CN102618560B - 油菜呼吸代谢相关基因BnAOX1及应用 - Google Patents
油菜呼吸代谢相关基因BnAOX1及应用 Download PDFInfo
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Abstract
本发明公开了一种油菜呼吸代谢相关基因BnAOX1及应用，通过筛选基因表达差异，发现BnAOX1基因在油菜高油亲本及高油F2混合群体中表达量高于低油亲本及低油F2混合群体。通过arabidopsis网站拟南芥AOX1基因的序列和测序数据库，寻找与之同源的油菜基因组序列拼接，并设计基因编码区序列的两侧引物。以亲本zy036cDNA第一链为模板进行RT-PCR扩增，将扩增得到的片段进行测序，获得BnAOX1基因的编码区序列，还包括在添加、取代、插入或缺失一个或多个核苷酸而产生的突变体等位基因或衍生物。利用拟南芥作为受体的转基因结果证实，此类基因的过量表达可以提高拟南芥种子的含油量，同时也增加了种子的粒重。因此，此类基因在油菜及其他油料作物的产油量育种中具有很好的应用前景。
Description
技术领域
本发明涉及植物基因工程领域，更具体涉及一种油菜呼吸代谢相关酶基因BnAOX1，同时还涉及一种油菜呼吸代谢相关酶基因BnAOX1在提高油料作物的产量和含油量中的应用。
背景技术
油菜籽是世界上食用植物油和饲用蛋白质的最主要来源之一，也是欧洲生物柴油最重要的原料。油菜是我国种植面积最大的油料作物，也是继水稻、小麦、玉米和大豆之后我国的第五大农作物。菜籽油是我国居民的主要食用油，占国产植物油消费总量的40％以上。我国作为世界第一植物油消费大国，植物油的自给率不足40％。为解决这些需求矛盾，除扩大种植面积外，提高油菜含油量和产量是提高其单位面积产油量的最有效途径。高产油量能够显著提高油菜生产效益，提高油菜籽的总体利润。
植物线粒体电子传递链有多条途径存在，其中主要有细胞色素主路和抗氰交替途径支路两条途径。抗氰呼吸(交替途径)指的是对氰化物不敏感的一条呼吸途径。交替氧化酶(alter native oxidase，AOX)是植物线粒体呼吸链中抗氰呼吸途径(cyanide-resistant respiration pathway)的末端氧化酶，它广泛存在于高等植物及部分真菌和藻类中。交替氧化酶作为交替途径的末端氧化酶，其内源底物包括还原泛醌和02。交替途径的呼吸电子流从主路(细胞色素途径)泛醌处分支出来，绕过复合物III和IV两个ATP形成位点，直接由AOX催化分子氧的4个电子还原成水，而未用来合成ATP的能量则以热的形式被散失掉。
交替氧化酶是一种双铁羧基蛋白(di-iron carboxylate protein)，它不仅具有其它双铁羧基蛋白共有的结构特点以及去除分子氧的功能，更重要的是它还可以通过改变自身结构等方式来主动调节抗氰呼吸途径的运行程度，进而调节细胞多方面的代谢和功能，以适应环境条件的改变，增强植物适应各种逆境的能力，调节植物生长速率，并与细胞凋亡和光合作用相关。AOX活性调控受多种因素影响，环境胁迫、植物受伤、冻害、干旱、渗透压及病菌等都可诱导AOX表达。水杨酸(SA)、过氧化氢、乙烯和主呼吸链抑制剂也同样能诱导AOX在植物中的表达。抗氰呼吸活性和AOX表达在产热植物的佛焰花序中较高。在非产热植物中，AOX表达水平与发育状态有关，如在衰老和果实成熟过程中都有AOX的表达。
AOX调节能量代谢，当能量代谢饱和时，AOX途径以散热方式释放能量，维持电子传递和TCA。Lambers(1982)提出的能量溢满理论认为：当主呼吸链活性受到抑制时，或细胞还原力偏高时，电子通过AOX传递可使TCA循环和糖酵解继续进行。AOX活性能为TCA循环底物丙酮酸激活，说明TCA循环的底物过高时可诱导AOX途径转移部分电子。环境变化导致AOX表达差异，不仅改变线粒体功能，也影响线粒体外细胞功能。氧胁迫诱导AOX表达并抗氧化，当细胞色素途径受抑时，主呼吸链产生活性氧(ROS)，AOX氧化可使ROS下降。在转基因烟草中AOX的超量表达时ROS水平下降，反之ROS水平则提高。Hansen等(2002)测量热呼吸发现环境变化能改变植物生长率，AOX对植物生长动态起平衡作用。