CN116555331A - 一种粉红色棉花纤维和种子的转基因材料及其应用 - Google Patents
一种粉红色棉花纤维和种子的转基因材料及其应用 Download PDFInfo
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Abstract
本发明公开了一种粉红色棉花纤维和种子的转基因材料及其应用，所述转基因材料的构建方法为：针对棉花密码子优化了CYP76AD1、L‑DOPA 4,5‑dioxygenase、Betanidin‑5‑O‑glucosyltransferase三个基因的CDS序列，构建包括这三个优化后CDS序列的重组载体，转化陆地棉材料，获得粉红色棉花纤维及种子的转基因植株。该粉红色转基因棉花纤维在成熟过程中呈现紫红色，随着纤维发育，紫红色逐渐变浅，最终成熟纤维呈现出不同程度深浅的粉红色。本发明证明了在棉花中共表达甜菜碱合成关键基因以产生天然粉红色可行性。能够在不影响纤维产量和质量的情况下提供彩色纤维，为彩色棉及种子研究提供了新思路，为创制更多颜色的棉花纤维奠定了基础。
Description
技术领域
本发明公开了一种粉红色棉花纤维和种子的转基因材料及其应用，属于基因工程领域。
背景技术
棉花是世界上最重要的天然纤维来源和经济作物之一，其中白色棉花占主导地位。由于颜色单调，因此棉花经棉纺厂纺织后会进行染色，但是纺织品染色产生的大量合成染料排放到环境中，极大地危害人类和其他生物的健康。为了减少污染和节约用水，迫切需要环保的替代品。彩色棉是一种具有天然纤维色泽的棉花，可以有效减少纺织过程中对环境造成的污染以及对人体的危害，是真正的绿色产品。随着人民生活需求、环保意识和健康观念的不断提高，彩色棉因其具有天然的颜色，在纺织生产上可以不需要漂白、消毒、染色等过程，减轻了纺织过程对环境造成的污染，降低了企业生产成本，减小了对身体的危害，成为真正意义上的绿色产品。随着现代生物科学技术的迅速发展，为彩色棉育种的深入研究创造了良好的环境。
甜菜红素是世界上广泛使用的一种水溶性含氮色素，甜菜、火龙果和其他植物中见到的鲜红色就是甜菜红素积累的结果，该色素无毒副作用且色泽鲜艳，鉴于其毒理学安全性和可能对健康有利的生物影响，甜菜红素是唯一允许用于食物中甜菜红素添加剂的来源。甜菜碱由橙黄色的甜菜红素和红紫色的甜菜红素这两类化合物组成，这些化合物是通过一系列的酶催化步骤合成的，包括产生颜色的羟基酶、双加氧酶和葡萄糖基转移酶。由于锦葵科植物通常不产生甜菜碱，因此本发明的目的在于在世界上最大的植物性纤维——陆地棉中进行了甜菜碱途径的基因工程操作，从而获得一种粉红色棉花纤维和种子的转基因材料。
发明内容
针对现有技术的不足，本发明的目的是提供一种粉红色棉花纤维和种子的转基因材料的构建方法及其应用，能够获得产生粉红色棉花纤维和种子的转基因材料。
为了实现上述目的，本发明的技术方案是：
一种粉红色棉花纤维和种子的转基因材料，所述转基因材料的构建方法为：针对棉花密码子优化了CYP76AD1、L-DOPA4,5-dioxygenase、Betanidin-5-O-glucosyltransferase三个基因的CDS序列，构建包括这三个优化后CDS序列的重组载体，转化陆地棉材料，获得粉红色棉花纤维及种子的转基因植株。具体的构建方法包括以下步骤：
(1)针对棉花密码子优化了CYP76AD1、L-DOPA4,5-dioxygenase、Betanidin-5-O-glucosyltransferase三个基因的CDS序列，合成包含由编码2A肽的序列依次串联的优化后的甜菜红素催化合成酶基因CYP76AD1、L-DOPA4,5-dioxygenase和Betanidin-5-O-glucosyltransferase，命名为CDB基因，构建到pUC57载体上，获得含有目的基因片段的pUC57-CDB质粒；
(2)以pUC57-CDB质粒为模板，进行PCR扩增，并回收目的基因片段；
(3)将WMV067载体进行酶切，获得线性载体；
(4)目的基因片段和酶切过的线性载体进行重组连接，构建重组载体；
