CN111334519A - 一种表达类胡萝卜素的融合基因、重组质粒及应用 - Google Patents
一种表达类胡萝卜素的融合基因、重组质粒及应用 Download PDFInfo
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- CN111334519A CN111334519A CN202010031666.9A CN202010031666A CN111334519A CN 111334519 A CN111334519 A CN 111334519A CN 202010031666 A CN202010031666 A CN 202010031666A CN 111334519 A CN111334519 A CN 111334519A
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Abstract
本发明提供了一种表达类胡萝卜素的融合基因、重组质粒及其应用,涉及转基因技术领域,将来自莱茵衣藻的β‑胡萝卜素酮化酶基因(CrBKT)、来自雨生红球藻的羟化酶基因(HpBHY)、八氢番茄红素合成酶基因(CrtB)以及促进有色体分化与类胡萝卜素储藏的基因Orange构建成融合基因,然后在种子特异性启动子NAPIN控制下通过农杆菌花序侵染法导入油料作物亚麻荠中,可使亚麻荠种子中合成并积累高含量类胡萝卜素(410.42μg/g干重)和虾青素(13.32μg/g干重),且在不影响不饱和脂肪酸的含量前提下,提高种子总抗氧化能力。
Description
技术领域
本发明属于转基因技术领域,具体涉及一种表达类胡萝卜素的融合基因、重组质粒及应用。
背景技术
类胡萝卜素在光合生物中具有辅助吸收光能和保护叶绿素等多种生理功能。植物的特异器官(如花和果)和绿藻能够合成和积累大量不参与光合作用的次生类胡萝卜素。次生类胡萝卜素是人类和动物获取必需类胡萝卜素 (β-胡萝卜素,叶黄素和玉米黄素)的主要来源。
虾青素是一种分子结构独特的红色酯溶性酮式类胡萝卜素,具有极强的抗氧化活性以及抗辐射、抗衰老、抗肿瘤和预防心血管等疾病的功能,由于其对人体健康方面的强大功能已被批准应用于食品添加剂。虾青素的生物合成只发生于少量细菌和绿藻中,且产量很低;植物中也只有夏侧金盏花 (Adonis aestivalis)可以在其花瓣中合成微量的虾青素。目前广泛应用于虾青素生产的是雨生红球藻,它是目前所知合成虾青素效率最高的生物,可以达到4%(40mg/g),然而雨生红球藻的生长条件苛刻、生物产量不高、生产成本高致使虾青素的总产量受到了抑制。由于虾青素的自然资源有限,科学家们都在通过基因工程方法寻求不同的植物来生产更高产量的天然虾青素,并在莴苣、马铃薯、小麦、油菜、烟草、番茄和水稻等植物中获得成功。
亚麻荠(Camelina sativa(L.))是十字花科亚麻荠属1年生草本植物,具有耐贫瘠、抗干旱、抗病虫害、耐盐碱等优良的农艺学特征;且亚麻荠种子油中含有油酸、亚油酸、亚麻酸等丰富人体必需的多不饱和脂肪酸,具有很高的营养价值,然而目前并不能够在亚麻荠的种子中成功表达类胡萝卜素。
发明内容
有鉴于此,本发明的目的在于一种表达类胡萝卜素的融合基因、重组质粒及其应用,可以在亚麻荠的种子种成功表达类胡萝卜素,同时还能在亚麻荠种子中合成虾青素,进一步提高亚麻荠的营养品质,为国内首例在亚麻荠种子中合成高价值虾青素。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种表达类胡萝卜素的融合基因,所述融合基因的结构包括:5’-八氢番茄红素合成酶基因CrtB-羟化酶基因HpBHY-β-胡萝卜素酮化酶基因CrBKT-促进类胡萝卜素合成基因Orange-3’;相邻两个基因间以2A序列连接。
优选的,所述融合基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种包含所述融合基因的重组质粒。
优选的,所述重组质粒以PBI121为基础载体,将所述融合基因构建进 NAPIN启动子下游。
本发明还提供了一种包含所述融合基因或所述重组质粒的重组菌。
优选的,所述重组菌以农杆菌为基础菌株。
本发明提供了一种在亚麻荠种子中表达类胡萝卜素的方法,包括以下步骤:利用农杆菌Floral Dip方法将所述重组菌侵染亚麻荠的花序部分。
优选的,在所述侵染后,还包括将所述亚麻荠的植株培育至果荚成熟,收集种子,并对种子进行转基因验证。
优选的,所述转基因验证包括观察,当所述种子呈红色时,表示转基因成功;当所述种子呈黄色时,表示转基因未成功。
优选的,所述转基因验证包括PCR验证,所述PCR验证的引物包括正向引物F和反向引物R,所述正向引物F的核苷酸序列如SEQ ID NO.2所示,所述反向引物R的核苷酸序列如SEQ ID NO.3所示。