产热植物通过AOX释放的热量可使花粉散发芳香气味吸引虫媒(Meeuse等，1975)。非产热植物组织中AOX的表达与此同产热无关(Borecky andVercesi，2005)。AOX可有效调控呼吸率保持细胞能量恒定以保证植物在环境变化时正常生长(Hansen等，2002)。AOX不仅使拟南芥在低温下发芽，而且AOX能防止植物组织在逆境中产生过氧化物而广泛影响细胞功能(Fiorani等，2005)。AOX在细胞质和一些碳代谢途径中发挥重要作用(Umbach等，2005)。各种环境胁迫都能改变植物抗氰途径的运行，但这些研究还很肤浅，其运行的信号调节机制和生理意义还不清楚。
发明内容
本发明的目的是在于提供了一种油菜呼吸代谢相关基因BnAOX1，本发明提供了这个基因的核苷酸序列，如SEQ ID NO：1所示的DNA序列，也包括与SEQID NO：1所示的DNA序列至少有70％同源性的本物种及其它物种中的基因序列。该基因属于植物交替氧化酶家族中的一员，它通过调节抗氰呼吸途径的运行程度，来调节细胞多方面的代谢和功能，以适应环境条件的改变，调节植物生长速率，并与细胞凋亡和光合作用相关。在拟南芥中该基因被抑制可导致部分基因表达量发生改变，但未见对其表型的影响报道。
本发明的另一个目的是在于提供了一种油菜呼吸代谢相关基因BnAOX1在提高油料植物含油量中的应用。将此基因应用于油料作物育种，可以提高油料作物种子的含油量，最大限度的提高油料作物的产油量。
本发明的再一个目的是在于提供了一种油菜呼吸代谢相关基因BnAOX1在提高拟南芥植物种子千粒重中的应用。通过模式植物拟南芥证实BnAOX1基因不但可调控种子含油量的变化，同时还增加了千粒重。
为了达到上述目的，本发明采用了以下技术措施：
本发明申请人通过分析油菜含油量差异显著的两亲本及F2代分离群体混合样本的基因表达差异，筛选出一类在亲本及F2代极端分离材料中同时存在表达差异的基因，其中包括BnAOX1基因。为了验证该基因是否影响种子含油量或种子其它性状，申请人通过基因全长的克隆，表达载体的构建以及模式植物拟南芥的转化。该油菜呼吸代谢相关基因BnAOX1在提高油料植物含油量和千粒重中应用。
一、基因的来源：
一种油菜呼吸代谢相关基因BnAOX1的制备方法，其步骤是：
以高低含油量品系zy036与y817(含油量分别为51％和35％)杂交构建F2代群体共得到169个后代单株为材料；分别收集各F2单株开花后25天左右的角果(带角果皮和胚珠)；各单株成熟种子进行含油量检测，将含油量检测大于47％和小于38.5％的单株的角果分别取等量200mg混合组成两极端含油量混合样本分别编号H(含油量大于47％的有11株)和L(含油量小于38.5％的有11株)，两个混样平均含油量相差11.1％。分析两亲本及两混样表达基因，找出亲本与F2代混样表达量同时表现差异的基因，BnAOX1基因即源于此。本研究中所用的亲本材料zy036和Y817，均由中国农科院油料所生物技术育种课题组技术人员在王汉中研究员带领下育成。zy036品系是由中双4号、中双7号、中双9号、华双3号、油研9号(中双4号、中双7号、中双9号、华双3号、油研9号)构建轮回选择群体，轮回选择两代后选优良单株进行小孢子培养轮回选择第三代，最终经高油定向选择得到。Y817是杂交品种中油杂1号的保持系(中油杂一号制种技术研究与应用，农村经济与科技，2001年第10期)。
二、基因的全长克隆：
通过筛选基因表达差异，发现BnAOX1基因在油菜高油亲本及高油F2混合群体中表达量高于低油亲本及低油F2混合群体。因此申请人根据arabidopsis网站拟南芥AOX1基因的序列和申请人自己的测序数据库，寻找与之同源的油菜基因组序列拼接，并设计基因编码区序列的两侧引物。以亲本zy036cDNA第一链为模板进行RT-PCR扩增，将扩增得到的片段进行测序，获得BnAOX1基因的编码区序列，还包括在添加、取代、插入或缺失一个或多个核苷酸而产生的突变体等位基因或衍生物。一种分离的蛋白质，其碱基序列为SEQ ID NO：1所示的核苷酸序列。一种分离的蛋白质，其蛋白质序列为SEQ ID NO：2所示的氨基酸序列。同时，利用arabidopsis网站上的基因序列设计引物扩增拟南芥中同源基因AtAOX1序列。利用拟南芥作为受体的转基因结果证实，此类基因的过量表达可以提高拟南芥种子的含油量，同时也增加了种子的粒重。因此，此类基因在油菜及其他油料作物的产油量育种中具有很好的应用前景。