(5)将重组载体转化大肠杆菌，挑取阳性克隆；
(6)将阳性克隆培养后，提取质粒；
(7)将提取的质粒在农杆菌的介导下转化陆地棉材料，再经过出苗-生根-炼苗-移栽/嫁接，获得粉红色棉花纤维及种子的转基因植株。
所述优化后的CYP76AD1基因的CDS序列如SEQ ID NO.1所示；所述优化后的L-DOPA4,5-dioxygenase基因的CDS序列如SEQ ID NO.2所示；所述优化后的Betanidin-5-O-glucosyltransferase基因的CDS序列如SEQ ID NO.3所示。
所述的陆地棉材料为中棉所49号。
一种含有优化后CYP76AD1、L-DOPA 4,5-dioxygenase、Betanidin-5-O-glucosyltransferase基因的CDS序列的重组载体。
所述重组载体由35S组成型启动子或E6纤维特异性启动子驱动。
所述优化后的CYP76AD1基因的CDS序列如SEQ ID NO.1所示；所述优化后的L-DOPA4,5-dioxygenase基因的CDS序列如SEQ ID NO.2所示；所述优化后的Betanidin-5-O-glucosyltransferase基因的CDS序列如SEQ ID NO.3所示。
一种所述的转基因材料在培育彩色纤维和种子的棉花新品种中的应用。
所述彩色为粉红色。
本发明的有益效果：
本发明设计的载体包括三个基因CYP76AD1、L-DOPA 4,5-dioxygenase、Betanidin-5-O-glucosyltransferase的编码序列(CDS序列)，通过密码子优化软件，根据陆地棉密码子偏好性，对三个基因的CDS序列进行了优化，并进行合成。甜菜碱基因分别由35S组成型启动子和E6纤维特异性启动子驱动，转化陆地棉高产品种(中棉所49号)中，再经过出苗-生根-炼苗-移栽/嫁接，获得粉红色棉花纤维及种子的转基因植株。该粉红色转基因棉花纤维在成熟过程中呈现紫红色，随着纤维发育，紫红色逐渐变浅，最终成熟纤维呈现出不同程度深浅的粉红色。本发明证明了在棉花中共表达甜菜碱合成关键基因以产生天然粉红色可行性。能够在不影响纤维产量和质量的情况下提供彩色纤维，为彩色棉及种子研究提供了新思路，为创制更多颜色的棉花纤维奠定了基础。同时，也为生产既环保又健康的彩棉纤维提供了不用化工染料染色的合成替代品。此外，还显示了在棉花中生产甜菜碱的潜力，提供了食品着色剂的来源。
附图说明
图1为WMV067载体构造图谱。
图2为35S CaMv启动子驱动的三个关键的甜菜碱基因载体构造图谱。
图3为E6启动子驱动的三个关键的甜菜碱基因载体构造图谱。
图4为转WMV067-35S::CDB基因棉花材料分子检测结果，片段大小1400bp。1为marker，2为野生型对照，3-13为转基因植株(其中12为阴性材料)，14为质粒阳性对照。
图5为转WMV067-E6::CDB基因棉花材料分子检测结果，片段大小520bp。15为marker，1-11为转基因植株(其中10为阴性材料)，12为质粒阳性对照，13和14为野生型对照。
图6为甜菜碱在转基因植株的叶、茎、棉铃、苞片、花、花药和种子中的积累。从上向下各行依次为：野生型植株、E6启动子驱动的转基因植株和35S启动子驱动的转基因植株。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1：茎尖转化载体构建
(1)通过密码子优化软件，根据陆地棉密码子偏好性，对三个基因CYP76AD1、L-DOPA4,5-dioxygenase、Betanidin-5-O-glucosyltransferase的CDS序列进行优化，合成包含由编码2A肽的序列依次串联的优化后的甜菜红素催化合成酶基因CYP76AD1(序列如SEQID NO.1所示)、L-DOPA4,5-dioxygenase(序列如SEQ ID NO.2所示)和Betanidin-5-O-glucosyltransferase(序列如SEQ ID NO.3所示)的CDS序列，简称CDB基因。将密码子优化合成的CDB基因构建到pUC57载体(通用载体)上，获得含有目的基因片段的pUC57-CDB质粒。
