CN116987701A - 不结球白菜BcERF070基因的基因工程应用 - Google Patents
不结球白菜BcERF070基因的基因工程应用 Download PDFInfo
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Abstract
不结球白菜BcERF070基因调控不结球白菜Vc含量中的应用，本研究验证了BcERF070基因在不结球白菜Vc合成途径中的重要作用，构建CRISPR/Cas9基因编辑载体并转化到不结球白菜中，与非转基因植株相比，转基因植株的BcERF070基因序列发生碱基替换，植株Vc含量随之下降，并进而实现了提早不结球白菜开花时间、缩短育种周期、提高蔬菜品质，为创造不结球白菜新种质提供技术支持。
Description
技术领域
本发明属于分子生物学和生物技术领域，具体涉及不结球白菜BcERF070基因调控不结球白菜Vc含量中的应用。
背景技术
植物中维生素C(vitamin C，以下简称Vc)又称L-抗坏血酸(L-ascorbic acid，AsA)，在植物自身的抗氧化系统、光合保护以及调节生长发育中起到至关重要的作用，更为我们提供了丰富而方便的Vc补充。针对植物中的Vc合成的研究一直在进行中，通过基因工程技术提高AsA含量，探索基因功能是当前研究的热门方向。
不结球白菜在植物学上属于十字花科芸薹属植物，常规育种方法受限于种质资源的匮乏，难有作为，而植物基因工程技术可以突破物种间的界限，转移外源的有用基因，为芸薹属作物改良提供了新途径。芸薹属植物常用的遗传转化方法有农杆菌介导法、基因枪法、电击法等，其中农杆菌介导法应用最广泛，方法最成熟。农杆菌介导法转基因技术就是使分别含有Ti质粒和Ri质粒的根癌农杆菌和发根农杆菌携带目的基因，利用农杆菌诱导植物伤口产生冠瘿瘤或发状根的特性，将质粒上T-DNA携带的目的基因插入到植物基因组中，再通过组织培养技术，再生出携带目的基因的转基因植株的技术方法。1988年，Zhang等发现芸薹属作物的再生与基因型密切相关。1990年Boulter在甘蓝型油菜的遗传转化研究中发现，根癌农杆菌有比发根农杆菌更高的转化频率，从此芸薹属蔬菜遗传转化体系的研究开始不断取得新进展，积累了丰富的研究材料。
如何通过基因工程获得突变体验证基因功能、调控不结球白菜中的VC含量，进而创造不结球白菜新种质具有重要的现实意义。
发明内容
本研究验证了该基因在Vc合成途径中的重要作用，成功获得该基因的基因编辑突变体植株，与野生型植株相比，基因编辑植株的BcERF070基因序列碱基发生替换，植株Vc含量随之下降。为之后通过基因工程提早不结球白菜开花时间、缩短育种周期、创造蔬菜新种质提供了理论依据和实践依据。
本发明的第一个目的是提供不结球白菜BcERF070基因的基因工程应用，敲除BcERF070基因，能够降低不结球白菜Vc含量。
本发明的第二个目的是提供不结球白菜BcERF070基因的基因工程应用，敲除BcERF070基因，能够提早不结球白菜开花时间。
进一步的，前述应用中，所述BcERF070基因的核苷酸序列如SEQ ID NO.1所示：ATGAAGCGAATCGTGAGGATATCATTCACCGACGTGGAGGCCACCGATTCTTCCAGCAGCGAAGACGATCAGACGAACACCGAATCACCGTCGCCACGAAAAGGGAAGAGGTTCGTCAAGGAGATCGTCATCGACCCATCCGATTCCGCCGAGGTGAGAAAGACGCGGTTTAAGATCAGGATTCCGGCGAGGCTTACGAAGAAGTTCCGAGGTGTGAGGCAGAGGCCGTGGGGGAAATGGGCGGCTGAGATCAGGTGCGGTAAAGCTCACGGTGGAATTCGCAACGGGGGACCTGTTCGTCTTTGGCTTGGGACATTCGAAACCGCCGAGGAAGCTGCTTTGGCTTACGACAAGGCCGCGATTCGGCTTATTGGGCCTCACGCGCCGATCAATTTCGGCCCAGAATCTCCGGCTGTGAAGCAAGATTCCGTTGCGGGGGACTGA
进一步的，前述应用中，所述敲除BcERF070基因为将BcERF070基因CRISPR/Cas9载体导入不结球白菜，导致BcERF070基因序列改变，失去作用。
在某一个特定的实施例中，敲除BcERF070基因具体包括如下步骤：
(1)BcERF070基因CRISPR载体构建
