CN114941009B - 一种用于茶树基因功能研究的vigs方法 - Google Patents
一种用于茶树基因功能研究的vigs方法 Download PDFInfo
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Abstract
本发明涉及一种用于茶树基因功能研究的VIGS方法,选择茶树实生苗,去除顶芽和部分真叶,进行真空侵染,所述真空侵染条件为:侵染压强为0.7‑0.8kPa、菌液浓度D600为1.2O;侵浸时间为0.5‑1h。本发明的方法表明,茶树VIGS方法能够沉默茶树内源基因,研究茶树基因功能,弥补茶树没有稳定遗传转化体系的不足,为未来研究茶树基因组奠定了基础。
Description
技术领域
本发明涉及基因工程领域,具体涉及一种用于茶树基因功能研究的VIGS方法。
背景技术
茶树属山茶科山茶属,为多年生常绿木本植物,在我国已经有几千年的栽培历史,是我国的一种重要的经济树种。茶因富含茶多酚、茶氨酸及咖啡碱等多种特征性次生代谢物,具有卓越的保健功效及优异的风味品质。为精确研究与茶树优良性状相关基因的功能,提升突破性茶树新品种的选育效率,创建新的茶树品种,亟需建立一套高效、稳定、简单的茶树基因功能研究体系。现阶段,茶树基因功能验证大多依赖拟南芥和烟草等模式植物的异源表达系统。然而,生物合成网络的复杂性和多样性,往往使得异源表达系统不适用于茶树特征基因功能鉴定。而茶树基因功能基础研究的缺失,也制约了生产上急需的突破性新品种定向选育,由于茶树体胚富含多酚类物质,且体胚的诱导、成熟和萌发时间较长,使得茶树组织培养过程中存在严重的褐化现象,且可重复性差,因此依赖于组织培养的茶树转基因体系普遍实施周期长、转化率低,难以获得转基因植株。
发明内容
为解决上述问题,本研究采用去顶芽和部分真叶的茶树实生幼苗(母本福鼎大白茶、父本龙井43)为转化受体,利用根瘤农杆菌GV3101真空侵染茶苗伤口,经表型观察和基因表达量分析获得茶树沉默基因植株。该茶树VIGS方法将为茶树基因功能研究和种质创新奠定坚实的基础。
一种用于茶树基因功能研究的VIGS方法,包括以下步骤;
1)目的基因的获得,
2)pTRV2-目的基因载体的构建,
3)pTRV2-目的基因载体热击法转化DH5α大肠杆菌感受态细胞,
4)冻融法将含有载体的质粒导入农杆菌GV3101,
5)选择侵染材料,
6)真空侵染,
7)暗培养和光照培养侵染茶苗后,常规培养;
所述侵染材料为长出3-4枚真叶且没有木质化的实生茶苗,侵染前去除实生茶苗的顶芽和所有叶片,所述真空侵染条件为:侵染压强为0.7-0.8kPa、菌液浓度D600为1.2O;侵染时间为0.5-1h,所述菌液为体积比1:1的含pTRV1载体与pTRV2-目的基因载体的重悬液的混合菌液。
所述真空侵染条件为:侵染压强为0.7kPa、菌液浓度D600为1.2O;侵染时间为0.5-1h。
所述重悬液配制方法,4.74g的MS、2ml的6-BA、30g的蔗糖、100ul的NAA、溶于1L蒸馏水中,调pH为5.6,121℃高温高压灭菌21min后,在超净台中加入0.2g乙酰丁香酮AS,-20℃保存。
所述侵染前将除去顶芽和所有叶片的实生茶苗置于混合菌液中浸泡1-2小时。
所述茶树品种为福鼎大白茶。
所述实生茶苗的培养方法为将茶树种子去除种皮放在催芽液中,在25℃,在16h光照/8h黑暗循环的人工气候室中萌发生长3-5周,所述催芽液为6-BA 100ul/L+赤霉素600ul/L+氯霉素5ml/L。
所述暗培养和光照培养采用保鲜膜覆盖侵染茶苗,在保持湿润环境下培养。
培养条件:培养室昼夜光周期为光/暗:16h/8h,温度为光/暗:25℃/18℃,相对湿度为65%,光源为日光灯。
所述目的基因为CsPOR1(原叶绿素求酸酯氧化还原酶1)基因,常规培养直至出现沉默的白化表型。
本发明标记基因的选择:利用CsPOR1(原叶绿素求酸酯氧化还原酶1)基因作为标记基因,该基因合成的酶是唯一能够将原叶绿素酸酯转化为叶绿素酸酯的酶,控制叶绿素a和叶绿素b合成,CsPOR1沉默后叶片会出现光漂泊现象,所以可以选择它作为标记基因。