本发明提供了一种表达类胡萝卜素的融合基因,将来自莱茵衣藻的β-胡萝卜素酮化酶基因(CrBKT)、来自雨生红球藻的羟化酶基因(HpBHY)、八氢番茄红素合成酶基因(CrtB)以及促进有色体分化与类胡萝卜素储藏的基因Orange(来自花椰菜)构建成融合基因,同时为了简化表达载体的构建和实现各基因在亚麻荠种子中的协同表达将四个关键酶基因用手足口病 2A自切割肽段连接,在种子特异性启动子NAPIN控制下通过农杆菌花序侵染法导入油料作物亚麻荠中。
转化成功的种子由于类胡萝卜素的积累而呈现暗红色,未转化成功的种子为淡黄色,两者颜色相差较大,可以通过肉眼直接鉴定转化是否成功,简单快捷。
本发明利用促进合成、降低产物降解的策略获得了能在亚麻荠种子中合成并积累高含量类胡萝卜素(410.42μg/g干重)的高品质亚麻荠株系,其类胡萝卜素含量是野生型(13.21μg/g干重)的31倍,更为重要的是虾青素含量由0 增加至13.32μg/g干重,进一步提高了亚麻荠的营养品质,为国内首例在亚麻荠种子中合成高价值虾青素的株系。同时该转化株系种子亚麻酸(C18:3)、亚油酸(C18:2)、油酸(C18:1)含量与野生型没有明显差异,而种子总抗氧化能力转化株系显著高于野生型。
利用亚麻荠作为宿主,创建高产虾青素的亚麻荠,一方面是因为亚麻荠是重要的油料作物,营养价值高,引入虾青素增强了亚麻荠籽油的营养水平,是高端用油的理想选择;另一方面简化了虾青素的提取工艺,含有虾青素的亚麻荠籽油可以直接通过常规物理榨油方式获得,这比传统的通过超临界 CO2临界萃取的方法简单快捷,且低成本高效率;因此以此种具有优良品质的亚麻荠生产高品质的兼具不饱和脂肪酸和虾青素的食用油具有极大的商业前景。
附图说明
图1为重组质粒的结构图谱;
图2为T0代转化子N-BBBO(右)与野生型(左)种子对比图;
图3为转化子N-BBBO PCR鉴定图;
图4为转化子N-BBBO(A)与野生型(B)类胡萝卜素UHPLC色谱图,其中峰1表示Neoxanthin,2表示Astaxanthin,3表示keto-lutein,4表示lutein, 5表示keto-lutein,6表示β-Cryptoxanthin,7表示Echinenone,8表示lycepene, 9表示α-carotene,10表示β-carotene;
图5为转化子N-BBBO与野生型种子总抗氧化能力比较图。
具体实施方式
本发明提供了一种表达类胡萝卜素的融合基因,所述融合基因的结构包括:5’-八氢番茄红素合成酶基因CrtB-羟化酶基因HpBHY-β-胡萝卜素酮化酶基因CrBKT-促进类胡萝卜素合成基因Orange-3’;相邻两个基因间以2A序列连接。
本发明所述融合基因的结构优选如图1所示,本发明所述八氢番茄红素合成酶基因CrtB优选来源于雨生红球藻(Haematococcus Pluvialis),所述羟化酶基因(HpBHY)优选来源于雨生红球藻(Haematococcus Pluvialis),所述β-胡萝卜素酮化酶基因(CrBKT)优选来源于莱茵衣藻(Chlamydomonas reinhartii),所述促进类胡萝卜素合成基因Orange优选来源于花椰菜,其中,所述基因的登录号分别为:CrtB(GenBank:CP001875.2),HpBHY(GenBank:KP866868.1),CrBKT(GenBank:AY860820.1),Orange (GenBank:AT5G61670)。本发明优选在所述CrtB、HpBHY和CrBKT三个基因的5’端ATG前融合有拟南芥RBCS2的叶绿体导肽序列(TP),各基因间通过2A序列链接(SEQ ID NO.4:cagctgctgaactttgatctgctgaaactggctggtgacgtggagtctaaccct),使四个基因的表达在同一阅读框但翻译时形成各自编码的肽。本发明所述2A序列优选为手足口病2A自切割肽段。本发明所述融合基因的核苷酸序列优选如SEQ ID NO.1所示。
本发明还提供了一种包含所述融合基因的重组质粒,所述重组质粒优选以PBI121为基础载体,将所述融合基因构建进NAPIN启动子下游。本发明所述融合基因的获得方法并没有特殊限定优选通过人工全合成的方式获得。本发明对所述构建的方法并没有特殊限定,利用本领域的常规载体构体方法即可。
本发明还提供了一种包含所述融合基因或所述重组质粒的重组菌,所述重组菌优选以农杆菌为基础菌株。本发明对所述农杆菌的来源并没有特殊限定,利用本领域的常规市售农杆菌感受态即可。
