%琼脂糖凝胶上进行电泳检测。Detection Primer-R: 5'-AGTACTCCGTCGGCTACTGCCA-3' (as shown in SEQ ID NO. 10). The reaction system (25 µL) was: 10×buffer 2.5 µL; d NTPs 0.5 µL; Taq enzyme 0.3 µL; ddH2 O 16.2 µL; Primer F 1.5 µL; Primer R 1.5 µL; DNA 2.5 µL. The reaction program was: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min; extension at 72°C for 10 min, and storage at 4°C. PCR products were detected by electrophoresis on a 1% agarose gel.
检测结果如图3所示，M：DNA分子质量标准；泳道1：以ddH2O为模板进行PCR；泳道2：以转化空载体的拟南芥DNA为模板进行PCR；泳道3：以pCAMBIA2300-VyGOLS质粒DNA为模板进行PCR；泳道4-6：以转化VyGOLS基因的转基因拟南芥DNA为模板进行PCR。从图中可以看出，本试验例成功获得了转VyGOLS基因拟南芥植株，分别命名为OE#1、OE#2、OE#3。The detection results are shown in Figure 3, M: DNA molecular mass standard; Lane 1: PCR using ddH2 O as template; Lane 2: PCR using Arabidopsis DNA transformed with empty vector as template; Lane 3: pCAMBIA2300- PCR was performed with VyGOLS plasmid DNA as template; lanes 4-6: PCR was performed with transgenic Arabidopsis thaliana DNA transformed with VyGOLS gene as template. As can be seen from the figure, theVyGOLS transgenic Arabidopsis plants were successfully obtained in this test example, which were named OE#1, OE#2, and OE#3, respectively.
试验例4 转基因拟南芥植株的抗旱性鉴定Test Example 4 Identification of drought resistance of transgenic Arabidopsis plants
VyGOLS转基因植株和转化空载体的拟南芥（EV表示）在MS 培养基上生长 7 天后，转移至营养钵中，正常浇水20天使其生长成健壮的幼苗。然后停止给拟南芥幼苗浇水即进行干旱处理，直到第 7 天部分拟南芥植株叶片出现明显的失水萎焉症状。之后对所有植株进行复水，48小时后观察植株生长状况。VyGOLS transgenic plants and Arabidopsis thaliana transformed with empty vector (indicated by EV) were grown on MS medium for 7 days, then transferred to nutrient pots and watered normally for 20 days to grow into robust seedlings. Then, the watering of Arabidopsis seedlings was stopped and drought treatment was performed until the leaves of some Arabidopsis plants showed obvious symptoms of water loss and wilting on the 7th day. All plants were then rehydrated, and the growth status of the plants was observed after 48 hours.
干旱处理前后及复水后拟南芥植株的表现型通过拍照记录，结果如图4所示，从图4中可以看出与转化空载体拟南芥相比，转VyGOLS基因拟南芥植株OE#1、OE#2、OE#3的抗旱能力明显增强。The phenotypes of Arabidopsis thaliana plants before and after drought treatment and after rehydration were recorded by photographing. The results are shown in Figure 4. It can be seen from Figure 4 that compared with the transformed empty vector Arabidopsis thaliana, theVyGOLS gene transgenic Arabidopsis thaliana plant OE The drought resistance of #1, OE#2, and OE#3 was significantly enhanced.
试验例5 转基因拟南芥植株生理生化特性分析Test Example 5 Analysis of Physiological and Biochemical Characteristics of Transgenic Arabidopsis Plants
失水率的测定：VyGOLS转基因植株和转化空载体植株正常生长3周后，分别取约0.2 g的莲座叶进行失水率的测定。将采取的莲座叶放置于干燥的滤纸上，每隔10 min测量一次叶片的鲜重（FW），直到测至50 min时失水率测定结束。将每一次测定的失水量与第一次测定的鲜重的比值作为失水率。Determination of water loss rate:After VyGOLS transgenic plants and transformed empty vector plants grew normally for 3 weeks, about 0.2 g of rosette leaves were taken respectively to measure the water loss rate. The collected rosette leaves were placed on dry filter paper, and the fresh weight (FW) of the leaves was measured every 10 min until the water loss rate was measured at 50 min. The ratio of the water loss of each measurement to the fresh weight of the first measurement was taken as the water loss rate.
