CN102295691B - Bccp2基因及其在提高植物及藻类油脂含量方面的应用 - Google Patents
Bccp2基因及其在提高植物及藻类油脂含量方面的应用 Download PDFInfo
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Abstract
本发明提供了BnaA.BCCP2.a基因及其在提高植物及藻类油脂含量方面的应用。该基因来源于植物甘蓝型油菜(Brassica napus)。该基因编码异质型乙酰辅酶A羧化酶(ACCase)的生物素羧基载体蛋白亚基(BCCP)。该发明涉及BnaA.BCCP2.a基因转化植物与藻类能提高其油脂含量。
Description
技术领域
本发明涉及BnaA.BCCP2.a基因,包含其的表达载体及高等植物和藻类,及其提高植物种子和藻类的油脂含量的应用。
技术背景
油菜是我国重要的油料作物,在过去的30年里,油菜仅次于大豆和油棕,已经成为世界上第三大油料作物。我国是油菜种植面积最大的国家,种植区域极为广泛,遍及全国各地,总产量居世界第一。油菜的油脂合成是一个非常复杂的多因素多基因控制的数量性状。前人通过采用常规育种的方法,已经得到含油量具有较大差异的油菜品种。但是随着生物技术的迅猛发展,利用基因工程进一步提高油菜油脂含量是改良油菜的又一重要手段。
微藻能源作为生物能源领域的战略储备,目前已引起国内外高度重视。它具有产能大、无污染、可再生、易培养、含有较多的脂类物质等优点。同时把它转化为生物燃料的效率较高,也具有较大的食用和化学利用价值。由于以上多方面的优势,微藻被认为是当今最有开发前途的能源之一【1】。小球藻是一种单细胞真核藻类,具有繁殖速度快、无性生殖、易培养、可以进行光自养培养、也可以进行无光照的异养培养、可规模化生产等特点,所以,小球藻是进行能源微藻研究的一个潜在的理想材料【2】。基因工程技术已被广泛应用于作物改良,并取得了许多显著的成效。而通过基因工程技术提高真核藻类总的油脂含量的研究报道很少【3】。根据在植物中的研究经验,通过基因工程技术来提高藻类的油脂含量的研究是一个不可回避的课题。
生物体中ACCase有2种类型。一种是异质型(heteromeric),其包含4个亚基,即生物素羧化酶(biotin carboxylase,BC)、生物素羧基载体蛋白(biotin carboxyl carrier protein,BCCP)以及羧基转移酶(carboxyltransferase,CT)的2个亚基α-CT和β-CT【4-7】。另一类ACCase称为同质型(homomeric),与异质型ACCase不同的是同质型ACCase的各功能域融合成一条多肽链,具有一个分子量为220-260kD的生物素包含亚基【8】,存在于酵母【9-10】、藻类【11】、动物【12-13】以及植物【14-17】的胞质溶胶中。这类单亚基ACCase含有3个功能域,在序列上对应于异质型ACCase的BC、BCCP、α-CT和β-CT。两种不同类型的ACCase在脂肪酸的合成中有不同的作用,异质型ACCase催化产生的丙二酰辅酶A用于脂肪酸的从头生物合成,而同质型ACCase催化产生的丙二酰辅酶A则用于脂肪酸链的延伸及类黄酮许多次生代谢产物的合成【18-19】。已经证明,异质性ACCase是脂肪酸生物合成的关键酶或限速酶,是碳流进入脂肪酸生物合成的重要调控位点【20-23】。由于ACCase是脂肪酸生物合成的限速酶,人们期望能通过超量表达ACCase基因提高油料作物的种子含油量。Gengenbach等发现在早期种子发育过程中ACCase基因表达量的增加与种子成熟时含油总量增加具有相关性【24】。Sellwood等通过反义表达技术抑制油菜同质型ACCase的活性,获得的转基因植株成熟种子的含油量显著低于对照【25】。Ohlrogge等将油菜种子贮藏蛋白napin特异性表达启动子连接到拟南芥同质型ACCase基因上,将其转入油菜中,获得的转基因植株T1代成熟种子的脂肪酸含量增加了5%,T2代脂肪酸含量增加了5-6.5%【26】。由于异质型ACCase由4个亚基组成,同时转化植物或小球藻比较困难。BCCP亚基是生物素羧基载体蛋白,负责在生物素羧化酶与羧基转移酶之间转运分子,在异质型ACCase行使功能的过程中起到桥梁的作用。目前尚未检索到来自油料作物甘蓝型油菜的编码BCCP2亚基的基因转化植物或小球藻并提高其油脂含量的研究【27】。本发明发现来源于甘蓝型油菜编码BCCP2亚基的BnaA.BCCP2.a基因转入植物和藻类后,可以使植物种子和藻类的油脂含量显著提高。