本发明提供了一种在亚麻荠种子中表达类胡萝卜素的方法,包括以下步骤:利用农杆菌花序侵染法将所述重组菌侵染亚麻荠的花序部分。本发明对所述花序侵染法的具体步骤并没有特殊限定,优选包括:取-70℃保存的含有重组质粒的重组菌,在含有50mg/L链霉素和50mg/L卡那霉素的LB固体培养上划线,28℃下培养约36h;从平板上挑取单菌落接种于3mL含有上述抗生素的LB液体培养基中,28℃条件下,220rpm振荡培养过夜;吸取 2mL农杆菌菌液加入250mL含有上述相应抗生素的LB液体培养基中,28℃条件下,220rpm振荡培养至OD600为0.8~1.0,5000rpm离心10min收集菌体,用等体积渗透培养基(1/2MS,5%蔗糖,0.05%Silwet L-77,pH=5.7) 悬浮菌体;转化时将种植亚麻荠植株的盆用保鲜膜包裹,植株倒立于真空桶中,花序部分浸入渗透培养基中;盖上真空桶盖,抽真空于80Kpa时开始计时,5min后将植株取出平放于铺了保鲜膜的地面并盖上黑色塑料袋;24h后将植株放置于温室培养。本发明所述侵染优选还包括在所述侵染一星期后重复浸染一次,而后将植株置于温室常规培养直至果荚成熟,收取成熟种子标记为T0代,用于后续实验。
本发明在所述侵染后,优选还包括将所述亚麻荠的植株培育至果荚成熟,收集种子,并对种子进行转基因验证。本发明所述转基因验证优选包括观察,当所述种子呈红色时,表示转基因成功;当所述种子呈黄色时,表示转基因未成功。本发明所述转基因验证优选还包括PCR验证,所述PCR验证的引物优选包括正向引物F和反向引物R,所述正向引物F的核苷酸序列优选如SEQ ID NO.2所示:GAACCTGGCAGTGTGGTTC,所述反向引物R 的核苷酸序列优选如SEQ ID NO.3所示:AAAGGACTGCAGGCGGGATG。本发明对所述PCR验证的体系并没有特殊限定,优选的所述体系内的模板 DNA优选来源于T0代种子播种后生长的植株叶片。本发明对所述播种和 DNA提取的方法并没有特殊限定。
下面结合实施例对本发明提供的表达类胡萝卜素的融合基因、重组质粒及其应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、研究材料
亚麻荠(Camelina sativa(L.))种质材料SC-N16,由山西农业大学作物遗传育种重点实验室提供参见苑丽霞,郝敬云,赵奎,等.2EMS诱变亚麻荠获得2S贮藏蛋白缺失突变体.华北农学报,2016,31(3):11-17。
将亚麻荠种子播种于直径9cm的育苗盆中,1粒/盆;盆中基质为腐殖质∶泥炭土∶珍珠岩=5∶4∶1;温室温度为16~22℃,光源为自然光,相对湿度为60%。待植株生长至10cm时进行摘心处理,以促进侧枝的生长和发育。植株生长约70d时进入花期准备转化,转化前一天将植株浇透水。共对20株(盆)植株进行处理,5株(盆)不处理作为对照。
2、研究方法
2.1构建载体
由昆明擎科生物科技有限公司全合成所述融合基因后,将其克隆于 PBI121的NAPIN启动子后形成目的质粒(简称N-BBBO)。
2.2农杆菌Floral Dip转化亚麻荠
取-70℃保存的含有重组质粒的农杆菌N-BBBO,在含有50mg/L链霉素和50mg/L卡那霉素的LB固体培养上划线,28℃下培养约36h。从平板上挑取单菌落接种于3mL含有上述抗生素的LB液体培养基中,28℃条件下, 220rpm/min振荡培养过夜。吸取2mL农杆菌菌液加入250mL含有上述相应抗生素的LB液体培养基中,28℃条件下,220rpm/min振荡培养至OD600为 0.8~1.0,5000rpm/min离心10min收集菌体,用等体积渗透培养基(1/2MS, 5%蔗糖,0.05%Silwet L-77,pH=5.7)悬浮菌体。转化时将种植亚麻荠植株的盆用保鲜膜包裹,植株倒立于真空桶中,花序部分浸入渗透培养基中。盖上真空桶盖,抽真空于80Kpa时开始计时,5min后将植株取出平放于铺了保鲜膜的地面并盖上黑色塑料袋。24h后将植株放置于温室培养,一星期后按照以上方法重复浸染一次后将植株置于温室常规培养直至果荚成熟,收取成熟种子标记为T0代,用于后续实验。
3、转基因植株的鉴定
3.1T0代种子颜色鉴定:由于类胡萝卜素为红色、黄色、橘红色色素,它在植物器官中合成后会改变植物器官的颜色,收获的T0代种子通过肉眼观察颜色鉴别转化成功种子,同时计算转化率。
类胡萝卜素在亚麻荠种子中积累,T0代转化成功种子与未转化种子在颜色上有明显差异,转化成功的种子呈现明显的暗红色,而未转化成功的种子呈现淡黄色(图2)。22株(盆)共收获种子3519粒,其中红色种子(转化成功)15粒,转化率为4/1000。
3.2PCR鉴定:为了进一步验证,将初期筛选到的红色T0代种子进行萌发,种植于温室,采集植株叶片并提取DNA,用CRBKT引物进行扩增正向引物SEQ ID NO.2:5'-GAACCTGGCAGTGTGGTTC-3';