载体的选择:本发明采用烟草脆裂病毒(tobaccorattlevirus,TRV)载体,属于RNA病毒,病毒粒体呈直杆状,有2种粒体,长粒体190~210nm×25nm,短粒体40~80nm×20~25nm。该病毒基因组中含有两条RNA链:RNA1和RNA2。RNA1能编码RNA依赖的RNA聚合酶(RNAdependentRNApolymerase,RdRp)基因、运动蛋白(movementprotein,Mp)以及富含半胱氨酸的蛋白质;RNA2能编码衣壳蛋白(coatprotein,Cp)以及克隆目标基因的酶切位点,病毒颗粒极其稳定。RNA1和RNA2两条链构成双元载体,因此TRV介导的基因沉默需要RNA1和RNA2的同时作用。当采用农杆菌侵染方法时,两个载体分别被转入到各自独立的农杆菌中,两者混合后对植物进行共侵染。与其他病毒载体相比,TRV载体侵染方便快捷,侵染后数天就可沉默目的基因,与通量克隆技术结合,TRV还能实现目的基因的高通量功能分析。
本发明优选真空抽滤法进行真空侵染,适当选择侵染压强和菌液浓度,配制最适的农杆菌生长的培养基、调节适宜于茶树生长的PH值、调整营养元素、植物生长调节剂对茶苗生长的影响,提高茶树VIGS方法的沉默效率;在暗培养和光照培养过程中的温度、湿度、光源等条件可自行调节,最终茶苗可以在5-6周时出现沉默的白化表型,达到研究茶树内源基因的目的。
本发明的茶树VIGS方法通过真空侵染的方式对茶苗进行侵染,侵染过程相对传统的遗传转化操作简单、用时少,不需要经过组织培养获得转基因植株,反向的弥补茶树没有完整遗传转化的不足。本发明中的茶树VIGS方法在真空侵染过程中控制合适的压强、OD600、侵染时间三者,将含目的片段的TRV(烟草脆裂病毒)载体导入茶树植株,使得茶树表现出沉默表型验证基因功能,该方法避免茶树内源基因在异源模式生物中不能完全表达或不表达的问题,为研究茶树基因功能和茶树种质创新开辟了一条新道路。
附图说明
图1为CsPOR1基因PCR扩增;
图2为农杆菌中含有ptrv1、pTRV2、pTRV2-CsPOR1载体DNA的PCR扩增;
图3为25d到45d TRV:00组和TRV:POR1组表型变化;
图4为TRV:00组、TRV:POR1组表型对比;
图5为55d TRV:00组和TRV:POR1组叶片表型对比;
图6为野生型、空载组、实验组茶树叶片中CsPOR1基因的相对表达量;
图7为空载组与实验组CsPOR1基因表达量随时间变化;
其中A为空载组叶片中CsPOR1基因表达量变化,B为实验组叶片中CsPOR1基因表达量变化。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,下面结合实施例对本发明中的技术方案进一步阐明,所描述的实施例仅是本发明的部分实施例,不是全部的实施例,不作为对本发明限制的依据。
VIGS实验所用试剂:
1.YEB培养基:称取10g/L酵母提取物(Yeast extract)、10g/L蛋白胨(Tryptone)5g/L NaCl,调pH为7.2,加蒸馏水定容到1L。配置YEB固体培养基时需加入15g琼脂,121℃高温高压灭菌21min后使用。
2.Kan(50mg/m L):称取50mg Kan溶于50ml无菌水,用0.22um过滤器过滤除菌,保存于-20℃。
3.Rif(25mg/m L):称取50mg Rif溶于100ml甲醇,用0.22um过滤器过滤除菌,分装到2ml EP管,保存在-20℃。
4.AS(100umol/m L):称取200mg AS溶于10m L二甲亚砜(DMSO),用0.22um过滤器过滤除菌,分装到2m L EP管,保存于-20℃。
5.农杆菌重悬液:取4.74g MS、2ml 6-BA、30g蔗糖、100ul NAA、溶于1L蒸馏水中,调pH为5.6,121℃高温高压灭菌21min后,在超净台中加入浓度为0.2g/L乙酰丁香酮AS,常温下保存。
6.营养液:吸取6-BA 100ul/L、赤霉素600ul/L、氯霉素5ml/L溶于超纯水。
实验中所用到的生物材料均为市售。
实施例1
1.目的基因的获得:拟南芥PORA基因在NCBI(https://www.ncbi.nlm.nih.gov/)上的序列号:NP_200230.1,通过PORA基因的序列号找到拟南芥的CDS序列,得到的拟南芥PORA基因的CDS序列通过TBtools软件转化成拟南芥PORA基因的蛋白序列,将得到的蛋白序列与茶树全蛋白组序列进行比对获得12组相关序列,同源性在70%以上的蛋白序列有三组(CSS0033593.1、CSS0008189.1、CSS0041527.1)将三组序列对应的CDS序列分别通过NCBI进行Bleast分析确定CSS0033593.1(茶树基因组中对应的序列号)对应的基因是目的基因CsPOR1(GenBank MH925309.1),CsPOR1基因的蛋白序列通过NCBI上的CD-Search功能确定CsPOR1的保守结构域是从第93bp-400bp,一共308个碱基符合pTRV载体目的基因的要求,选取这一片段作为目的基因。选择CsPOR1基因作为标记基因的原因:POR是唯一能够将原叶绿素酸酯转化为叶绿素酸酯的酶,控制叶绿素a和叶绿素b合成,将其沉默叶片表现出光漂白现象。