电解质渗漏率（电导率）的测定：将叶片装入离心管中，用超去离子水定容至 10mL，室温下振荡1小时后测定溶液的电导值，记为煮前C1。随后将溶液连同叶片置于沸水中煮沸10 min后，等温度降至室温后测定电导值，记为C2。将C1与C2的比值（C1/C2）作为相对电解质渗漏值。Determination of electrolyte leakage rate (conductivity): Put the leaves into a centrifuge tube, dilute the volume to 10mL with super deionized water, shake at room temperature for 1 hour, and measure the conductivity value of the solution, which is recorded as C1 before cooking. Subsequently, the solution and the leaves were boiled in boiling water for 10 min, and the conductance value was measured after the temperature dropped to room temperature, which was recorded as C2. The ratio of C1 to C2 (C1/C2) was taken as the relative electrolyte leakage value.
检测结果如图5所示，（A）转基因拟南芥植株中VyGOLS基因的表达量检测；（B）转VyGOLS基因拟南芥植株在干旱处理18d后的存活率统计；（C）转VyGOLS基因拟南芥叶片相对失水率；（D）转VyGOLS基因拟南芥植株在干旱处理18d后的相对电导率。从图中可以看出，转基因拟南芥植株中VyGOLS基因表达量较高，存活率相对于转化空载体的植株显著提高；相对于转化空载体植株，转VyGOLS基因拟南芥植株中失水率和电导率显著降低。The detection results are shown in Figure 5. (A) Detection of VyGOLS gene expression in transgenic Arabidopsis plants; (B) statistics of survival rate of VyGOLS transgenic Arabidopsis plants after drought treatment for 18 days; (C) VyGOLS gene transfection The relative water loss rate of Arabidopsis thaliana leaves; (D) The relative electrical conductivity of VyGOLS transgenic Arabidopsis thaliana plants after 18 days of drought treatment. It can be seen from the figure that the VyGOLS gene expression level in transgenic Arabidopsis thaliana plants is higher, and the survival rate is significantly higher than that of plants transformed with empty vector; and electrical conductivity decreased significantly.
试验例6 转基因拟南芥抗旱相关基因表达分析Test Example 6 Expression analysis of genes related to drought resistance in transgenic Arabidopsis
用plus植物总RNA提取试剂盒提取干旱处理后的转基因拟南芥叶片总RNA。普通反转录用PrimeScriptII1st Strand cDNA Synthesis Kit（TaKaRa）合成cDNA第一链。具体操作步骤如下：在PCR管中加入：Random 6 mers（50 μM） 1μL，dNTP Mixture（10 mM each）1 μL，Total RNA 2 μg，RNase free dH2O补齐至10 μL，充分混匀，瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5 min，冰上急冷。以拟南芥AtActin为内参基因，检测转基因拟南芥植株中抗旱相关基因AtCOR15A、AtERD15、AtRD29A、AtP5CS1基因的表达。所设计引物如下所示：The total RNA of the transgenic Arabidopsis thaliana leaves after drought treatment was extracted with the plus plant total RNA extraction kit. General reverse transcription uses PrimeScriptII 1st Strand cDNA Synthesis Kit (TaKaRa) to synthesize the first strand of cDNA. The specific operation steps are as follows: Add: Random 6 mers (50 μM) 1 μL, dNTP Mixture (10 mM each) 1 μL, Total RNA 2 μg, RNase free dH2O to 10 