反向引物SEQ ID NO.3:5'-AAAGGACTGCAGGCGGGATG-3'。
PCR体系:Taq-酶10μL,反向引物:1μL,正向引物:1μL,DNA:1μL, ddH2O:7μL;
PCR程序:94℃预变性3min;94℃变性30s,56℃退火30s,72℃延伸 20s,33个循环;72℃延伸2min。
PCR扩增结果如图3所示。
3.3类胡萝卜素提取及测定
3.3.1提取方法:准确称量T0亚麻荠种子(0.2~0.3g)于2.0mg EP管中,每个样品设置3个重复,加入2mm钢珠2颗,200μL丙酮溶液,置于快速研磨仪中(频率65Hz,99s)研磨5次,再加500μL丙酮溶液,放置于超声波清洗器超声洗脱30min。高速离心(14800rmp,10min)取上清,用 0.22μm滤器过滤后上样测定。
3.3.2检测方法:仪器:Agilent Technologies 1290Infinity;色谱柱: ZORBAXEclipse(3.0×50mm 1.8-Micron,Agilent,USA);流速:1mL/min;进样量:5μL;
流动相程序:0~1.0min:水:20%,乙腈:60%,异丙醇:5%,甲醇: 15%;
1.0-2.0min:水:0%,乙腈:80%,异丙醇:5%,甲醇:15%;
2.0-8.0min:水:0%,乙腈:80%,异丙醇:5%,甲醇:15%;
得到的色谱图通过与标准品出峰时间及色谱峰图来确定每个峰的成分,如图4所示。
3.3.3类胡萝卜素定量:采用外标法对样品中的类胡萝卜素含量进行定量。结果如表1所示:野生型种子中仅含有叶黄素,含量为13.21μg/g(干重,DW),而转化子N-BBBO种子中含有10种已确定类胡萝卜素,包含新黄质(Neoxanthin)、酮式叶黄素(ketolutein)、虾青素(Astaxanthin)、叶黄素(lutein)、隐黄质(β-Cryptoxanthin)、海胆酮(Echinenone)、番茄红素(lycopene)、α胡萝卜素(α-carotene)、β胡萝卜素(β-carotene),总类胡萝卜素含量为410.42μg/gDW,是野生型的31倍,虾青素含量也由0 增加至13.32μg/gDW。
表1转化子N-BBBO与野生型类胡萝卜素含量比较
**表示在p<0.05水平上的显著性差异,数据表示为平均值±标准误,n=3。
3.4脂肪酸提取及测定
3.4.1提取方法:准确称量T0代亚麻荠种子(0.2~0.3g),充分研磨。加入6mL氯仿甲醇(体积比2:1)混合液体,摇床(150rpm/min)放置1h,再加入1.5mL的0.7%的氯化钾溶液混匀,1000rpm/min离心5min,吸取上层液体置于玻璃试管中,烘干液体。向试管中加入10mL2%的硫酸甲醇溶液, 85℃水浴1h,直至油滴全部溶解溶液至无色,室温降温5min后加入1.5mL 的饱和氯化钠溶液,1mL正己烷,2000rmp离心10min,取上清于样品瓶中上样测定。
3.4.2测定方法:仪器:AgilentTechnologies 7890/5975;色谱柱:DB-5(30 m×250μm×0.25μm,Agilent,USA);氦气流速:1.2mL/min;进样量:1μL;分流比:20:1;进样口的温度:250℃;柱温的程序:初始温度为170℃,随后以5℃/min的速度升到250℃,并在此温度保持5min。通过比较样品和化合物库(NIST)的出峰时间以及每个峰的质荷比来确定脂肪酸的组分,脂肪酸的相对含量通过各成分峰面积比值确定。
结果如表2所示,N-BBBO转化子总脂肪酸含量为118.42mg/g与野生型(131.31mg/g)相当,且两者的亚麻酸(C18:3)、亚油酸(C18:2)、油酸(C18:1)也没有明显差异,说明类胡萝卜素的积累对脂肪酸的合成没有影响。
表2转化子N-BBBO与野生型脂肪酸成分及含量比较
SFA表示饱和脂肪酸,PUFA表示不饱和脂肪酸,TFA表示总脂肪酸。数据表示为平均值±标准误,n=3。
3.5种子总抗氧化能力测定
用苏州格锐思生物科技有限公司总抗氧化能力试剂盒进行种子总抗氧化能力的测定。
测定结果如图5所示,N-BBBO转化子种子总抗氧化能力为8.32μmol/g,野生型为3.02μmol/g,转化子种子总抗氧化能力明显高于野生型。高含量的总类胡萝卜素,尤其是虾青素的积累明显提高了种子总抗氧化能力。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院昆明植物研究所
<120> 一种表达类胡萝卜素的融合基因、重组质粒及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5657