发明内容
本发明的一方面提供了异质型乙酰辅酶A羧化酶的生物素羧基载体蛋白亚基BCCP,其氨基酸序列如SEQ ID NO:2所示。
本发明的另一方面提供了编码所述异质型乙酰辅酶A羧化酶的生物素羧基载体蛋白亚基BCCP的基因。优选地,其为来源于甘蓝型油菜的DNA片段的BnaA.BCCP2.a,其核苷酸序列如SEQ ID NO:1所示。
本发明的另一个方面提供了含本发明所述基因(优选BnaA.BCCP2.a)的植物表达载体。可使用任何一种可以引导外源基因在植物中表达的表达载体。这些植物表达载体包括但不限于,双元农杆菌载体,例如pBIN19、pBI121、pBI221,pCambia 1300,pGreen等的植物表达载体。
本发明的载体也可含有适当的启动子。在本发明中可使用任何一种强启动子。这些启动子包括但不限于花椰菜花叶病毒(CaMV 35S)、Ubiqutin、Actin启动子。它可单独使用或与其它的植物启动子结合使用。
本发明的表达载体可通过使用Ti质粒,Ri质粒,植物病毒载体,直接的DNA转化,微注射,电穿孔等方式导入植物细胞和藻类细胞。
本发明在一个方面提供了包含本发明所述基因的植物或藻类,其选自藻类,拟南芥、烟草、油菜、向日葵、大豆、番茄、蓖麻、棉花、芝麻和花生等。优选地,所述藻类选自小球藻。更具体地,所述小球藻选自椭圆小球藻(Chlorella ellipsoidea)。
本发明还提供了提高植物种子或藻类中总脂肪酸含量的方法,其特征在于将本发明所述基因(优选BnaA.BCCP2.a)转化到所述植物或藻类中。其中所述藻类和植物如上所述。
本发明还提供了提高植物种子或藻类中油酸或亚油酸含量的方法,其特征在于将本发明所述基因(优选BnaA.BCCP2.a)转化到植物或藻类中。其中所述藻类和植物如上所述。
可使用本发明的方法转化的真核生物选自由藻类和植物等真核生物组成的组。其中所述藻类和植物如上所述。
本发明还涉及本发明所述基因(优选BnaA.BCCP2.a)在提高植物或藻类中总脂肪酸或油脂含量中的应用。其中所述藻类和植物如上所述。
附图说明
图1植物表达载体pBI121-BnaA.BCCP2.a的构建
图2含nos终止子的载体图构建过程
图3空载(UN-CK)小球藻表达载体图构建
图4小球藻表达载体BnaA.BCCP2.a的构建
图5转基因藻株的PCR检测。M:Marker,1-6:阳性藻株,CK:对照藻株。
图6转基因藻株的RT-PCR检测。M:Marker,1-4:阳性藻株,CK:对照藻株。
图7转基因藻株的Southern检测。1-4:阳性藻株,5:对照藻株。
图8T2代转基因株系与野生型拟南芥脂肪酸GC-MS测定结果比较,(a)CK:对照,(b)转基因株系b2-6。
实施例1、油菜BnaA.BCCP2.a基因的克隆与序列结构分析
以大田种植的甘蓝型油菜8953(Brassica napus,来自于南京农业大学)为材料,取其授粉后30d的幼嫩种子(约100mg),置于液氮中研磨至材料完全破碎,按照Bio-Med(北京)试剂盒操提取RNA,以带polyT的引物进行逆转录获得cDNA,并进行RT-PCR(具体操作参见KitDRR019A,Takara Biotech.(Dalian)Co Ltd)。polyT的引物序列SEQ ID NO:3。
RT-PCR反应条件如下:
于65℃温浴10分钟,立即于冰上淬冷,然后加入
然后进行下列操作
1. 30℃ 10min