(2)目的片段扩增：以pUC57-CDB质粒为模板，设计引物F和R，用诺唯赞新一代高保真DNA聚合酶进行PCR反应。
PCR反应体系为：2×Phanta Max Master Mix 25μL；引物F 2μL(10μM)；引物R 2μL(10μM)；模板质粒(pUC57-CDB)1μL(150ng/μL)；ddH2O 20μL。
引物F：5’-AGAGAACACGGGGGACgtcgacatggatcatgcgaccctcgcc-3’(SEQ ID NO.4)
引物R：5’-GGGGAAATTCGAGCTCACTAGTtcactatcactggaggcttggc-3’(SEQ ID NO.5)
PCR反应程序如下：95℃5min；95℃20s，55℃25s，72℃4min(1min/1000bp)，34个循环；72℃10min，10℃保存。
对PCR扩增产物进行1.5％琼脂糖凝胶电泳(126v，25min)，CDB序列全长为3939bp。利用胶回收试剂盒纯化并获得带接头序列的目的片段产物，-20℃冰箱保存。
(3)酶切：WMV067载体(序列如SEQ ID NO.6所示)的多克隆位点选择合适的酶切位点，该载体有35S启动子以及壮观霉素抗性基因aadA(图1)。选用50μL酶切体系，用NewEngland BioLabs(NEB)公司限制性内切酶SalI和SpeI对载体进行双酶切。酶切体系为：WMV067载体1μL(3000ng)；限制性内切酶SalI 3μL；限制性内切酶SpeI 3μL；CutSmartBuffer 5μL；加ddH2O补足至50μL。酶切体系置于37℃恒温水浴锅酶切3h，同时以空载体质粒为对照进行琼脂糖凝胶电泳(126v，25min)，确认条带后，对成功切开的线性载体进行胶回收纯化。
(4)同源重组：用诺唯赞ClonExpressII one Step Cloning Kit同源重组试剂盒将(2)中所得到的目的片段和(3)中成功切开的线性载体重组连接，反应体系为10μL，具体包含：5×CEII Buffer 2μL；同源重组酶Exnase II 1μL；酶切过的线性载体1μL(50ng/μL)；带接头的目的片段1μL(35ng/μL)；加ddH2O补足至10μL。体系配制完成后，37℃水浴30min，置于冰上5min终止反应。将重组好的载体命名为WMV067-35S::CDB(如图2所示)。将载体WMV067-35S::CDB的35S启动子序列切下来，再将E6启动子序列连上去，得到重组载体WMV067-E6::CDB(如图3所示)。
(5)转化大肠杆菌：将重组载体WMV067-35S::CDB和WMV067-E6::CDB通过42℃热激转化到大肠杆菌感受态DH5α中，加600μL LB液体培养基，放37℃摇床进行扩大培养。1h后涂布在Kana抗性的LB固体培养基，24h后挑选单克隆进行阳性检测。
(6)提取质粒：挑取阳性克隆加入含有相应抗生素(壮观霉素)的5mL LB液体培养基中，置于37℃摇床培养后提取质粒。
(7)转化农杆菌：将提取的质粒WMV067-35S::CDB和WMV067-E6::CDB转化到EHA105农杆菌感受态中，加600μL LB液体培养基，放28℃摇床进行扩大培养。2h后涂布在Rif(利福平)+Kana(卡那霉素)抗性的LB固体培养基中，60h后挑选单克隆进行阳性检测。挑取阳性克隆(命名为J12-35S::CDB和J12-E6::CDB)加入含有相应抗生素(壮观霉素)的5mL LB液体培养基中，置于28℃摇床培养后，用50％的甘油保菌并存放于-80℃冰箱。
实施例2：茎尖转化棉花
(1)农杆菌制备：