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cccgggacaa agagtaaaga agaacaatgg catcttctat gctgtcttct gcgactatgg 60
ttgcgtctcc ggctcaggct actatggtgg caccgttcaa cggtctgaag tcctccgcgg 120
ctttcccggc tactcgtaaa gcgaacaacg acattaccag cattacctct aacggtggtc 180
gtgtgaactg catgaacaac ccgtctctgc tgaaccatgc ggtggaaacc atggcggtgg 240
gttccaaatc cttcgcgacc gcgtctaagc tgttcgatgc aaagacccgt cgttctgtgc 300
tgatgctgta tgcatggtgc cgccattgtg acgacgttat cgacgaccag actctgggct 360
tccaggcacg tcagccggcg ctgcagactc cggaacagcg tctgatgcag ctggagatga 420
agactcgcca ggcttatgca ggctctcaga tgcacgaacc ggcgttcgct gctttccagg 480
aagttgcgat ggcgcatgat atcgctccgg catacgcgtt tgatcatctg gaaggcttcg 540
ctatggacgt gcgcgaggcg cagtactccc agctggacga taccctgcgc tactgctacc 600
acgttgctgg tgttgtgggc ctgatgatgg cacagatcat gggtgttcgt gataacgcga 660
ccctggatcg cgcatgtgac ctgggtctgg cattccagct gactaacatt gcgcgtgaca 720
ttgttgacga tgcgcacgca ggccgttgtt atctgccggc gtcttggctg gaacacgaag 780
gcctgaacaa agaaaactac gctgcaccgg aaaaccgtca ggcactgtct cgcatcgctc 840
gccgcctggt gcaggaagca gagccgtact atctgagcgc gactgcgggc ctggctggcc 900
tgccgctgcg tagcgcttgg gcgattgcta ccgctaagca ggtgtaccgc aaaatcggtg 960
tgaaggttga gcaggcaggt cagcaggcgt gggatcagcg tcagtccacc actaccccgg 1020
aaaaactgac cctgctgctg gcagctagcg gccaggcgct gacttctcgt atgcgtgcac 1080
atccgccgcg tccggcacac ctgtggcagc gtccgctgtc tagacagctg ctgaactttg 1140
atctgctgaa actggctggt gacgtggagt ctaaccctgg tccgatggcg tcctctatgc 1200
tgtcttctgc tactatggtg gcatctccgg ctcaggcaac tatggtggca ccgtttaacg 1260
gtctgaagtc ttccgcagct ttcccggcaa ctcgcaaggc aaacaacgat attactagca 1320
ttacttccaa cggtggccgt gttaactgca tgctgagcaa gctgcagtct attagcgtta 1380
aggctcgccg tgtggagctg gctcgtgaca ttacccgtcc gaaagtttgt ctgcacgcgc 1440
agcgttgtag cctggtgcgt ctgcgtgtgg cggctccgca gactgaagag gcactgggta 1500
ccgttcaggc tgcaggtgcg ggcgacgaac actccgctga tgtggctctg cagcagctgg 1560
atcgtgcgat tgcagaacgt cgtgcgcgtc gcaaacgcga acagctgagc tatcaggcag 1620
cggctatcgc tgcttctatt ggtgtgtccg gtattgcgat cttcgcgacc tatctgcgtt 1680