2. 42℃ 45min
3. 99℃ 5min
4. 5℃ 5min
取适量上述逆转录产物以正向引物(SEQ ID NO:4)和反向引物(SEQID NO:5)进行PCR扩增。
PCR反应条件如下:
1. 95℃ 5min
2. 94℃ 30s
3. 56℃ 30s
4. 72℃ 2min 2 29cycles
5. 72℃ 5min
6. 4℃ pause
RT-PCR产物电泳(1%琼脂糖凝胶浓度)后切胶回收目的片段(BnaA.BCCP2.a,约0.8kb),连入pEASY-Blunt载体(购自全式金公司),测序验证获得了BnaA.BCCP2.a的全长cDNA(SEQ ID NO:1)。
实施例2、含有BnaA.BCCP2.a基因的植物表达载体的构建
根据BnaA.BCCP2.a的cds序列,设计左端含BamH I酶切位点引物(SEQ ID NO:6),右端含Sac I酶切位点引物(SEQ ID NO:7),采用TaKaRa公司高保真Pyrobest Taq酶,扩增出含BamH I和Sac I酶切位点的片段,用BamH I和Sac I双切。同时,将pBI121(Clontech)也用BamHI和Sac I双切。然后将这两个双切产物,高温灭活BamH I和Sac I后,加入T4连接酶过夜连接,转化DH5α,涂布LBK板,随机挑取单菌落,提质粒。取连入BnaA.BCCP2.a片段的质粒命名为pBI121BnaA.BCCP2.a,即为所构建植物表达载体pBI121BnaA.BCCP2.a。测序验证,pBI121BnaA.BCCP2.a载体见图1。
实施例3、小球藻表达载体p29-UN-BnaA.BCCP2.a的构建
1.以pGreen0029(购自the John Innes Centre)为基础载体,构建含Ubiquitin启动子和nos终止子的对照载体pG29-UN-CK。
用TaKaRa公司高保真Pyrobest Taq酶从载体pGreen0029上扩增nos终止子,用引物SEQ ID NO:8(正向引物带Not I酶切位点)和SEQ IDNO:9(反向引物带Sac I酶切位点)。
PCR反应体系如下:
PCR反应条件如下:
1. 95℃ 5min
2. 94℃ 30s
3. 55℃ 40s
4. 72℃ 1min 回复至2,32cycles
5. 72℃ 10min
6. 4℃ pause
扩增产物直接使用限制性内切酶Not I和Sac I进行双酶切,与同样双酶切的pGreen0029连接,构建中间载体pGreen0029-nos(图2),测序验证。
用高保真酶Pyrobest Taq (来源同上)从含Ubi启动子的载体pC1303(购自Cambia公司)上扩增Ubi启动子,所用引物SEQ ID NO:10(正向引物带HindIII酶切位点)和SEQ ID NO:11(反向引物带BamHI酶切位点)。
PCR反应体系如下:
PCR反应条件如下:
1. 95℃ 5m
2. 94℃ 30s
3. 55℃ 40s
4. 72℃ 4min 回复至2,32cycles
5. 72℃ 10min
6. 4℃ pause
扩增产物直接使用限制性内切酶HindIII和BamH I进行双酶切,与同样双酶切的pGreen0029-nos连接,构建含Ubiquitin启动子和nos终止子的对照载体pGreen0029-Ubi-Nos(简写UN-CK),测序验证,其载体图见图3。
2.BnaA.BCCP2.a基因小球藻表达载体的构建
用高保真酶Pyrobest Polymerase从载体pBI121BnaA.BCCP2.a中扩增BnaA.BCCP2.a,所用引物SEQ ID NO:12(正向引物带Spe I酶切位点)和SEQ ID NO:13(反向引物带Not I酶切位点)。反应体系及条件同上。利用Spe I、Not I酶切位点,双切后获得BnaA.BCCP2.a基因,定向克隆于同样酶切的小球藻中间表达载体UN-CK,获得小球藻表达质粒p29-UN-BCCP2,转化大肠杆菌DH-5α,随机挑取单克隆,经PCR、测序验证得到p29-UN-BCCP2的表达载体,其载体图见图4。