μL in a PCR tube, mix well, and centrifuge briefly Bring the solution to the bottom of the PCR tube. React at 65°C for 5 min on a PCR machine, and quench on ice. Using Arabidopsis AtActin as an internal reference gene, the expression of drought resistance-related genesAtCOR15A ,AtERD15 ,AtRD29A andAtP5CS1 in transgenic Arabidopsis plants was detected. The designed primers are as follows:
qRT-AtActin-F：5’-CGGTGGTTCTATCTTGGCATC-3’（如SEQ ID NO.11所示）；qRT-AtActin-F: 5'-CGGTGGTTCTATCTTGGCATC-3' (as shown in SEQ ID NO. 11);
qRT-AtActin-R：5’-GTCTTTCGCTTCAATAACCCTA-3’（如SEQ ID NO.12所示）；qRT-AtActin-R: 5'-GTCTTTCGCTTCAATAACCCTA-3' (as shown in SEQ ID NO. 12);
qRT-AtCOR15A-F：5’-CAGCGGAGCCAAGCAGAGCAG-3’（如SEQ ID NO.13所示）；qRT-AtCOR15A -F: 5'-CAGCGGAGCCAAGCAGAGCAG-3' (as shown in SEQ ID NO. 13);
qRT-AtCOR15A-R：5’-CATCGAGGATGTTGCCGTCACC-3’（如SEQ ID NO.14所示）；qRT-AtCOR15A -R: 5'-CATCGAGGATGTTGCCGTCACC-3' (as shown in SEQ ID NO. 14);
qRT-AtERD15-F：5’-CCAGCGAAATGGGGAAACCA-3’（如SEQ ID NO.15所示）；qRT-AtERD15 -F: 5'-CCAGCGAAATGGGGAAACCA-3' (as shown in SEQ ID NO. 15);
qRT-AtERD15-R：5’-ACAAAGGTACAGTGGTGGC-3’（如SEQ ID NO.16所示）；qRT-AtERD15 -R: 5'-ACAAAGGTACAGTGGTGGC-3' (as shown in SEQ ID NO. 16);
qRT-AtRD29A-F：5’-GTTACTGATCCCACCAAAGAAGA-3’（如SEQ ID NO.17所示）；qRT-AtRD29A -F: 5'-GTTACTGATCCCACCAAAGAAGA-3' (as shown in SEQ ID NO. 17);
qRT-AtRD29A-R：5’-GGAGACTCATCAGTCACTTCCA-3’（如SEQ ID NO.18所示）；qRT-AtRD29A -R: 5'-GGAGACTCATCAGTCACTTCCA-3' (as shown in SEQ ID NO. 18);
qRT-AtP5CS1-F：5’-CGACGGAGACAATGGAATTGT-3’（如SEQ ID NO.19所示）；qRT-AtP5CS1 -F: 5'-CGACGGAGACAATGGAATTGT-3' (as shown in SEQ ID NO. 19);
qRT-AtP5CS1-R：5’-GATCAGAAATGTGTAGGTAGC-3’（如SEQ ID NO.20所示）。qRT-AtP5CS1 -R: 5'-GATCAGAAATGTGTAGGTAGC-3' (as shown in SEQ ID NO. 20).
实时荧光定量PCR按照TaKaRa SYBR® Premix Ex Taq™ II（Perfect RealTime）说明在Bio-Rad IQ5 Real-Time PCR Detection System（Bio-Rad Laboratories,HercμLes, CA）上进行。25 μL的反应体系：1 μL的反转录模板；正反向引物各1 μL； 12.5 μL 的2×SYBR® Premix Ex Taq™（2×）；9 μL的nuclease-free water。反应程序为：95℃，30 s；40 cycles of 95℃ for 5 s；57℃ for 30 s；72℃ for 30 s。结果采用2-ΔΔC(t)法进行分析。Real-time PCR was performed on a Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Herc μLes, CA) according to TaKaRa SYBR® Premix Ex Taq™ II (Perfect RealTime) instructions. 25 μL reaction system: 1 μL of reverse transcription template; 1 μL of forward and reverse primers; 12.5 μL of 2×SYBR® Premix Ex Taq™ (2×); 9 μL of nucleus-free water. The reaction program was: 95°C for 30 s; 40 cycles of 95°C for 5 s; 57°C for 30 s; 72°C for 30 s. The results were analyzed using the 2-ΔΔC(t) method.