根据在线网站http://cbi.hzau.edu.cn/cgi-bin/CRISPR设计sgRNA，将SEQ IDNO.1所示BcERF070的序列输入网站，得到一系列长为20bp的序列，设计sgRNA：GTGTGAGGCAGAGGCCGTGG(SEQ ID NO.2)，5’-NGG-3’为PAM(所述N代表任一碱基)。
上述SEQ ID NO.1所示BcERF070基因核苷酸序列中，下划线处为SEQ ID NO.2所示sgRNA，加粗处为PAM。
选择PAM结构前二十个碱基作为前引物，反向互补作为后引物，设计sgRNA引物，分别是，从5’-3’：
sgRNA-1-F:GATTGTGTGAGGCAGAGGCCGTGG(SEQ ID NO.3)
sgRNA-1-R:AAACCCACGGCCTCTGCCTCACAC(SEQ ID NO.4)
5μl sgRNA-1-F+5μl sgRNA-1-R直接退火；
程序：第一步，95℃，3min；第二步，95℃，1min；第三步，回到步骤二，每次下降1℃，重复40次；第四步，55℃，30min；第五步，55℃，1min；第六步，回到步骤五，每次下降1℃，重复30次；最后4℃保存。
将退火产物连到Bpi 1单酶切的PMD18-T质粒(含有psgR-Cas9-At，由浙江大学提供)上，体系为退火产物2μl、载体2μl、5×buffer 2μl、T4连接酶1μl、H2O 3μl，连接产物转化大肠杆菌；提取带有片段的PMD18-T质粒后酶切(酶切位点Kpn1和HindIII)连接到Kpn1和HindIII酶切过的pCAMBIA1301上，连接产物转化大肠杆菌；测序鉴定确保表达载体中编码区阅读框架正确，得到CRISPR载体。
(2)植物表达载体质粒转化农杆菌
将(1)得到的CRISPR载体分别加入到感受态农杆菌，混匀后冰浴10min，随后液氮速冻5min，迅速移至37℃水浴中5min，冰浴5min，加入900ul新鲜的LB液体培养基，于28℃，250rpm振荡培养2～3h，然后6000rpm离心5min，留取100ul左右上清吹打重悬菌液，涂布于含50mg/L卡那霉素和20mg/L利福平的LB固体培养基，28℃暗培养2-3d直到平板上长出单菌落，PCR鉴定后获得携带有CRISPR/Cas9基因编辑载体的农杆菌GV3101。
(3)农杆菌介导法转化不结球白菜
①无菌苗的获得：将种子用75％酒精(无水乙醇与灭菌水体积比为3:1配制)振荡2分钟，再用次氯酸钠(吸取5.6％次氯酸钠溶液1mL，灭菌水9ml混合)振荡15分钟消毒，无菌水清洗后用滤纸吸干水分播种到发芽培养基，放到光培箱培养5～7天，至幼苗子叶完全展开。
②获得无菌外植体：将步骤①剪取带柄子叶及下胚轴，作为外植体平铺于预培养基，光培箱培养3天。
③外植体与农杆菌共培养：将②得到的农杆菌OD600调为0.2，在28℃摇床，250rpm振荡3h后，侵染外植体8min，用无菌滤纸吸去多余的农杆菌液，置于共培养基(垫无菌滤纸)，于光培箱暗培养3天。
④获得植株：将经过共培养的外植体转移到分化培养基，光培箱培养20-30天后将单个芽转移至筛选培养基进行筛选培养，约20d后选择绿色的不定芽转移到生根培养基，约20d根系健壮后炼苗移栽。
⑤转基因植株检测：体式镜荧光检测根部或桑格测序法。
步骤①发芽培养基条件为1/2MS，琼脂7g/L，蔗糖30g/L，pH值为5.8，不添加激素。
步骤①选用酒精和次氯酸钠为消毒液，所述酒精浓度为75％，可通过以下方法制备所得：无水乙醇与灭菌水体积比为3:1配制；所述次氯酸钠溶液有效氯含量为0.56％，可通过以下方法制备所得：吸取5.6％次氯酸钠溶液1mL，灭菌水9ml混合。
步骤①中次氯酸钠消毒时间为15分钟，且消毒后用无菌水清洗5次，每次10秒。
步骤①中播种后在光培箱中培养5天，此时幼苗子叶已完全展开。
步骤②中选取的外植体材料为带柄子叶和下胚轴，下胚轴长度为0.5cm，带有1～2mm子叶柄的子叶及5mm左右的下胚轴更容易生成愈伤分化出芽。
步骤②中获得外植体后光培箱培养时间为3天，此过程有助于伤口产生愈伤细胞，减少共培养后褐化几率。
步骤②中预培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L噻苯隆(TDZ)、0.25mg/L萘乙酸(NAA)、7.5mg/L AgNO3，pH值为5.8。
本发明步骤③中为先将步骤②得到的农杆菌在4000rpm，10min离心一次之后，去上清液，采用农杆菌悬浮液重悬浮。
本发明步骤③中农杆菌悬浮液为1/10Ms液体培养基，配制方法为：0.474g/L MS培养基、3g/L蔗糖，pH为5.2。
本发明步骤③中农杆菌悬浮液在使用前要添加乙酰丁香酮(As)，终浓度为100mmol。