实施例4、BnaA.BCCP2.a基因拟南芥菜的转化和筛选
将pBI121BnaA.BCCP2.a转入农杆菌LBA4404中,然后侵染拟南芥(Columbia),其中YEP液体培养基(酵母提取物10g/L,蛋白胨10g/I,NaCl 5g/L,pH 7.0)和1/2MS液体培养基的配制参见文献【29】
1、挑取单菌落的LBA4404菌株(Biovector Co.,LTD)接种于5mL的YEP液体培养基(Tet 3μg/mL,Str 30μg/mL,Kan 50μg/mL)中,28℃振荡培养过夜;
2、取1mL菌液转接于100mL新鲜的YEP液体培养基中,28℃振荡培养;
3、当菌液摇至OD600为0.8左右时,8000rpm(6000g)离心10分钟收集菌体;
4、用含有2.5%Silwet L-77和5%蔗糖的1/2MS液体培养基作为侵染液,将菌液稀释至OD600为0.6~0.8,浸泡拟南芥数分钟,再将培养液喷于植株上。
5、盖上塑料膜保湿培养。1~2日后去除覆膜,培养至种子成熟。
其间可在一周后重复侵染过程一次以增加侵染率。等待种子成熟时收获,晾干后去果荚,保存于4℃也可长期保存于-20℃。
转基因拟南芥的筛选和鉴定
将收获的T0代种子于4℃浸泡3天打破休眠,在20%消毒水中浸泡20min后用无菌水漂洗3遍,播种于含相应抗生素(Kan 30μg/mL)的MS培养基(配制参见参见文献【29】)上。22℃长日照培养10天左右可见子叶伸出,此时可见抗性植株呈深绿色,子叶健康;而未转化植株呈黄色或褐色,子叶萎蔫。此时将阳性植株移栽至装有蛭石和营养土的培养钵中培养至收获。用T1代种子重复上述步骤,获得转基因T2代种子。
实施例5、转BnaA.BCCP2.a基因拟南芥种子油脂含量分析
1.脂肪酸的提取。
把获得的转基因T2代种子,在研钵中研磨成均匀的细粉,称取100mg,加入100μL的d17:0(sigma,浓度100μM/mL),5mL 5%KOH-CH3OH,70℃水浴3h后加入HCl酸化至其pH值达2.0。再加入4mL 14%BF3-CH3OH(购自Aldtich公司)溶液,70℃水浴1.5h。加入2mL0.9%NaCl溶液,混匀后静止片刻。加入2mL氯仿∶正己烷(V/V 1∶4)抽提,吸取抽提液,重复抽提一次,合并两次的抽提液用N2吹干。最后溶于150μl乙酸乙酯。
2.终产物GC-MS检测分析实验。
所用GC-/MS仪为TurboMass(PerkinElmer公司),柱子:BPX-70,30m×0.25mm×0.25vm,柱温120℃,气化室温度230℃。取1μL终产物上样,分流比10∶1。
3.GC-MS结果分析。
研究表明,在拟南芥转基因株系中含油量(即总脂肪酸含量)增加了4.3-10.7%,且各脂肪酸所占总脂肪酸的比例也发生了变化,油酸(C18:1)和亚油酸(C18:2)分别提高了0.5-1.3%和1.6-2.4%,亚麻酸(C18:3)降低了0.9-3.0%(0.01<P<0.05,差异显著)(表1,图8)。
表1 T2代转基因株系和野生型拟南芥总脂肪酸含量的比较(μg/seed)
注:脂肪酸总量及脂肪酸组分的评价是分不同批次进行的,每次拟南芥菜的生长状态会有差异,因此每次评价时每个转基因拟南芥菜株系单独设立对照,使每个转基因株系与对照尽量生长状态一致。WT:为野生型;b2-6,b2-5,b6-3,b6-7:为转基因不同株系。括号内为样本标准误差。
实施例6、BnaA.BCCP2.a基因在小球藻中的转化和检测
1.小球藻的转化
所用小球藻为椭圆小球藻(Chlorella ellipsoidea)(购自中国科学院水生生物所),参照文献【2】培养小球藻至适宜的生长状态,离心收集藻细胞后进行高渗处理,利用p29-UN-BCCP2和pG29-UN-CK质粒(对照),采用相同的电激转化条件(参见文献【2】)对椭圆小球藻进行电激,将电激后小球藻细胞涂于含30mg/L G418固体培养基(参见文献[2])中培养,约4~6周后检测平板上长出的藻落。