检测结果如图6所示，从图6中可以看出，在干旱条件下，与转化空载体拟南芥相比，转基因拟南芥植株中抗旱相关基因表达量明显升高，表明本发明中的野葡萄VyGOLS基因，能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达，促进转基因植株抗旱性增强。The detection results are shown in Figure 6. It can be seen from Figure 6 that, under drought conditions, compared with the transformation of the empty vector Arabidopsis, the expression of drought resistance-related genes in the transgenic Arabidopsis plants is significantly increased, indicating that the present invention The wild grapeVyGOLS gene can increase the accumulation of stress resistance-related substances and the expression of drought resistance-related genes in transgenic plants, and promote the enhanced drought resistance of transgenic plants.
<110> 河南科技大学<110> Henan University of Science and Technology
<120> 野葡萄VyGOLS基因及其编码蛋白在干旱胁迫中的应用<120> Application ofVyGOLS gene and its encoded protein in wild grape under drought stress
<160> 20<160> 20
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<211> 1011<211> 1011
<212> DNA<212> DNA
<213> 野葡萄<213> Wild grapes
<221>VyGOLS基因<221>VyGOLS gene
<400> 1<400> 1
gcgaaaccgc cccctttttg ttggtacccg ggaaaccggc cattacggcc ggggaggaac 60gcgaaaccgc ccccttttttg ttggtacccg ggaaaccggc cattacggcc ggggaggaac 60
aaaggcaaag tagggtggca tccacagtgt tgctggttta ctttcccaac cctcctcacc 120aaaggcaaag tagggtggca tccacagtgt tgctggttta ctttcccaac cctcctcacc 120
accaactctc tccttaacat ttttcttgca ccaacttgaa atctcacccc aaataaacca 180accaactctc tccttaacat ttttcttgca ccaacttgaa atctcacccc aaataaacca 180
caacaatggc cccaggagtg cccgcagatg tgtttacagc cggcggaaag gtttccaccc 240caacaatggc cccaggagtg cccgcagatg tgtttacagc cggcggaaag gtttccaccc 240
tcaacgcagg ctactcaaag ggggcctacg tcacattttt agctggaaac ggggactatg 300tcaacgcagg ctactcaaag ggggcctacg tcacattttt agctggaaac ggggactatg 300
tgaaaggggt tgttgggttg gctaagggtt tgcgcaaggt gaagagcgcg taccctcttg 360tgaaaggggt tgttgggttg gctaagggtt tgcgcaaggt gaagagcgcg taccctcttg 360
tggttgcaat gttgccggat gtgcctgagg agcaccgtga gatcttaaag tctcagggct 420tggttgcaat gttgccggat gtgcctgagg agcaccgtga gatcttaaag tctcagggct 420
gcataattcg tgaaattgag cccatctacc ctcctgagaa ccaaatccag tttgcaatgg 480gcataattcg tgaaattgag cccatctacc ctcctgagaa ccaaatccag tttgcaatgg 480
catactacgt catcaactat tccaaactcc gtatttggaa tttcgaggaa tacagcaaga 540catactacgt catcaactat tccaaactcc gtatttggaa tttcgaggaa tacagcaaga 540
tggtgtattt ggatgctgat atccaagttt acgacaacat agaccacctt atggacgccc 600tggtgtattt ggatgctgat atccaagttt acgacaacat agaccacctt atggacgccc 600
cggacggcta cttttacgcg gtaatggact gcttctgtga gaagacatgg agtcacactc 660cggacggcta cttttacgcg gtaatggact gcttctgtga gaagacatgg agtcacactc 660
cccagtactc cgtcggctac tgccagcagt gcccggacaa ggtgacttgg cccgctgaga 720cccagtactc cgtcggctac tgccagcagt gcccggacaa ggtgacttgg cccgctgaga 720
tgggttcacc tccacctttg tacttcaacg ctgggatgtt cgtctttgag cctagccgtt 780tgggttcacc tccacctttg tacttcaacg ctgggatgtt cgtctttgag cctagccgtt 780
tgacttatga aagccttctc catactctac ggatcactcc tccgaccgcc tttgccgagc 840tgacttatga aagccttctc catactctac ggatcactcc tccgaccgcc tttgccgagc 840
aagatttctt gaacatgttc ttccaacaca tgtacaagcc catccctctc gtatacaact 900aagatttctt gaacatgttc ttccaacaca tgtacaagcc catccctctc gtatacaact 900
tggttctagc aatgctgtgg cgccacccgg agaacgttga gctcgaccag gtcaaggtgg 960tggttctagc aatgctgtgg cgccacccgg agaacgttga gctcgaccag gtcaaggtgg 960