将-80℃保存的J12-35S::CDB和J12-E6::CDB农杆菌分别涂在含有卡那霉素和利福平的LB固体培养基上，在28℃恒温箱培养2天，得到活化的J12-35S::CDB和J12-E6::CDB农杆菌，侵染前一天再次涂板LB固体培养基，28℃恒温箱培养1天。次日用接种环收集平板上的农杆菌，悬浮在液体侵染培养基(成分：MS盐和B5维生素的混合物(M404)4.2g/L、葡萄糖30g/L、4-吗啉乙磺酸4.2g/L、6-苄基腺嘌呤0.1mg/L、萘乙酸0.1mg/L)中。用无菌吸管吹打菌块，直至农杆菌细胞均匀的分散在悬浮液中，用分光光度计测量农杆菌悬浮液，光吸收值OD660nm约为0.9，得到J12-35S::CDB悬浮液和J12-E6::CDB悬浮液，备用。
(2)遗传转化受体准备：
将受体中棉所49号脱绒后在40℃烘箱中烘干，取300粒脱绒种子置于200mL三角瓶中，加入200mL医用酒精(75％，v/v)，摇晃30s，弃去废液，之后加入200mL质量浓度为6％的双氧水，剧烈摇晃15min后，弃去废液，之后用无菌水清洗3-4次，加入MSB液体培养基中，过夜培养，24h后种子露白，用解剖刀剖开种皮，剥开子叶，暴露叶原基下部的茎尖分生组织，进行下一步侵染。
(3)农杆菌侵染及共培养：
以叶原基下部的茎尖分生组织作为受体外植体，将携带有含目的基因质粒J12-35S::CDB和J12-E6::CDB的农杆菌分别导入外植体，使农杆菌携带的目的基因的T-DNA插入棉花基因组。具体方法为：将外植体放入加入10mL液体侵染培养基的无菌三角瓶中。每瓶处理100-150个外植体后，弃去侵染培养基，加入15mLJ12-35S::CDB或J12-E6::CDB悬浮液，放入超声波清洗仪中处理，超声处理频率为40kHz，时长40s。放入摇床室温摇50min。
侵染后将外植体放在无菌滤纸上吸干表面菌液，并在超净工作台上吹10min，放入共培养基(成分：葡萄糖30g/L、MES(吗啉乙磺酸)4g/L、6-BA(苄基腺嘌呤)0.1mg/L、NAA(萘乙酸)0.1mg/L)中，23℃黑暗共培养3-4天。
(3)恢复培养：
将共培养后的外植体移植到恢复培养基(成分：MS盐和B5维生素的混合物(M404)4.2g/L、葡萄糖30g/L、琼脂粉4g/L、6-苄基腺嘌呤0.1mg/L、萘乙酸0.1mg/L、头孢霉素100mg/L)中，50个外植体转移到一个培养皿，胚根插入到恢复培养基中。在35℃、光周期为光明16h/黑暗8h的条件下培养3天后转移到25℃、光周期为光明16h/黑暗8h的条件下继续培养4天。
(4)芽诱导和筛选：
恢复培养后，将外植体切去根部，移入芽诱导培养基(成分：MS盐和B5维生素的混合物(M404)4.2g/L、葡萄糖30g/L、琼脂粉4g/L、6-苄基腺嘌呤0.1mg/L、萘乙酸0.1mg/L、壮观霉素和头孢霉素各100mg/L)在25℃、光周期为光明16h/黑暗8h的条件下培养，2周继代一次，共培养3次。
(5)生根培养：
将外植体插入到生根培养基(成分：MS盐和B5维生素的混合物(M404)4.2g/L、葡萄糖10g/L、琼脂粉4g/L、头孢霉素100mg/L)中，在25℃、光周期为光明16h/黑暗8h的条件下培养。每2周进行一次继代培养(炼苗)，待抗性芽伸长后进行嫁接或移栽，得到的即为T0代转化植株。
(6)转基因棉花植株鉴定：
将T0代转化植株生根后移栽到育苗基质中，常规肥水管理，等待其生长，取幼嫩叶片进行PCR检测。
分别以转基因棉花(T0代转化植株)、质粒(WMV067-35S::CDB和WMV067-E6::CDB)和野生型棉花DNA作为模板进行PCR扩增；针对目的基因的引物对由F1和R1组成，由生工生物工程(上海)有限公司合成。
35S::CDB-F1：5’-atcatggccgacaagcagaa-3’(SEQ ID NO.7)
35S::CDB-R1：5’-tctcgttggggtctttgctc-3’(SEQ ID NO.8)
E6::CDB-F1：5’-TTTCAAATACCCATTTTTGA-3’(SEQ ID NO.9)
E6::CDB-R1：5’-CATTCTCCCACACTGCTAAACAG-3’(SEQ ID NO.10)
PCR反应体系：10×PCR Mix 10μL、F1 1μL(10μM)、R1 1μL(10μM)、DNA 1μL(20ng/μL)，加RNase Free ddH2O至20μL。