本发明步骤③中农杆菌OD600为0.2，之后在28℃摇床，250rpm振荡3h，外植体侵染时间为8分钟，此时转化效率高又不会对外植体造成过多伤害。
本发明步骤③中共培养基使用时垫无菌滤纸。
本发明步骤③中共培养时间为3天，且在黑暗条件下进行，可以有效控制农杆菌的生长。
本发明步骤③中共培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L TDZ、0.25mg/L NAA、7.5mg/L AgNO3，pH值为5.2。
本发明步骤④中分化时间为20～30天，继代培养和生根培养各20d左右。
本发明步骤④中分化培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L TDZ、0.25mg/L NAA、5mg/L AgNO3、160mg/L Carb、160mg/L特美汀、0.2mM VC，pH值为5.8，该培养基简单有效，一次分化培养后就可以形成愈伤和芽，并且玻璃化及褐化少。
本发明步骤④中筛选培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、2mg/L 6-BA、0.1mg/L NAA、7.5mg/L AgNO3、160mg/L Carb、160mg/L特美汀、0.2mM VC、5mg/L Hyg(潮霉素)，pH值为5.8。
本发明步骤④中生根培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、0.2mg/LNAA、7.5mg/L AgNO3、160mg/L Carb、160mg/L Tim，pH值为5.8。
本发明步骤①～④中光培箱环境为(25±5℃)16h光照8h黑暗，光照度5000lx。
有益效果
Vc对人体及植物本身都具有非常重要的作用，本发明通过基因工程探究不结球白菜体内的Vc合成通路，对不结球白菜进行遗传改良。
本发明通过基因编辑技术实现了植物体内的BcERF070基因的碱基替换，不结球白菜体内Vc含量降低，并进而提早不结球白菜开花时间，缩短育种周期。可操作性强，实验周期短，且目标性状单一，获得的转基因植株与非转基因对照相比，BcERF070基因发生突变后，Vc含量相比较对照组(未编辑植株)下降了37.5％，验证了BcERF070基因在植物Vc合成途经中的重要作用。本发明通过基因工程技术调控不结球白菜的VC含量，为创造白菜类蔬菜的新种质和遗传改良提供了新思路和技术支持，具有重要的现实意义。
附图说明
图1为组培外植体出芽。
图2为潮霉素筛选抗性芽。
图3为抗潮霉素植株移栽后。
图4为转基因植株的PCR测序结果。
图5为转基因苏州青植株Vc含量测定结果。
图6为定植20d后再生材料开花情况。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1.BcERF070基因CRISPR载体构建
根据在线网站http://cbi.hzau.edu.cn/cgi-bin/CRISPR设计sgRNA，将SEQ IDNO.1所示BcERF070的序列输入网站，得到一系列长为20bp的序列，设计sgRNA，5’-NGG-3’为PAM。选择PAM结构前二十个碱基作为前引物，反向互补作为后引物，设计sgRNA引物，分别是，从5’-3’：
sgRNA-1-F:GATTGTGTGAGGCAGAGGCCGTGG(SEQ ID NO.3)
sgRNA-1-R:AAACCCACGGCCTCTGCCTCACAC(SEQ ID NO.4)
5μl F+5μl R直接退火；
程序：第一步，95℃，3min；第二步，95℃，1min；第三步，回到步骤二，每次下降1℃，重复40次；第四步，55℃，30min；第五步，55℃，1min；第六步，回到步骤五，每次下降1℃，重复30次；最后4℃保存。
将退火产物连到Bpi 1单酶切的PMD18-T质粒(Amp+抗性)(含有psgR-Cas9-At，由浙江大学提供)上，体系为退火产物2μl、载体2μl、5×buffer 2μl、T4连接酶1μl、H2O 3μl，连接产物转化大肠杆菌；提取带有片段的PMD18-T质粒后酶切(酶切位点Kpn1和HindIII)连接到Kpn1和HindIII酶切过的pCAMBIA1301(Kana抗性)上，连接产物转化大肠杆菌；测序鉴定确保表达载体中编码区阅读框架正确，得到CRISPR/Cas9载体，记为CRISPR载体。实施例2.植物表达载体质粒转化农杆菌