目的基因序列:
2.pTRV2-CsOR1载体的构建
使用TransZol Up Plus RNA试剂盒(天根生化科技有限公司,北京)并按照试剂盒的说明提取总RNA。在1.0%(w:v)琼脂糖凝胶上检查RNA质量。利用Prime ScriptTMⅡ1ststrand cDNA Synthesis Kit(索莱宝技术有限公司,北京)进行反转录合成cDNA。CsPOR1(GenBank MH925309.1)基因的部分转录序列用(君诺德生物技术有限公司,武汉)高保真DNA聚合酶扩增目的片段(见图1)。
PCR反应体系为:PrimeSTARMaxPremix(2×)25μL、正向引物和反向引物(CsPOR1F:GAGGGTGGGTGATACCATATTC/CsPOR1R:GCTGCTGTTTGTGCTCTTATAG)各2.5μL、cDNA3.0μL,补充灭菌蒸馏水至50μL。PCR反应条件:94℃4min;95℃40s,55℃30s,72℃1min,34个循环;72℃10min,4℃保存。
PCR的产物与克隆载体pMD18-T(君诺德生物技术有限公司,武汉)相连,热击法转化DH5α大肠杆菌感受态细胞(科文生物科技有限公司,长沙),涂板过夜培养,经卡那霉素(Kanamycin,50mg/L)抗性和通用引物M13-47/48(F:CGCCAGGGTTTTCCCAGTCACGAC/R:AGCGGATAACAATTTCACACAGGA)扩增菌液PCR,筛选的阳性重组子送公司测序,测序结果通过与从GenBank数据库获得的序列信息进行比较而得到证实。
为了构建pTRV2-CsPOR1载体,扩增具有EcoRI和XbaI限制性位点的301bpCsPOR1片段。用EcoR和XbaI限制性酶消化并回收的PCR产物和pTRV2载体与T4DNA连接酶(科文生物科技有限公司,长沙)在16℃连接过夜,然后转化入大肠杆菌DH5α。摇样保种在-20℃冰箱保存,为之后提质粒导入农杆菌GV3101做准备,后续检验工作在质粒导入农杆菌后进行。
3.农杆菌感受态细胞制备:
取出保存在-80℃超低温冰箱的农杆菌菌种,用接种针蘸取菌液,在含Rif(20mg·L-1)抗生素的YEP平板培养基上划线,使其能够挑出单菌落;将接好菌的YEP固体培养基倒置,放于28℃恒温培养箱中培养从平板培养基上挑取单菌落,接种于5mL含Rif(20mg·L-1)抗生素的YEP液体培养基中,在28℃,200rpm条件下,振荡培养;从上述YEP培养基中取1mL菌液,接种于50mL含Rif(20mg·L-1)的YEP液体培养基中,在28℃,200rpm条件下,振荡培养,直至OD600为0.5~0.6;取1.5ml离心管,每管中加入18uL的甘油(浓度为15%),将离心管置于冰上预冷,将灭过菌的CaCl2(0.1mol·L-1)溶液置于冰上预冷;将OD600值为0.5~0.6的菌液分装到10mL离心管中,4℃,5000rpm离心10min,倒掉上清液;向10ml离心管中加入200uL预冷的CaCl2溶液,用移液枪轻轻吹打菌体使其重悬混匀;从10ml离心管中取100uL重悬液,加入提前预冷的的1.5mL离心管中,使其与甘油混匀,然后液氮速冻;将制好的农杆菌感受态细胞放于-80℃超低温冰箱中保存。
4.pTRV2-CsPOR1载体质粒导入农杆菌
取出保存在-80℃超低温冰箱的农杆菌感受态细胞放于冰上10min,让其解冻;
分别量取5uL的pTRV1质粒、pTRV2质粒、pTRV2-CsPOR1重组质粒DNA加入农杆菌感受态细胞中,轻轻弹动混匀,放于冰上30min,然后液氮速冻5min;
将离心管放于37℃水浴锅中2min,使其溶解;向离心管中加入900uL含Rif(20mg·L-1)抗生素的YEP液体培养基,28℃,200rpm,振荡培养4h;室温5000×g离心1min后,倒掉上清液;向离心管中加入100uLYEP液体培养基,轻轻混匀;
取适量菌液涂布于含有Rif(20mg·L-1)和Kan(100mg·L-1)的YEP固体培养基上,将上述YEP固体培养基倒置放于28℃恒温培养箱培养2天,挑取单菌落,保种备用。
5.pTRV2-CsPOR1载体阳性检测