tcgctatgca catgactgtg ggtggtgcgg ttccgtgggg cgaagttgcg ggtactctgc 1740
tgctggttgt tggtggcgct ctgggcatgg agatgtatgc gcgttatgcg cacaaagcga 1800
tctggcacga gtccccgctg ggttggctgc tgcacaaaag ccatcacacc ccgcgtaccg 1860
gtccgttcga ggctaacgat ctgtttgcaa tcatcaacgg cctgccggcg atgctgctgt 1920
gcaccttcgg cttctggctg ccgaacgtgc tgggcgcggc ttgtttcggc gcaggtctgg 1980
gtatcactct gtacggcatg gcgtacatgt tcgtgcatga tggtctggtt catcgtcgtt 2040
tcccgactgg cccgatcgcg ggtctgccgt atatgaaacg cctgactgtt gctcatcagc 2100
tgcatcattc tggcaaatat ggcggtgcgc cgtggggtat gttcctgggt ccgcaggaac 2160
tgcagcatat tccgggtgcg gctgaggaag tggaacgtct ggtgctggaa ctggactggt 2220
ccaaacgtca gctgctgaac ttcgatctgc tgaaactggc gggtgacgtg gaatctaacc 2280
ctggtccgat ggctagctct atgctgtcct ccgctaccat ggttgcgtcc ccggcacagg 2340
cgactatggt ggctccgttc aacggcctga aatctagcgc tgcatttccg gcgactcgta 2400
aggcgaacaa cgacatcact tctatcacca gcaacggcgg tcgcgttaac tgtatgggtc 2460
cgggtatcca gccgaccagc gcgcgtccgt gttctcgtac taaacactct cgttttgcac 2520
tgctggcggc agcactgacc gctcgtcgtg tgaaacagtt caccaagcag tttcgttctc 2580
gccgcatggc agaggacatt ctgaagctgt ggcagcgtca gtatcatctg ccgcgtgaag 2640
attctgacaa gcgtaccctg cgtgagcgcg ttcatctgta tcgtccgccg cgtagcgacc 2700
tgggcggcat cgcggttgcg gttaccgtta ttgctctgtg ggcaaccctg tttgtttatg 2760
gcctgtggtt cgttaagctg ccgtgggcgc tgaaagttgg tgagaccgct acttcttggg 2820
cgaccatcgc agcggttttc ttctctctgg aattcctgta cactggtctg ttcatcacca 2880
ctcacgatgc gatgcacggt actatcgcac tgcgtaaccg tcgtctgaac gattttctgg 2940
gtcagctggc aatttccctg tacgcgtggt ttgactattc tgtgctgcac cgtaaacatt 3000
gggaacatca taaccatacc ggcgaaccgc gtgttgaccc ggacttccat cgtggcaacc 3060
cgaacctggc agtgtggttc gcgcagttta tggtgagcta catgactctg tcccagtttc 3120
tgaagatcgc ggtttggtcc aacctgctgc tgctggcggg tgcgccgctg gcaaaccagc 3180
tgctgtttat gaccgcagca ccgatcctgt ctgcgttccg cctgttctat tacggcactt 3240
atgttccgca ccatccggag aagggccata ctggcgcgat gccgtggcag gtttcccgca 3300
cttcttccgc atcccgcctg cagtcctttc tgacttgcta tcatttcgac ctgcactggg 3360
agcatcatcg ttggccgtat gctccgtggt gggagctgcc gaaatgccgt cagatcgcac 3420