2 对转基因藻株进行PCR检测
挑取平板上的单藻落细胞接种于15mg/L G418液体培养基(参照文献【2】)中培养2周(浓度达到约为1×108个/mL),5000r/min离心10min收集藻细胞,在液氮中将细胞磨碎,参照文献[2]中的方法提取小球藻总DNA,得到的DNA溶于灭菌的双蒸水中放置-80℃保存。
用Easy Taq(来源全式金公司)扩增基因BnaA.BCCP2.a,所用引物SEQ ID NO:12(正向引物)和SEQ ID NO:13(反向引物)。
PCR反应体系如下:
PCR反应条件如下:
1. 95℃ 5min
2. 94℃ 30s
3. 55℃ 40s
4. 72℃ 1min 回复至2,32cycles
5. 72℃ 10min
6. 4℃ pause
图5为部分转基因藻株PCR检测结果,共获得了42个转基因阳性藻株。
3.对转基因藻株进行RT-PCR检测
挑取PCR检测阳性的藻株单藻落细胞,培养收集方法同上,RNA提取的方法参见Bio-Med(北京)试剂盒。以试剂盒(FSK-100,TOYOBOCo.,LTD.)自带的Random primer进行逆转录获得cDNA,并进行RT-PCR(具体操作参见FSK-100,TOYOBO Co.,LTD.)。
RT-PCR反应体系如下:
进行下列操作
1. 30℃ 10min
2. 42℃ 30min
3. 90℃ 5min
4. 5℃ 5min
取适量上述逆转录产物以引物SEQ ID NO:14(正向引物)和SEQ IDNO:15(反向引物)进行PCR扩增,反应体系和反应条件参见对转基因藻株进行的PCR检测,图6为部分转基因藻株RT-PCR检测结果。
4.对转基因藻株进行Southern检测
Southern杂交是分子生物学的经典实验方法,其基本原理是将待检测的DNA样品固定在固相载体上,与标记的核酸探针进行杂交,在与探针有同源序列的固相DNA的位置上显示出杂交信号。通过Southern杂交可以判断被检测的DNA样品中是否有与探针同源的片段。本实验操作主要参考文献【28】。
4.1探针标记
使用引物SEQ ID NO:12和SEQ ID NO:13从载体p29-UN-BCCP2(图4)PCR扩增基因BnaA.BCCP2.a,反应体系和反应条件参见对转基因藻株进行的PCR检测。探针标记使用Random Primer DNA Labeling Kitver.2.0(TAKARA BIOTECHNOLOGY(DALLAN)CO.,LTD.Code D6045)。PCR产物纯化回收后取10ng-1μg作为模板放于0.2mL离心管中,加Random Primer 2μL,ddH2O补齐16μL,95℃变性5min,立即置冰浴5min。
在另一个1.5mL离心管中加入:
将变性模板DNA加入到上管中,室温或37℃1h。
4.2小球藻总DNA提取和酶切
挑取转BnaA.BCCP2.a单藻落细胞和对照(转UN-CK的藻落细胞)分别接种于15mg/L G418液体培养基中培养2周,5000r/min离心10min收集藻细胞,在液氮中将细胞磨碎,参照文献[2]中的小球藻总DNA提取方法,获得的DNA用限制性内切酶酶进行酶切。BCCP2-1-3用Xba I和EcoR I双酶切;BCCP2-7-5用Xba I和EcoR I双酶切,BCCP2-8-3用XbaI和Nco I双酶切;BCCP2-21-5用Xba I和Xho I双酶切;UN CK-1-1XbaI和EcoR I双酶切。限制性内切酶均购自Takara公司。
反应体系:100μL反应体系中包括DNA 10~20μg、buffer 10μL、限制性内切酶4μL,其余用ddH2O补平。
反应条件:37℃过夜,补加1μL酶继续酶切3-5小时,然后1%琼脂糖电泳检测酶切效果,DNA需切成弥散的一条带,没有主带。酶切体系加ddH2O至体积200μL,加等体积氯仿抽提后乙醇醇沉,70%乙醇洗,晾干后加水不超过50μL,65℃热10min备用。