tgcactactg tgctgctgga tcaaagccat ggagatacac tgggaaagaa gcaaacatgg 1020tgcactactg tgctgctgga tcaaagccat ggagatacac tgggaaagaa gcaaacatgg 1020
agagagagga catcaagatg ttggtagcca aatggtggga catttacaat gataagtctc 1080agagagagga catcaagatg ttggtagcca aatggtggga catttacaat gataagtctc 1080
tggatttcaa ggctgaggac agtgttccag agggagaagg attctctagg ccatcgatca 1140tggatttcaa ggctgaggac agtgttccag agggagaagg attctctagg ccatcgatca 1140
tggcttccat gcctgagcct gcaatctcct atattcccgc accctctgct gcttgaagat 1200tggcttccat gcctgagcct gcaatctcct atattcccgc accctctgct gcttgaagat 1200
tacaaatctt taggagagag tgtattgaag ctcagggtgt gatctatctc tttttctatt 1260tacaaatctt taggagagag tgtattgaag ctcagggtgt gatctatctc tttttctatt 1260
taataccttt tccaaaggct acttggt 1287taataccttt tccaaaggct acttggt 1287
<211> 336<211> 336
<212> PRT<212> PRT
<213> 野葡萄<213> Wild grapes
<221> VyGOLS蛋白<221> VyGOLS protein
<400> 2<400> 2
MET Ala Pro Gly Val Pro Ala Asp Val Phe Thr Ala Gly Gly LysMET Ala Pro Gly Val Pro Ala Asp Val Phe Thr Ala Gly Gly Lys
1 5 10 151 5 10 15
Val Ser Thr Leu Asn Ala Gly Tyr Ser Lys Gly Ala Tyr Val ThrVal Ser Thr Leu Asn Ala Gly Tyr Ser Lys Gly Ala Tyr Val Thr
20 25 30 20 25 30
Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly LeuPhe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu
35 40 45 35 40 45
Ala Lys Gly Leu Arg Lys Val Lys Ser Ala Tyr Pro Leu Val ValAla Lys Gly Leu Arg Lys Val Lys Ser Ala Tyr Pro Leu Val Val
50 55 60 50 55 60
Ala MET Leu Pro Asp Val Pro Glu Glu His Arg Glu Ile Leu LysAla MET Leu Pro Asp Val Pro Glu Glu His Arg Glu Ile Leu Lys
65 70 75 65 70 75
Ser Gln Gly Cys Ile Ile Arg Glu Ile Glu Pro Ile Tyr Pro ProSer Gln Gly Cys Ile Ile Arg Glu Ile Glu Pro Ile Tyr Pro Pro
80 85 90 80 85 90
Glu Asn Gln Ile Gln Phe Ala MET Ala Tyr Tyr Val Ile Asn TyrGlu Asn Gln Ile Gln Phe Ala MET Ala Tyr Tyr Val Ile Asn Tyr
95 100 105 95 100 105
Ser Lys Leu Arg Ile Trp Asn Phe Glu Glu Tyr Ser Lys MET ValSer Lys Leu Arg Ile Trp Asn Phe Glu Glu Tyr Ser Lys MET Val
110 115 120 110 115 120
Tyr Leu Asp Ala Asp Ile Gln Val Tyr Asp Asn Ile Asp His LeuTyr Leu Asp Ala Asp Ile Gln Val Tyr Asp Asn Ile Asp His Leu
125 130 135 125 130 135
MET Asp Ala Pro Asp Gly Tyr Phe Tyr Ala Val MET Asp Cys PheMET Asp Ala Pro Asp Gly Tyr Phe Tyr Ala Val MET Asp Cys Phe
140 145 150 140 145 150
Cys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr Ser Val Gly TyrCys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr Ser Val Gly Tyr
155 160 165 155 160 165
Cys Gln Gln Cys Pro Asp Lys Val Thr Trp Pro Ala Glu MET GlyCys Gln Gln Cys Pro Asp Lys Val Thr Trp Pro Ala Glu MET Gly
170 175 180 170 175 180
Ser Pro Pro Pro Leu Tyr Phe Asn Ala Gly MET Phe Val Phe GluSer Pro Pro Pro Leu Tyr Phe Asn Ala Gly MET Phe Val Phe Glu
185 190 195 185 190 195
Pro Ser Arg Leu Thr Tyr Glu Ser Leu Leu His Thr Leu Arg IlePro Ser Arg Leu Thr Tyr Glu Ser Leu Leu His Thr Leu Arg Ile
200 205 210 200 205 210
Thr Pro Pro Thr Ala Phe Ala Glu Gln Asp Phe Leu Asn MET PheThr Pro Pro Thr Ala Phe Ala Glu Gln Asp Phe Leu Asn MET Phe
215 220 225 215 220 225
Phe Gln His MET Tyr Lys Pro Ile Pro Leu Val Tyr Asn Leu ValPhe Gln His MET Tyr Lys Pro Ile Pro Leu Val Tyr Asn Leu Val
230 235 240 230 235 240