gtggcgcagc tctggctcag ctgctgaact tcgatctgct gaagctggca ggcgacgttg 3480
aatccaaccc tgggcccatg gcttcttctg catttgcttt tccttcttac ataataacca 3540
aaggaggact ttcaactgat tcttgtaaat caacttcttt gtcttcttct agatctttgg 3600
ttacagatct tccatcacca tgtctgaaac ccaacaacaa ttcccattca aacagaagag 3660
caaaagtgtg tgcttcactt gcagagaagg gtgaatatta ttcaaacaga ccaccaactc 3720
cattacttga cactattaac tacccaatcc acatgaaaaa tctttctgtc aaggaactga 3780
aacaactttc tgatgagctg agatcagacg tgatctttaa tgtgtcgaaa accggtggac 3840
atttggggtc aagtcttggt gttgtggagc ttactgtggc tcttcattac attttcaata 3900
ctccacaaga caagattctt tgggatgttg gtcatcagtc ttatcctcat aagattctta 3960
ctgggagaag aggaaagatg cctacaatga ggcaaaccaa tggtctctct ggtttcacca 4020
aacgaggaga gagtgaacat gattgctttg gtactggaca cagctcaacc acaatatctg 4080
ctggtttagg aatggcggta ggaagggatt tgaaggggaa gaacaacaat gtggttgctg 4140
tgattggtga tggtgcgatg acggcaggac aggcttatga agccatgaac aacgccggat 4200
atctagactc tgatatgatt gtgattctta atgacaacaa gcaagtctca ttacctacag 4260
ctactttgga tggaccaagt ccacctgttg gtgcattgag cagtgctctt agtcggttac 4320
agtctaaccc ggctctcaga gagttgagag aagtcgcaaa gggtatgaca aagcaaatag 4380
gcggaccaat gcatcagttg gcggctaagg tagatgtgta tgctcgagga atgataagcg 4440
gtactggatc gtcactgttt gaagaactcg gtctttacta tattggtcca gttgatgggc 4500
acaacataga tgatttggta gccattctta aagaagttaa gagtaccaga accacaggac 4560
ctgtacttat tcatgtggtg acggagaaag gtcgtggtta tccttacgcg gagagagctg 4620
atgacaaata ccatggtgtt gtgaaatttg atccagcaac gggtagacag ttcaaaacta 4680
ctaatgagac tcaatcttac acaacttact ttgcggaggc attagtcgca gaagcagagg 4740
tagacaaaga tgtggttgcg attcatgcag ccatgggagg tggaaccggg ttaaatctct 4800
ttcaacgtcg cttcccaaca agatgtttcg atgtaggaat agcggaacaa cacgcagtta 4860
cttttgctgc gggtttagcc tgtgaaggcc ttaaaccctt ctgtgcaatc tattcgtctt 4920
tcatgcagcg tgcttatgac caggttgtcc atgatgttga tttgcaaaaa ttaccggtga 4980
gatttgcaat ggatagagct ggactcgttg gagctgatgg tccgacacat tgtggagctt 5040
tcgatgtgac atttatggct tgtcttccta acatgatagt gatggctcca tcagatgaag 5100