4.3胶的制备及转膜
配制0.8%的琼脂糖凝胶,点入酶切回收产物50μL,在1×TAE电泳缓冲液中,30V电压下电泳至溴酚蓝到胶下端,用ddH2O将胶洗两遍,浸于数倍体积的碱性缓冲液中中轻摇15min,换一次碱性缓冲液浸泡并轻轻摇荡20min。同时剪一张比凝胶大1mm的尼龙膜(CAT.No:RPN303B,Amersham Biosciences UK Limited),预先在双蒸水中浸泡使其湿透,浸润后的尼龙膜浸入碱性转移缓冲液中至少15min。在0.4M的NaOH和1M NaCl碱性转移缓冲液条件下,用真空转膜仪(美国伯乐Bio-rad 785型真空转印仪)将琼脂糖凝胶上的DNA转到带正电荷的尼龙膜上,转膜条件为真空度50m bar转1h-2h。转膜完成后将膜浸泡于中和缓冲液II中室温15min。置于滤纸上晾干,紫外交联(1.245J/cm)8min,80℃烘烤1h,最后将尼龙膜立即用于杂交或用保鲜膜包好放在-20℃冰箱待用。
4.4预杂交及杂交
在杂交瓶中加入杂交液10-20mL(0.5M Na3PO4(pH7.2),1mMEDTA(pH8.0),7%SDS),将膜的背面贴紧杂交瓶壁,正面朝向杂交液,注意不要产生气泡,放入杂交炉中,使杂交体系升到65℃。预杂交2-3h后,倒出预杂交的杂交液,换入5mL左右的杂交液。在上述制作的探针中加0.4N的NaOH使其变性,迅速加到杂交瓶中,65℃杂交过夜。
4.5洗膜与压片
取出尼龙膜在65℃依次使用下列条件洗膜:
2×SSC,0.5%SDS,15min
1×SSC,0.5%SDS,15min
0.5×SSC,0.1%SDS,15min
在洗膜的过程中,不断振摇,并保持膜一直是湿润的状态,不断用放射性检测仪探测膜上的放射强度。期间洗膜2次后用monitor检测杂交信号的强弱,降低到20μCi/h左右时即可。洗完的膜用滤纸吸干膜表面的水分,并用保鲜膜包裹。
将膜正面向上,放入暗盒中(加双侧增感屏),在暗室的红光下,在膜的正面向上贴覆一张X光片,合上暗盒,置于-80℃低温冰箱中曝光。根据信号强弱决定曝光时间,一般时间在2天左右。
4.6显影及定影
从-70℃中取出的暗盒在显影室打开后,取出X光片,显影液中浸泡1min,期间不断晃动光片,取出沥掉一些水分,浸到定影液中1min,ddH2O冲洗两遍并照相,见附图7。点样顺序如下:1:BCCP2-1-3(Xba I和EcoR I),2:BCCP2-7-5(Xba I和EcoR I),3:BCCP2-8-3(Xba I和Nco I),4:BCCP2-21-5(Xba I和Xho I),5:UN CK-1-1(Xba I和EcoR I)。
4.7结果分析
质粒载体图4没有Xho I、EcoR I和Nco I的酶切位点,有一个Xba I的酶切位点。使用Xba I分别与Xho I、EcoR I和Nco I组合进行双酶切,在转化藻株的酶切产物中一端是固定的,另一端是随机的。因此可以认为Southern杂交出的条带数等同于去除内源基因后的转基因拷贝数。图7中的泳道5是对照藻株,没有明显条带说明没有杂到内源基因,泳道1和2有两个条带,说明在藻株BCCP2-1-3和BCCP2-7-5的基因组里面插入了两个拷贝的基因。泳道3和4各有三个条带,说明在藻株BCCP2-8-3和BCCP2-21-5的基因组里面插入了三个拷贝的基因。
实施例7、BnaA.BCCP2.a基因提高小球藻脂肪酸含量的GC-MS分析
对PCR、RT-PCR和southern检测为阳性的藻株,先使用50mL三角瓶培养4-6天后,按干重0.1g/L的起始浓度在150mL新培养基扩大培养,7天后离心收集生长达到平台期的小球藻(OD540=16),用去离子水洗涤,-80℃冷冻干燥,冻干后在85℃烘箱过夜烘干,在研钵中研磨成均匀的细粉。称取50mg小球藻粉,加入80μL的十七烷酸(Heptadecanoic Acid,d17:0,购自Sigma公司,溶于甲醇浓度100μM/mL)作为脂肪酸含量测定的内参。5mL5%KOH-CH3OH,70℃水浴5h后加入HCl酸化至其pH值达2.0。再加入4mL 14%BF3-CH3OH(购自Aldtich公司)溶液,70℃水浴1.5h。加入2mL 0.9%NaCl溶液,混匀后静止片刻。加入2mL氯仿∶正己烷(V/V 1∶4)抽提,吸取抽提液,N2吹干。最后溶于150μL乙酸乙酯。