将实施例1得到的CRISPR/Cas9载体分别加入到感受态农杆菌，混匀后冰浴10min，随后液氮速冻5min，迅速移至37℃水浴中5min，冰浴5min，加入900ul新鲜的LB液体培养基，于28℃，250rpm振荡培养2～3h，然后6000rpm离心5min，留取100ul左右上清吹打重悬菌液，涂布于含50mg/L卡那霉素和20mg/L利福平的LB固体培养基，28℃暗培养2～3d直到平板上长出单菌落，PCR鉴定后获得携带有CRISPR/Cas9载体的农杆菌GV3101。以上工作需在超净工作台上进行无菌操作。
实施例3.农杆菌介导法转化苏州青
1.无菌苗的获得：将种子用75％酒精(无水乙醇与灭菌水体积比为3:1配制)振荡2分钟，再用次氯酸钠(吸取5.6％次氯酸钠溶液1mL，灭菌水9ml混合)振荡15分钟消毒，无菌水清洗清洗5次，每次10秒，后用滤纸吸干水分播种到发芽培养基，放到光培箱培养5天，至幼苗子叶完全展开。
发芽培养基为1/2MS，琼脂7g/L，蔗糖30g/L，pH值为5.8，不添加激素。
2.获得无菌外植体：将获得的幼苗剪取带柄子叶及下胚轴作为外植体，下胚轴每段5mm，带柄子叶带有1～2mm子叶柄，平铺于预培养基，光培箱培养3天。预培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L噻苯隆(TDZ)、0.25mg/L萘乙酸(NAA)、7.5mg/LAgNO3，pH值为5.8。
3.外植体与农杆菌共培养：为先将步骤②得到的农杆菌在4000rpm，10min离心一次之后，去上清液，采用农杆菌悬浮液重悬浮。农杆菌悬浮液为添加终浓度为100mmol的乙酰丁香酮的1/10Ms液体培养基(0.474g/L MS培养基、3g/L蔗糖，pH为5.2)，将农杆菌OD600调为0.2，在28℃摇床，250rpm振荡3h后，侵染外植体8min，用无菌滤纸吸去多余的农杆菌液，置于共培养基(垫无菌滤纸)，于光培箱暗培养3天。共培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L TDZ、0.25mg/L NAA、7.5mg/L AgNO3，pH值为5.2。
4.获得植株：将经过共培养的外植体转移到分化培养基，光培箱培养20～30天后外植体出芽(图1)，将单个芽转移至筛选培养基进行筛选培养，约20d后选择绿色的不定芽(图2)转移到生根培养基，约20d根系健壮后炼苗移栽获得T0代植株(图3)。分化培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、3mg/L TDZ、0.25mg/L NAA、5mg/L AgNO3、160mg/LCarb、160mg/L特美汀、0.2mM VC，pH值为5.8；筛选培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、2mg/L 6-BA、0.1mg/L NAA、7.5mg/L AgNO3、160mg/L Carb、160mg/L特美汀、0.2mM VC、5mg/L Hyg(潮霉素)，pH值为5.8；生根培养基配比为：MS培养基、7g/L琼脂、30g/L蔗糖、0.2mg/L NAA、7.5mg/L AgNO3、160mg/L Carb、160mg/L Tim，pH值为5.8。
实施例4转基因植株的PCR鉴定
取实施例3获得的5株抗潮霉素植株新生叶片提取基因组DNA，克隆BcERF070基因，用PCR方法进行检测，野生型植株为阳性对照模板，水为阴性对照模板，所用引物(5’至3’)为：
F：ATGAAGCGAATCGTGAGGAT(SEQ ID NO.5)
R：TCAGTCCCCCGCAACGGAAT(SEQ ID NO.6)
PCR反应程序为：预变性94℃30s；变性98℃10s；退火53℃Tm 30s；延伸72℃1min30s，34个循环；再延伸72℃1min；10℃保存。PCR检测结果送测序，结果返回后与SEQ IDNO.1所示BcERF070基因序列比对看是否发生碱基缺失或替换，#45及#51在目标片段上相比较于BcERF070基因序列在SEQ ID NO.2所示sgRNA发生了碱基的替换(图4)，确定株系#45及#51为阳性转基因植株。
实施例5转基因苏州青植株Vc含量测定
以相同时期的非转基因阴性对照(ck)和实施例5得到的转基因植株(#45及#51)叶片为材料，用高效液相色谱仪测定植物体内AsA含量。具体方法:称取0.2g植物叶片，加入1.5ml0.1％草酸中研磨，转移至2ml离心管中，12000rpm4℃离心15min，上清液供测定用。色谱条件为流动相0.1％乙酸，流速1ml/min，进样10μl，柱温30℃，检测波长245nm。获得的转基因植株与非转基因对照相比，Vc含量相比较对照组下降了37.5％(图5)。