三、转基因载体的构建、转化及验证：
通过上述方法获得了油菜BnAOX1基因，采用一种提高油菜基因表达活性的转基因拟南芥，其特征在于与受体对照(非转基因植株)相比，转基因植物表现为基因表达量增加，其具体措施是：
一种PCR8/GW/TOPO质粒(invitrogen公司购买)，可以与表达载体质粒重组构建基因表达质粒的实验方法(invitrogen公司购买)。
一种质粒表达载体Pearleygate100(invitrogen公司购买)，它含有35S启动子和翻译控制件。
一种可在植物中表达的宿主菌(如GV3101，LBA4404等)，本发明优选的是农杆菌(GV3101，invitrogen公司购买)。
一种能够过量表达某基因的转基因拟南芥，其特征在于拟南芥中所转基因表达量增加。其应用过程包括下列步骤：
1)将克隆得到的油菜基因与PCR8/GW/TOPO质粒连接，利用PCR8/GW/TOPO质粒与质粒表达载体Pearleygate100可以体外重组的特性将油菜基因转移至表达载体；
2)将步骤1)中制备的表达载体转入根癌农杆菌GV3101，再导入拟南芥植株中。
3)转基因阳性植株PCR鉴定，除草剂(Bar，草苷膦)筛选后正常条件生长并收获种子测定含油量。结果表明，转基因拟南芥种子的含油量与对照拟南芥相比有较大幅度的提高。
本发明中所用的术语“转基因植物”是指含有导入的基因并能够稳定地增强或抑制所导入的基因表达并产生具有特定的生物学性状的植物。克隆本发明中所述的油菜基因的方法是本领域中所常采用的方法如：利用CTAB法提取植物叶片DNA，提取mRNA的方法也有多种成熟的技术，如采用TRIzol Reagent，Invetrogen公司或Total RNA extraction，qiagen公司，可从商业途径获得。构建cDNA文库也是常用的分子生物学技术。构建本发明中所述的载体和将载体转染入植株也是本领域中所常采用的方法。其中所涉及的质粒(入门载体PCR8/GW/TOPO，质粒表达载体Pearleygate100)，转染用媒体(如根癌农杆菌GV3101和所用试剂(蔗糖等)可从商业途径获得。聚丙烯酰胺凝胶电泳是分子标记的多态性分析的最常用手段，所有试剂如丙烯酰胺、甲叉丙烯酰胺等均可从商业途径获得。
转基因拟南芥分析：通过在野生拟南芥中过量表达BnAOX1、AtAOX1基因，转基因拟南芥整体植株表型与野生型没有明显区别。收获种子后发现，转AOX1基因的植株种子明显大于野生型对照。通过脉冲式核磁共振仪测定含油量，结果表明转基因植株与对照相比，含油量均有不同程度的提高，转基因拟南芥种子含油量与野生型拟南芥相比有明显提高，最高幅度可达20％以上。而千粒重也有所增加，增加幅度最高达25％(见表1)。上述结果表明，不仅油菜AOX1基因可以提高种子含油量，同时还可以增加粒重，而且与此类油菜基因序列有70％同源性的其它物种基因同样可以提高含油量。
本发明的优点是：本发明中油菜BnAOX1基因为首次报导与种子含油量、粒重相关。交替氧化酶(AOX1)具有其它双铁羧基蛋白共有的结构特点以及去除分子氧的功能，属于植物交替氧化酶家族中的一员。它通过调节抗氰呼吸途径的运行程度，来调节细胞多方面的代谢和功能，以适应环境条件的改变，增强植物适应各种逆境的能力，调节植物生长速率，并与细胞凋亡和光合作用相关。在拟南芥中该基因被抑制可导致部分基因表达量发生改变，但未见对其表型的影响报道。通过在拟南芥中的转基因研究证实，此类基因确实可以提高种子含油量，同时还可以增加种子粒重。此类基因可以应用于油菜高含油量育种，通过使用组成型启动子35S在油菜品种中超量表达此类基因，得到含油量提高且千粒重增加的油菜新品种，加快油菜含油量育种的步伐。同时，将其扩展到其他油料作物如大豆、花生、芝麻等的育种应用中，最大限度的提高油料作物的产油量。
附图说明
图1为一种油菜呼吸代谢相关基因BnAOX1的拟南芥表达载体示意图
图2为一种油菜呼吸代谢相关基因BnAOX1的拟南芥T1代的转基因鉴定结果。