结果表明在小球藻转基因株系中总脂肪酸含量增加11.4-14.3%,各脂肪酸所占总脂肪酸的比例也发生了变化,油酸(C18:1)的含量变化范围为-0.9-57.9%,亚油酸(C18:2)提高了1.1-80.1%(0.01<P<0.05,差异显著),亚麻酸(C18:3)含量变化幅度不大(表2)。
表2.T2代小球藻转基因株系和转空载株系总脂肪酸含量的比较(μg/mg)
注:括号内为样本标准误差
参考文献:
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Claims (16)
1.异质型乙酰辅酶A羧化酶的生物素羧基载体蛋白亚基BCCP,其氨基酸序列如SEQ ID NO:2所示。
2.编码权利要求1的异质型乙酰辅酶A羧化酶的生物素羧基载体蛋白亚基BCCP的基因。
3.权利要求2所述的基因,其为来自甘蓝型油菜(Brassica napus)的BnaA.BCCP2.a基因,其核苷酸序列如SEQ ID NO:1所示。
4.包含权利要求2或3的基因的表达载体。
5.权利要求4的表达载体,其为植物表达载体。
6.权利要求5的表达载体,其是双元农杆菌载体。
7.权利要求5的表达载体,其是pBIN19、pBI121、pBI221,pCambia1300或pGreen。
8.提高植物种子总脂肪酸含量的方法,其特征在于将权利要求2或3的基因的转化到植物中。
9.提高藻类总脂肪酸含量的方法,其特征在于将权利要求2或3的基因的转化到藻类中。
10.提高植物种子中油酸或亚油酸含量的方法,其特征在于将权利要求2或3的基因的转化到植物中。
11.提高藻类中油酸或亚油酸含量的方法,其特征在于将权利要求2或3的基因的转化到藻类中。
12.权利要求8或10的方法,其中所述植物选自拟南芥、烟草、油菜、向日葵、大豆、番茄、蓖麻、棉花、芝麻和花生。
13.权利要求9或11的方法,其中所述藻类为小球藻。
14.权利要求13的方法,其中所述小球藻选自椭圆小球藻(Chlorellaellipsoidea)。
15.权利要求2或3的基因在提高植物种子中总脂肪酸含量中的应用。
16.权利要求2或3的基因在提高藻类中总脂肪酸含量中的应用。
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Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus- cloning and analysis of expression during oilseed rape embryogenesis;Kieran M. ELBOROUGH et al;《Biochem. J.》;19961231;103-112 * |
FERNADO LOPEZ-CASILLAS et al.Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase.《Pro.Natl.Acad.Sci》.1988,第85卷 |
Kieran M. ELBOROUGH et al.Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus- cloning and analysis of expression during oilseed rape embryogenesis.《Biochem. J.》.1996,103-112. |
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