维生素C标准曲线制定：100ml/L抗坏血酸标准液经过过滤，分别上样2.5μl，5μl，7.5μl，10μl，12.5μl，15μl，以峰面积为纵坐标，标准浓度为横坐标绘制标准曲线。
实施例6统计开花情况
观察并记录3株同一批再生幼苗对照组(未发生编辑)和2株实施例5得到的转基因植株即基因编辑植株开花情况，结果显示：定植20d左右，基因编辑植株均出现花蕾(基因编辑植株1、2)，其中一株甚至出现提前开花现象(基因编辑植株2)，而对照组均未抽薹开花，基因编辑植株相比较对照组出现提前抽薹开花的现象(图6)；定植40d后，对照组共3株开花率为33.3％，基因编辑植株均出现开花现象；对照组在定植约两个月时全部开花。可以看出，BcERF070基因发生碱基替换后，能够提早不结球白菜开花时间。
本发明验证了ERF070基因在植物Vc合成途径中的重要作用，获得了ERF070转基因植株，为辅助不结球白菜基因在调控开花时间功能的验证提供了数据支持，具有重要的现实意义。
序列表
<110> 南京农业大学
<120> 不结球白菜BcERF070基因的基因工程应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 444
<212> DNA
<213> 不结球白菜(Brassica campestris ssp. chinensis Makino)
<400> 1
atgaagcgaa tcgtgaggat atcattcacc gacgtggagg ccaccgattc ttccagcagc 60
gaagacgatc agacgaacac cgaatcaccg tcgccacgaa aagggaagag gttcgtcaag 120
gagatcgtca tcgacccatc cgattccgcc gaggtgagaa agacgcggtt taagatcagg 180
attccggcga ggcttacgaa gaagttccga ggtgtgaggc agaggccgtg ggggaaatgg 240
gcggctgaga tcaggtgcgg taaagctcac ggtggaattc gcaacggggg acctgttcgt 300
ctttggcttg ggacattcga aaccgccgag gaagctgctt tggcttacga caaggccgcg 360
attcggctta ttgggcctca cgcgccgatc aatttcggcc cagaatctcc ggctgtgaag 420
caagattccg ttgcggggga ctga 444
<210> 2
<211> 20
<212> DNA
<213> 不结球白菜(Brassica campestris ssp. chinensis Makino)
<400> 2
gtgtgaggca gaggccgtgg 20
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gattgtgtga ggcagaggcc gtgg 24
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaacccacgg cctctgcctc acac 24
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaagcgaa tcgtgaggat 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcagtccccc gcaacggaat 20
Claims (4)
1.不结球白菜BcERF070基因的基因工程应用，其特征在于，敲除BcERF070基因，能够降低不结球白菜Vc含量。
2.不结球白菜BcERF070基因的基因工程应用，其特征在于，敲除BcERF070基因，能够提早不结球白菜开花时间。
3.根据权利要求1或2所述的应用，其特征在于，所述BcERF070基因的核苷酸序列如SEQID NO.1所示。
4.根据权利要求1或2所述的应用，其特征在于，所述敲除BcERF070基因为将BcERF070基因CRISPR/Cas9载体导入不结球白菜，导致BcERF070基因序列改变，失去作用。
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