图3为一种油菜呼吸代谢相关基因BnAOX1的野生型对照种子的大小比较示意图。
其中图3A为野生对照，图3B为转基因株系的种子。
具体实施方式
实施例1：BnAOX1、AtAOX1cDNA编码区序列
一种油菜呼吸代谢相关基因BnAOX1的制备方法，其步骤是：
在NCBI数据库中检索已发表的拟南芥基因序列，BLAST NCBI网站的油菜EST序列，并结合拟南芥序列设计基因编码序列的两侧引物(BnAOX1F：正向引物：[5’-atgatgatgagtcgctacgg-3’]，反向引物：[5’-tcaatgatccaattggag-3’]用来从油菜15天角果cDNA中扩增对应序列。拟南芥中的扩增引物分别为：AtAOX1(arabidopsis网站)正向引物：[5’-atgatgatgagtcgtcgcta-3’]，反向引物：[5’-tcaatgatatccaatgggagc-3’]，直接在拟南芥10天角果cDNA中扩增。
1、提取油菜、拟南芥mRNA。
RNA的提取(TRIZOL TM Kit提取RNA)，液氮研磨100mg材料。
A.加1mlTRIZOL，室温(20-25℃，以下相同)放置5min。
B.加入200ul氯仿，剧烈振荡30s，室温放置2min。
C.12000g，15min，4℃，取上清至新管中，加入500ul异丙醇，混匀后室温放置15min。
D.12000g，15min，4℃，去上清，加入1ml70％(体积比)乙醇。
E.7500g，7min，4℃，去上清，空气干燥。
F.DEPC-H2O溶解。
2、cDNA第一链的反转录采用RevertAid H Minus First Strand cDNASynthesis Kit(Fermentas)，操作参照所用试剂盒说明进行。
3、以cDNA为模板进行PCR扩增，得到了BnAOX1及AtAOX1基因序列如：一种油菜呼吸代谢相关基因BnAOX1，一种分离的蛋白质，其碱基序列为SEQ ID NO：1所示的核苷酸序列。一种分离的蛋白质，其蛋白质序列为SEQ ID NO：2所示的氨基酸序列所示以及NCBI网站数据库中已发表的拟南芥基因序列。
实施例2：转基因表达载体的构建及拟南芥的转化：
将PCR扩增得到的基因序列与TOPO入门载体(invitrogen公司)连接后，转化到感受态细胞DH5α(invitrogen公司)中，壮观酶素筛选，载体引物(T7引物，TAATACGACTCACTATAGGG)与基因引物(基因上游引物，引物序列见实施例1)扩增鉴定正向插入克隆，质粒经小量制备后与Pearleygate100(invitrogen公司)进行重组，并转化到感受态细胞DH5α中，卡那霉素筛选，其插入片段经载体引物(35S启动子序列引物，CACGTCTTCAAAGCAAGTGGA)与基因引物(基因下游引物，引物序列见实施例1)PCR鉴定，示意图见图1。
拟南芥的转化过程：
试剂配制：
渗透培养基(1L)：1/2xMurashige-Skoog；5％(质量比)蔗糖；0.5克MES；用KOH调至pH5.7；再加：10ul 1mg/ml的6-BA(6-苄氨基嘌呤)母液；200微升Silwet L-77。
转化步骤：
(1)制备好已转化了相应质粒的农杆菌(GV3101，市场可购买)菌液10ml，在转化前一天晚上，转入大瓶培养过夜，第二天取出使用时农杆菌液0.D600当在1.2到1.6之间。
(2)室温5000rpm离心15分钟。
(3)弃上清，将农杆菌沉淀悬浮于相应体积的渗透培养基里，使O.D600在0.8左右。
(4)将整个植株直接浸泡至农杆菌悬浮液30s。
(5)避光培养过夜，然后正常培养至结子，收获种子作下一步筛选鉴定。
实施例3：转基因拟南芥的筛选和验证：
转化子的筛选：
将春化过的拟南芥种子种于浇过饱和花宝二号(商业购买)营养液的人工土中，并用保鲜膜罩上。两天后光照，三天后揭膜。
人工培养室条件：相对湿度80％，恒温20-240C，光照强度80-200umol/M2/S，光照周期为8h黑暗、16h光照培养。一周左右，喷除草剂(草苷膦)筛选阳性植株。
PCR鉴定：
(1)用于PCR的转化植株总DNA的提取：
A.70％(体积比)乙醇擦洗叶片，称取大约100mg