移液枪吸取3ul含目的基因的农杆菌菌液加到50ul PCR反应体系(17ul ddH2o、5ul引物、25ul 2xTaq plus Mix、3ul模板)进行合成cDNA利用特异性引物(CsPOR1F:CCAAGGCTCTAGCTGAAACA/CsPOR1R:GTCAAGTGAGGCAAGGTCTAA)对菌液进行PCR鉴定,能够扩增出与阳性对照相同的目标片段(图2),表明pTRV2-CsPOR1载体构建成功并导入农杆菌GV3101中。图2第二、三条带表示pTRV1、pTRV2载体的部分基因,说明两个载体成功导入农杆菌GV3101,可用于真空侵染实验。
6.植物材料和试剂的准备
茶树品种选用‘福鼎大白茶’(母本五年生福鼎大白茶、父本四年生龙井43号),将茶树种子去除种皮放在催芽液(6-BA 100ul/L、赤霉素600ul/L、氯霉素5ml/L)中,在25℃,在16h光照/8h黑暗循环的人工气候室中生长,五周后将实生苗用于侵染实验(此时植物茎段高度为2-3cm)。每个处理使用四十个植株,每个植物去除顶芽和所有叶片,带有伤口的茶苗放在菌液中先浸泡1-2小时,后将茶苗进行侵染。试剂的准备:YEP固体培养基(用于农杆菌的培养)、LB(Luria Bertan)液体培养基(用于农杆菌和TRV载体的共同培养),MS粉、蔗糖、琼脂、卡那霉素、利福平、乙酰丁香酮等试剂。
7.真空侵染茶树幼苗及注射法侵染茶树幼苗
(1)真空侵染法:挑取新鲜培养的含pTRV1、pTRV2和目的基因片段的pTRV2-CsPOR1的农杆菌GV3101单菌落,分别接种到3mL-5ml的YEP液体培养基中,28℃下220-250转/min培养24h;去1ml小样接入40~50mL YEP液体培养基中,28℃下220-250转/min培养12h,至菌液OD600为1.0~1.5左右;6000转离心10min收集菌体细胞,以适当体积的重悬液重悬至终浓度为1.2(OD600);
pTRV1分别与含有目的基因片段的pTRV2-CsPOR1,以及不含目的片段的空载体pTRV2的重悬液按体积比1∶1混匀,等体积混合的重悬液于室温下静置1-2h,采用真空侵染方法侵染去掉顶芽和叶片的茶苗。于昼/夜温度23/22℃,光/暗周期16h/8h的人工气候室内培养侵染后的茶树植株。在相同的环境下培养未经侵染处理和空载体(1∶1混和pTRV1和pTRV2)的对照植株,5-6周观察实验组和对照组的表型变化,并检测目的基因的表达情况,对每种材料处理40个单株。
(2)注射法:农杆菌培养方法和真空侵染法相同,菌液两两混合后在常温下放置两小时后,进行注射侵染,设置OD600值为1.0、1.2、1.5三组实验。操作如下:用去除针头的注射器吸等体积1:1混合的菌液,然后用不带针头的注射器挫伤茶苗子叶和叶片,最后用力将菌液注射到伤口处,直至出现浸渍状,每组处理40个单株。
图3为25d空载组和实验组对照图;图4为45d空载组和实验组对照图;图5为空载组和对照组叶片对照图。
在真空侵染实验中,本申请设计了压强和菌液浓度两个变量,每个变量三个梯度一共九组(见表1)实验其中侵染压强0.7kPa、菌液浓度1.2时获得基因沉默植株5株(12.5%),侵染压强0.8kPa、菌液浓度1.2获得基因沉默植株1株,其余组合没有获得基因沉默的白化苗,所以侵染压强为0.7-0.8kPa、菌液浓度1.2为优选侵染条件;而侵染压强为0.7kPa、菌液浓度1.2为最佳侵染条件。
表1真空侵染条件筛选
注射法侵染的茶树实验组茶苗全部没有表现出沉默表型,所以该方法对本实验无效。
综上两种侵染方法的结果分析,所我们选择真空侵染法作为茶树VIGS实验的侵染方法。8.通过荧光定量qRT-PCR检测CsPOR1目的基因表达量
采用TRNzol–A+(天根生化科技有限公司,北京)试剂盒提取样品总RNA利用PrimeScriptTMⅡ1ststrandcDNASynthesisKit(索莱宝技术有限公司,北京)进行反转录合成cDNA,将cDNA稀释10倍用于荧光定量PCR检测,以确定CsPOR1基因的表达情况,利用PrimerPremier5.0软件设计CsPOR1基因、pTRV1、pTRV2的特异性引物,以茶树Actin基因作为内参(表2)。qRT-PCR试验在BioRadCFXConnectTM实时定量PCR仪(Bio-Rad公司)上进行操作。qRT-PCR所用为南京诺唯赞通用型高灵敏度染料法定量PCR检测试剂盒,根据试剂盒上的使用说明,进行qRT-PCR反应体系配置。
表2载体与对应引物