Leu Ala MET Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp GlnLeu Ala MET Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Gln
245 250 255 245 250 255
Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp ArgVal Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg
260 265 270 260 265 270
Tyr Thr Gly Lys Glu Ala Asn MET Glu Arg Glu Asp Ile Lys METTyr Thr Gly Lys Glu Ala Asn MET Glu Arg Glu Asp Ile Lys MET
275 280 285 275 280 285
Leu Val Ala Lys Trp Trp Asp Ile Tyr Asn Asp Lys Ser Leu AspLeu Val Ala Lys Trp Trp Asp Ile Tyr Asn Asp Lys Ser Leu Asp
290 295 300 290 295 300
Phe Lys Ala Glu Asp Ser Val Pro Glu Gly Glu Gly Phe Ser ArgPhe Lys Ala Glu Asp Ser Val Pro Glu Gly Glu Gly Phe Ser Arg
305 310 315 305 310 315
Pro Ser Ile MET Ala Ser MET Pro Glu Pro Ala Ile Ser Tyr IlePro Ser Ile MET Ala Ser MET Pro Glu Pro Ala Ile Ser Tyr Ile
320 325 330 320 325 330
Pro Ala Pro Ser Ala AlaPro Ala Pro Ser Ala Ala
335 336 335 336
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-VyGOLS-F<221> qRT-VyGOLS-F
<400> 3<400> 3
ggggactatg tgaaaggggt t 21ggggactatg tgaaaggggt t 21
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-VyGOLS-R<221> qRT-VyGOLS-R
<400> 4<400> 4
ggatttggtt ctcaggaggg 20ggatttggtt ctcaggaggg 20
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-VyGAPDH-F<221> qRT-VyGAPDH-F
<400> 5<400> 5
cccttgtcct cccaactct 19cccttgtcct cccaactct 19
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-VyGAPDH-R<221> qRT-VyGAPDH-R
<400> 6<400> 6
ccttctcagc actgtccct 19ccttctcagc actgtccct 19
<211> 32<211> 32
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> VyGOLS-ORF-XbaI-F<221> VyGOLS-ORF-XbaI-F
<400> 7<400> 7
gggtctagaa tggccccagg agtgcccgca ga 32gggtctagaa tggccccagg agtgcccgca ga 32
<211> 32<211> 32
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> VyGOLS-ORF-KpnI-R<221> VyGOLS-ORF-KpnI-R
<400> 8<400> 8
gggggtacct caagcagcag agggtgcggg aa 32gggggtacct caagcagcag agggtgcggg aa 32
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> 检测引物-F<221> Detection Primer-F
<400> 9<400> 9
gaagatgcct ctgccgacag tg 22gaagatgcct ctgccgacag tg 22
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> 检测引物-R<221> Detection Primer-R
<400> 10<400> 10
agtactccgt cggctactgc ca 22agtactccgt cggctactgc ca 22
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtActin-F<221>qRT-AtActin-F
<400> 11<400> 11
cggtggttct atcttggcat c 21cggtggttct atcttggcat c 21
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtActin-R<221> qRT-AtActin-R
<400> 12<400> 12
gtctttcgct tcaataaccc ta 22gtctttcgct tcaataaccc ta 22
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtCOR15A-F<221> qRT-AtCOR15A-F
<400> 13<400> 13
cagcggagcc aagcagagca g 21cagcggagcc aagcagagca g 21
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtCOR15A-R<221> qRT-AtCOR15A-R
<400> 14<400> 14
catcgaggat gttgccgtca cc 22catcgaggat gttgccgtca cc 22
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtERD15-F<221>qRT-AtERD15-F
<400> 15<400> 15
ccagcgaaat ggggaaacca 20ccagcgaaat ggggaaacca 20
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtERD15-R<221>qRT-AtERD15-R
<400> 16<400> 16
acaaaggtac agtggtggc 19acaaaggtac agtggtggc 19
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtRD29A-F<221> qRT-AtRD29A-F
<400> 17<400> 17
gttactgatc ccaccaaaga aga 23gttactgatc ccaccaaaga aga 23