B.加入600ul抽提缓冲液(0.2M Tris-Cl，0.25NaCl，25mM EDTA，0.5％(质量比)SDS，pH 7.5)，室温快速研磨。
C.1.5ml Ependorff管中涡旋混匀5-10s。
D.12000rpm，25min，室温。取上清，加等体积异丙醇，-20摄氏度沉淀过夜。
E.12000rpm，15min，室温。加入70％(体积比)乙醇200ul泡洗DNA沉淀。
F.12000rpm，15min，室温。去乙醇。倒置于纸巾上，待乙醇挥发干净。
G.加无菌水100ul溶解粗提DNA沉淀。用分光光度计测定或电泳估测其浓度。
H.以总DNA为模板，进行PCR。
(2)PCR程序：
PCR反应混合液的配比同质粒PCR鉴定，依据植物表达载体中35S启动子序列和BnAOX1、AtAOX1基因下游引物5’-tcaatgatccaattggag-3’，5’-tcaatgatatccaatgggagc-3’，反应的时间和温度如下：94℃3min，94℃45s，59℃45s，72℃2min 30s，30cycles，72℃5min。
检测结果显示，大多数转化植株均能够扩增出预期大小的电泳条带，而阴性对照则没有，表明已经获得多株转基因拟南芥株系。
实施例4.：转基因拟南芥种子含油量及千粒重检测
转基因纯合株系生长于21-23℃温室中，收获种子后测其含油量及千粒重的变化(图3)。结果显示，转基因拟南芥种子含油量与野生型拟南芥相比有明显提高，最高幅度可达20％以上。而千粒重也有所增加，增加幅度最高达25％。
表1过量表达BnAOX1、AtAOX1基因的拟南芥含油量及千粒重测定结果
SEQUENCE LISTING
<110> 中国农业科学院油料作物研究所
<120> 油菜呼吸代谢相关基因BnAOX1及应用
<130> 油菜呼吸代谢相关基因BnAOX1及应用
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atgatgatga gtcgctacgg agccaaggta atggtaacta ctggaccaag gctcttctct 60
tccagtgccg tgtctcccaa ccatcttttg aattctaggg ttccggcgag agagttcagc 120
aagatgacat ttgagaaaaa gaagaaaacg gaggataacc aatgggagaa aggttcaagt 180
ggtggcaagg gtgatcaagg gaacaaaggg gagcaattaa tcgttagcta ttggggagtg 240
aagcctatga aaatcaccaa agatgatgga actgaatgga aatggagctg ctttcggcca 300
tgggagacgt ataaagcaga tctgacaata gatttgaaga agcatcatgt tccatcgact 360
ttaccggaca aattagctta ctggacggtt aaatctctcc gatggcctac cgatttgttc 420
ttccagaggc ggtacggatg ccgagcgatg atgttggaaa cggtagcagc ggttccagga 480
atggtcggag gaatgttagt acactgcaaa tcgctgaggc ggtttgaaca aagcggcggt 540
tggatcaaag cactccttga agaagcagag aacgagagaa tgcacttgat gacattcatg 600
gaagtcgcaa aacccaattg gtacgaacgt gccctcgtga tcgccgttca aggcgttttc 660
ttcaacgcat atttcctcgg atacataatc tcgcccaagt ttgctcaccg tatggttgga 720
taccttgagg aagaagcaat ccactcttac actgaattcc tcaaggaact tgataatggt 780
aacatcgaaa acgttcctgc accagccata gccattgatt actggaggct ccctgctgat 840