qRT-PCR的条件和体系如下:一个变性循环(94℃,5min),然后40个扩增循环(94℃,30秒,50℃,45秒),以及信号采集(72℃,113秒),qRT_PCR选择50ul体系:Nuclease-freeWater20ul、Biorun Pfu PCR Mix 25ul、Template 1ul、Primer(+)(100μM)2ul、Primer(-)(100μM)2ul。
基因的相对表达水平用2–△Ct来分析。使用SPSS软件对数据进行方差分析,误差棒代表三个独立实验数据的±标准差。不同字母表示P<0.05差异显著、P<0.01差异极显著。空载组和实验组叶片中CsPOR1基因相对表达量,如图6所示。结果表明,茶树VIGS技术能够有效沉默CsPOR1基因,该方法可用于茶树同源基因功能的研究。
从图中可以看出,在一定的培养时间内,随着培养时间越长,实验组叶片中CsPOR1表达量越低,55d后达到最低值(实验结果见图7),叶片中CsPOR1表达量仅为空载组的30.5%,表明茶树VIGS技术诱导的基因沉默,在一定的时间内随时间的延长,沉默效率越高,可以通过该技术能在短时间内研究茶树中特异表达基因的功能。
本发明通过探究最佳侵染压强、菌液浓度等因素对茶树同源基因沉默效率的影响,弥补茶树没有完整遗传转化的不足。同时,在培养过程中的温度、湿度、光照时间等培养条件完全可控,大大缩短获得沉默植株的时间,节约实验成本,避免茶树内源基因在异源模式生物中不能完全表达或不表达的问题,为研究茶树基因功能和茶树种质创新开辟了一条新道路。
Claims (9)
1.一种用于茶树基因功能研究的VIGS方法,包括以下步骤;
1)目的基因的获得,
2)pTRV2-目的基因载体的构建,
3)pTRV2-目的基因载体热击法转化DH5α大肠杆菌感受态细胞,
4)冻融法将pTRV2-目的基因载体导入农杆菌GV3101,
5)选择侵染材料,
6)真空侵染,
7)暗培养和光照培养侵染茶苗后,常规培养;
所述侵染材料为长出3-4枚真叶且没有木质化的实生茶苗,侵染前去除实生茶苗的顶芽和所有叶片,所述真空侵染条件为:侵染压强为0.7-0.8kPa、菌液浓度D600为1.20;侵染时间为5-6min,所述菌液为体积比1:1的含pTRV1载体与pTRV2-目的基因载体的重悬液的混合菌液。
2.根据权利要求1所述的方法,所述真空侵染条件为:侵染压强为0.7kPa、菌液浓度D600为1.20;侵染时间为5-6min。
3.根据权利要求1所述的方法,所述重悬液配制方法,4.74g的MS、2ml的6-BA、30g的蔗糖、100ul的NAA、溶于1L 蒸馏水中,调pH为5.6,121℃高温高压灭菌21min后,在超净台中加入 0.2g 乙酰丁香酮AS,-20℃保存。
4.根据权利要求1所述的方法,所述侵染前将除去顶芽和所有叶片的实生茶苗置于混合菌液中浸泡1-2小时。
5.根据权利要求1所述的方法,所述茶树品种为福鼎大白茶。
6.根据权利要求5所述的方法,所述实生茶苗的培养方法为将茶树种子去除种皮放在催芽液中,在25℃,在16 h光照/ 8 h黑暗循环的人工气候室中萌发生长3-5周,所述催芽液为6-BA 100ul/L+赤霉素 600ul/L+氯霉素 5ml/L。
7.根据权利要求1所述的方法,所述暗培养和光照培养采用保鲜膜覆盖侵染茶苗,在保持湿润环境下培养2天。
8.根据权利要求7所述的方法,培养条件:培养室昼夜光周期为光/暗:16h /8h,温度为光/暗:25℃/18℃,相对湿度为65%,光源为日光灯。
9.根据权利要求1所述的方法,所述目的基因为CsPOR1基因,常规培养直至出现沉默的白化表型。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006072742A2 (fr) * | 2005-01-05 | 2006-07-13 | Institut De Recherche Pour Le Developpement (Ird) | Vecteurs viraux pour la surexpression ou pour l'extinction de genes chez les plantes et leurs applications |
CN101558158A (zh) * | 2004-09-24 | 2009-10-14 | 巴斯福植物科学有限公司 | 具有对环境胁迫增加的耐性的植物细胞和植物 |