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtRD29A-R<221> qRT-AtRD29A-R
<400> 18<400> 18
ggagactcat cagtcacttc ca 22ggagactcat cagtcacttc ca 22
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtP5CS1-F<221> qRT-AtP5CS1-F
<400> 19<400> 19
cgacggagac aatggaattg t 21cgacggagac aatggaattg t 21
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequences
<221> qRT-AtP5CS1-R<221> qRT-AtP5CS1-R
<400> 20<400> 20
gatcagaaat gtgtaggtag c 21gatcagaaat gtgtaggtag c 21
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910872930.9ACN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910872930.9ACN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
| Publication Number | Publication Date |
|---|---|
| CN110452917Atrue CN110452917A (en) | 2019-11-15 |
| CN110452917B CN110452917B (en) | 2022-08-02 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910872930.9AExpired - Fee RelatedCN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
| Country | Link |
|---|---|
| CN (1) | CN110452917B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444738A (en)* | 2021-06-22 | 2021-09-28 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN115094084A (en)* | 2022-06-06 | 2022-09-23 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding of high-seed activity rice |
| CN118726419A (en)* | 2024-08-13 | 2024-10-01 | 宁夏大学 | MsGOLS2 gene from alfalfa and its application in regulating plant salt tolerance |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259996A (en)* | 1997-04-28 | 2000-07-12 | 味之素株式会社 | Raffinose synthetase gene, process for producing, same, and transformed plant |
| CN107586809A (en)* | 2017-10-09 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | A kind of method of living things catalysis synthesis soyabean oligosaccharides |
| CN110079537A (en)* | 2019-05-27 | 2019-08-02 | 河南科技大学 | Vitis mitogen response regulation factor Ⅴ vRR gene and its coding albumen and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259996A (en)* | 1997-04-28 | 2000-07-12 | 味之素株式会社 | Raffinose synthetase gene, process for producing, same, and transformed plant |
| CN107586809A (en)* | 2017-10-09 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | A kind of method of living things catalysis synthesis soyabean oligosaccharides |
| CN110079537A (en)* | 2019-05-27 | 2019-08-02 | 河南科技大学 | Vitis mitogen response regulation factor Ⅴ vRR gene and its coding albumen and application |
| Title |
|---|
| GENBANK: ""PREDICTED: Vitis vinifera galactinol synthase 1 (LOC1 00260266), mRNA"，Accession Number：XM_002265911.3", 《GENBANK》* |
| TAO ZHOU ET AL.: ""Molecular Cloning and Characterization of GhGolS1, a Novel Gene Encoding Galactinol Synthase from Cotton (Gossypium hirsutum)"", 《PLANT MOL BIOL REP》* |
| 冯睿杰 等: ""葡萄肌醇半乳糖苷合成酶基因VvGolS2-4的克隆及表达特性分析"", 《华北农学报》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444738A (en)* | 2021-06-22 | 2021-09-28 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN113444738B (en)* | 2021-06-22 | 2022-07-05 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN115094084A (en)* | 2022-06-06 | 2022-09-23 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding of high-seed activity rice |