cagatctctt taacatggtt gcaactgctg ttgcgattga tgatcgtcct tcttgtttcc 5160
gttaccctag aggtaacggt attggagttg cattacctcc cggaaacaaa ggtgttccaa 5220
ttgagattgg gaaaggtaga attttaaagg aaggagagag agttgcgttg ttgggttatg 5280
gctcagcagt tcagagctgt ttaggagcgg ctgtaatgct cgaagaacgc ggattaaacg 5340
taactgtagc ggatgcacgg ttttgcaagc cattggaccg tgctctcatt cgcagcttag 5400
ctaagtcgca cgaggttctg atcacggttg aagaaggttc cattggaggt tttggctcgc 5460
acgttgttca gtttcttgct ctcgatggtc ttcttgatgg caaactcaag tggagaccaa 5520
tggtactgcc tgatcgatac attgatcacg gtgcaccagc tgatcaacta gctgaagctg 5580
gactcatgcc atctcacatc gcagcaaccg cacttaactt aatcggtgca ccaagggaag 5640
ctctgttttg agagctc 5657
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaacctggca gtgtggttc 19
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaggactgc aggcgggatg 20
<210> 4
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cagctgctga actttgatct gctgaaactg gctggtgacg tggagtctaa ccct 54
Claims (10)
1.一种表达类胡萝卜素的融合基因,其特征在于,所述融合基因的结构包括:5’-八氢番茄红素合成酶基因CrtB-羟化酶基因HpBHY-β-胡萝卜素酮化酶基因CrBKT-促进类胡萝卜素合成基因Orange-3’;相邻两个基因间以2A序列连接。
2.根据权利要求1所述融合基因,其特征在于,所述融合基因的核苷酸序列如SEQ IDNO.1所示。
3.一种包含权利要求1或2所述融合基因的重组质粒。
4.根据权利要求3所述重组质粒,其特征在于,所述重组质粒以PBI121为基础载体,将所述融合基因构建到NAPIN启动子下游。
5.一种包含权利要求1或2所述融合基因或权利要求3或4所述重组质粒的重组菌。
6.根据权利要求5所述重组菌,其特征在于,所述重组菌以农杆菌为基础菌株。
7.一种在亚麻荠种子中表达类胡萝卜素的方法,其特征在于,包括以下步骤:利用农杆菌花序侵染法将权利要求5或6所述重组菌侵染亚麻荠的花序部分。
8.根据权利要求7所述方法,其特征在于,在所述侵染后,还包括将所述亚麻荠的植株培育至果荚成熟,收集种子,并对种子进行转基因验证。
9.根据权利要求8所述方法,其特征在于,所述转基因验证包括观察,当所述种子呈红色时,表示转基因成功;当所述种子呈黄色时,表示转基因未成功。
10.根据权利要求8所述方法,其特征在于,所述转基因验证包括PCR验证,所述PCR验证的引物包括正向引物F和反向引物R,所述正向引物F的核苷酸序列如SEQ ID NO.2所示,所述反向引物R的核苷酸序列如SEQ ID NO.3所示。
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CN102888425A (zh) * | 2012-07-02 | 2013-01-23 | 中国科学院昆明植物研究所 | 利用转基因植物生产虾青素的方法 |
CN105907780A (zh) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | 一种在作物种子胚乳生产虾青素的转基因育种方法 |
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CN105907780A (zh) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | 一种在作物种子胚乳生产虾青素的转基因育种方法 |
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