WO2010080071A1 (en) * | 2009-01-09 | 2010-07-15 | Temasek Life Sciences Laboratory Limited | Functional analysis of jatropha curcas genes |
WO2010144058A1 (en) * | 2009-06-10 | 2010-12-16 | Temasek Life Sciences Laboratory Limited | Virus induced gene silencing (vigs) for functional analysis of genes in cotton. |
CN113736819A (zh) * | 2021-09-02 | 2021-12-03 | 浙江大学 | 一种蝴蝶花基因沉默体系的构建方法 |
-
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101558158A (zh) * | 2004-09-24 | 2009-10-14 | 巴斯福植物科学有限公司 | 具有对环境胁迫增加的耐性的植物细胞和植物 |
WO2006072742A2 (fr) * | 2005-01-05 | 2006-07-13 | Institut De Recherche Pour Le Developpement (Ird) | Vecteurs viraux pour la surexpression ou pour l'extinction de genes chez les plantes et leurs applications |
WO2010080071A1 (en) * | 2009-01-09 | 2010-07-15 | Temasek Life Sciences Laboratory Limited | Functional analysis of jatropha curcas genes |
WO2010144058A1 (en) * | 2009-06-10 | 2010-12-16 | Temasek Life Sciences Laboratory Limited | Virus induced gene silencing (vigs) for functional analysis of genes in cotton. |
CN113736819A (zh) * | 2021-09-02 | 2021-12-03 | 浙江大学 | 一种蝴蝶花基因沉默体系的构建方法 |
Non-Patent Citations (3)
Title |
---|
Guodong Li et al..Establishment of a Virus-Induced Gene-Silencing (VIGS) System in Tea Plant and Its Use in the Functional Analysis of CsTCS1.《Int. J. Mol. Sci.》.2022,第24卷第1-14页. * |
Jinhe Wang et al..Transcription factor CsDOF regulates glutamine metabolism in tea plants (Camellia sinensis).《Plant Science》.2020,第302卷第1-11页. * |
尹桥秀.拟盘多毛孢果胶裂解酶PL1B基因作为SIGS靶标的研究.《中国知网硕士电子期刊》.2022,(第5期),第1-104页. * |
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Application publication date: 20220826 Assignee: SANJIANG DONG AUTONOMOUS COUNTY XIANCHI TEA CO.,LTD. Assignor: Guizhou University Contract record no.: X2023980045628 Denomination of invention: A VIGS Method for Studying the Gene Function of Tea Trees Granted publication date: 20230502 License type: Common License Record date: 20231102 |