| CN115094084B (en)* | 2022-06-06 | 2023-10-24 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding high-seed-activity rice |
| CN118726419A (en)* | 2024-08-13 | 2024-10-01 | 宁夏大学 | MsGOLS2 gene from alfalfa and its application in regulating plant salt tolerance |
| Publication number | Publication date |
|---|---|
| CN110452917B (en) | 2022-08-02 |
| Publication | Publication Date | Title |
|---|---|---|
| CN110845590B (en) | Wild grape VyPPR gene and application of encoding protein thereof in drought stress | |
| CN107435047B (en) | A key gene of low phosphorus tolerance in plant phosphorus signaling network GmPHR25 and its application | |
| CN110128516B (en) | Barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding | |
| CN112876551B (en) | Transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and application thereof | |
| CN110452917B (en) | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress | |
| US20240263188A1 (en) | Application of bfne gene in tomato plant architecture improvement and biological yield increase | |
| CN110643618A (en) | Jatropha MYB transcription factor JcMYB16 gene and its application in improving plant drought resistance | |
| CN117343938B (en) | Mint McTRY gene and its expressed protein and application | |
| CN102719449A (en) | Clone of apple resistance-related gene MdSIMYB1 and application thereof | |
| CN115948417A (en) | Barley HvFRF1 gene, protein, expression vector and application | |
| CN104903444A (en) | Nucleic acid imparting high-yielding property to plant, method for producing transgenic plant with increased yield, and method for increasing plant yield | |
| CN103360484A (en) | Upland cotton protein GhMADS22, and coding gene and application thereof | |
| CN110129291A (en) | Humidity Tolerance Regulation Gene HvACO1, Protein and Its Application in Breeding in Barley | |
| CN116589545B (en) | Application of ONAC096 gene in controlling drought resistance of rice | |
| CN112391405A (en) | Application of tea tree hexokinase CsHXK3 gene in regulation of plant growth and development and enhancement of cold resistance | |
| CN112322600A (en) | Salt tolerance gene MsSnRK2.3 in alfalfa and its encoded protein and application | |
| CN110093352B (en) | Gene sequence of a transcription factor AgMYB1 related to anthocyanin synthesis in celery and its application | |
| CN111171127A (en) | Astragalus sinicus LHY gene and application thereof | |
| CN113234720B (en) | Wheat long non-coding RNA lncR156 and its application in regulating wheat response to drought stress | |
| CN105400804B (en) | A kind of FvNCED3 genes for strengthening Fraxinus velutina salt tolerance and its application | |
| CN104945493B (en) | A kind of soybean protein GmIDD influencing plant growth period and its encoding gene and application | |
| CN108148843A (en) | Chinese milk vetch LEAFY genes and its application | |
| CN108103075B (en) | A switchgrass gene PvC3H29 delaying plant senescence and its application | |
| CN115948454B (en) | Application of a grape VvDREB2c gene in improving plant heat tolerance | |
| CN105112395A (en) | Kiwi fruit AdPDC1 gene and application |
| 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | Granted publication date:20220802 | |
| CF01 | Termination of patent right due to non-payment of annual fee |