PCR反应程序为：预热98℃10s；98℃10s，55℃30s，72℃1min，30个循环反应；72℃延伸5min；4℃保存。
部分PCR扩增结果见图4和5。由图4和5可知，两个重组载体均得到转基因阳性植株。将阳性植株移栽到温室，开花期全程自交收种，得到T0代转基因种子，第二年在保护区内播种T0代收获的种子，即为T1代转基因植株。
实施例3：大田试验
在大田条件下对T1代转基因植株和野生型植株进行了表型分析(图6)。由图6可知，所有的转基因株系都积累了甜菜碱，但是积累模式根据启动子的不同有差别。在35S启动子驱动的转基因株系中，叶、茎、棉铃、苞片、花、花药和种子呈现紫红色或粉红色，野生型植株则是正常绿色。在E6启动子驱动的转基因植株中，粉红色仅在纤维中可见，而在种子、叶、茎、苞片、花和花药等其他组织中则不明显，表型颜色与野生型植株一致都是绿色。相比之下，35S启动子驱动的植株在整个植株中都显示出紫红色或粉红色，最引人注目的是在其整个发育过程中都有粉红色的纤维。
Claims (9)
1.一种粉红色棉花纤维和种子的转基因材料，其特征在于，所述转基因材料的构建方法为：针对棉花密码子优化了CYP76AD1、L-DOPA 4,5-dioxygenase、
Betanidin-5-O-glucosyltransferase三个基因的CDS序列，构建包括这三个优化后CDS序列的重组载体，转化陆地棉材料，获得粉红色棉花纤维及种子的转基因植株。
2.根据权利要求1所述的转基因材料，其特征在于，具体的构建方法包括以下步骤：
(1)针对棉花密码子优化了CYP76AD1、L-DOPA 4,5-dioxygenase、
Betanidin-5-O-glucosyltransferase三个基因的CDS序列，合成包含由编码2A肽的序列依次串联的优化后的甜菜红素催化合成酶基因CYP76AD1、L-DOPA 4,5-dioxygenase和Betanidin-5-O-glucosyltransferase，命名为CDB基因，构建到pUC57载体上，获得含有目的基因片段的pUC57-CDB质粒；
(2)以pUC57-CDB质粒为模板，进行PCR扩增，并回收目的基因片段；
(3)将WMV067载体进行酶切，获得线性载体；
(4)目的基因片段和酶切过的线性载体进行重组连接，构建重组载体；
(5)将重组载体转化大肠杆菌，挑取阳性克隆；
(6)将阳性克隆培养后，提取质粒；
(7)将提取的质粒在农杆菌的介导下转化陆地棉材料，获得粉红色棉花纤维及种子的转基因植株。
3.根据权利要求1所述的转基因材料，其特征在于，所述优化后的CYP76AD1基因的CDS序列如SEQ ID NO.1所示；所述优化后的L-DOPA 4,5-dioxygenase基因的CDS序列如SEQ IDNO.2所示；所述优化后的Betanidin-5-O-glucosyltransferase基因的CDS序列如SEQIDNO.3所示。
4.根据权利要求1所述的转基因材料，其特征在于，所述的陆地棉材料为中棉所49号。
5.一种含有优化后CYP76AD1、L-DOPA 4,5-dioxygenase、
Betanidin-5-O-glucosyltransferase基因的CDS序列的重组载体。
6.根据权利要求5所述的重组载体，其特征在于，所述重组载体由35S组成型启动子或E6纤维特异性启动子驱动。
7.根据权利要求5所述的重组载体，其特征在于，所述优化后的CYP76AD1基因的CDS序列如SEQ ID NO.1所示；所述优化后的L-DOPA 4,5-dioxygenase基因的CDS序列如SEQIDNO.2所示；所述优化后的Betanidin-5-O-glucosyltransferase基因的CDS序列如SEQ IDNO.3所示。
8.一种如权利要求1-4任一项所述的转基因材料在培育彩色纤维和种子的棉花新品种中的应用。
9.根据权利要求8所述的应用，其特征在于，所述彩色为粉红色。
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