gcaacgcttc gtgatgttgt catggtggtt cgtgctgatg aagcacatca ccgtgacgtt 900
aaccattatg catccgatat tcattaccaa ggtcgtgaac ttaaggaagc tccagctcca 960
attggatatc attga 975
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<211> 324
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Asp Gln Gly Asn Lys Gly Glu Gln Leu Ile Val Ser Tyr Trp Gly Val
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Lys Lys His His Val Pro Ser Thr Leu Pro Asp Lys Leu Ala Tyr Trp
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Thr Val Lys Ser Leu Arg Trp Pro Thr Asp Leu Phe Phe Gln Arg Arg
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Phe Leu Gly Tyr Ile Ile Ser Pro Lys Phe Ala His Arg Met Val Gly
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Leu Asp Asn Gly Asn Ile Glu Asn Val Pro Ala Pro Ala Ile Ala Ile
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Asp Tyr Trp Arg Leu Pro Ala Asp Ala Thr Leu Arg Asp Val Val Met
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Val Val Arg Ala Asp Glu Ala His His Arg Asp Val Asn His Tyr Ala
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Ser Asp Ile His Tyr Gln Gly Arg Glu Leu Lys Glu Ala Pro Ala Pro
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Ile Gly Tyr His
Claims (4)
1.一种分离的基因，其序列为SEQ ID NO:1所示的核苷酸序列。
2.一种分离的蛋白质，其序列为SEQ ID NO:2所示的氨基酸序列。
3.权利要求1所述的分离的基因或权利要求2所述的分离的蛋白质在提高拟南芥植物种子千粒重中的应用。
4.权利要求1所述的分离的基因或权利要求2所述的分离的蛋白质在提高拟南芥植物种子含油量中的应用。
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| PCT/CN2012/070283 WO2012100682A1 (en) | 2011-01-27 | 2012-01-20 | Respiratory metabolism-associated gene bnaox1 from brassica napus and use thereof |
| CA2825924A CA2825924C (en) | 2011-01-27 | 2012-01-20 | Respiratory metabolism-associated gene bnaox1 from brassica napus and use thereof |
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