CN113564181A - 油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量中的应用 - Google Patents
油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量中的应用 Download PDFInfo
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Abstract
本发明公开了油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量和脂肪酸组成中的应用,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4所示。通过获得过表达和突变体材料分析转化材料的含油量,发现BnNTT1基因促进甘蓝型油菜种子含油量的积累,同时还调控脂肪酸组成。因此可使用农杆菌介导的遗传转化方法将含有BnNTT1基因的过表达载体转化到作物中,获得BnNTT1基因过表达的作物品种。
Description
技术领域
本发明属于基因工程和作物育种技术领域,具体涉及油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量和脂肪酸组成中的应用。
背景技术
植物食用油提供了人体所需的大部分脂肪酸,其中饱和脂肪酸是人类生命活动中的主要能量来源;此外,植物油脂还是制造化妆品、油漆、肥皂、润滑剂等化工产品的重要原料。油菜则是世界三大油料作物之一,菜籽油消费量位居世界第三,仅次于棕榈油和大豆油。据研究表明,油菜种子含油量每提高1个百分点,单位面积油菜的产油量就能提高2.3-2.5百分点左右,因此提高种子含油量是提高油菜单位面积产油量的关键措施之一。此外,高油育种也一直是油菜育种的主要目标之一。
油脂是油和脂肪的统称,油是不饱和高级脂肪酸甘油酯,脂肪是饱和高级脂肪酸甘油酯。脂肪酸的生物合成受到多种酶、底物和转录因子的调控,是一个复杂的生理生化反应。ACCase就是一个脂肪酸从头合成的关键作用酶,研究表明异质型的ACCase可能调控胡麻种子发育前期油脂的合成积累;另外,一些转录因子也在脂肪酸的合成过程中起到了关键的调控作用,包括LEC1、LEC2、FUS3、WRI1和Dof等,这些与脂肪酸合成相关的转录因子组成了复杂的网络系统,来共同调控种子发育过程中油脂的合成与积累。
本发明从油菜中克隆了保守的三磷酸核苷酸转运蛋白基因BnNTT1。研究表明,在组成型启动子35S的作用下将该基因在油菜中过表达可以显著提高种子的含油量;而利用CRISPR/Cas9基因编辑技术创建的突变体油菜种子的含油量显著下降。以上结果表明BnNTT1基因在调控油菜种子含油量中起到重要作用,可以为油菜高油育种提供理论参考依据,同时为油脂合成这一研究提供新的思路。
发明内容
本发明的目的在于提供油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量和脂肪酸组成中的应用,该基因能与ATP/ADP结合,将胞浆内的ATP转运到叶绿体中,并将ADP交换到细胞质中。该基因在油菜C6和A7染色体上各有两个拷贝,分别是BnaC06.NTT1a(BnaC06g19090D)、BnaC06.NTT1b(BnaC06g40660D)、BnaA07.NTT1a(BnaA07g38360D)和BnaA07.NTT1b(BnaA07g35740D),基因的核苷酸序列分别如序列表SEQ ID NO:1、SEQ IDNO:2、SEQ ID NO:3和SEQ ID NO:4所示,其中BnaC06.NTT1a由1851bp组成、BnaC06.NTT1b由1848bp组成、BnaA07.NTT1a由1851bp组成,BnaA07.NTT1b由1845bp组成;该基因编码的蛋白质序列分别如序列表SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示,其中BnaC06.NTT1a编码616个氨基酸、BnaC06.NTT1b编码615个氨基酸、BnaA07.NTT1a编码616个氨基酸,BnaA07.NTT1b编码614个氨基酸。可以采用PCR技术从植物基因组、mRNA和cDNA中扩增得到本发明的基因。
在构建重组植物表达载体时,在其转录起始核苷酸前加一种组成型启动子花椰菜花叶病毒CaMV 35S,构建含有三磷酸核苷酸转运蛋白基因BnNTT1的重组表达载体,为了便于对转基因植物进行筛选,表达载体上加入了抗生素标记物。
本发明提供一种提高作物含油量的方法:使用农杆菌介导的遗传转化方法将含有三磷酸核苷酸转运蛋白基因BnNTT1的过表达载体转化到作物的基因组中,获得基因BnNTT1超表达的作物品种。
本发明通过获得过表达和突变体材料,分析了转化材料的含油量,发现BnNTT1正调控含油量的积累,同时还调控脂肪酸组成。
本发明中所述的表达载体是指现有技术中已知的、能够在植物中进行表达的任何载体,例如适用于构建本发明所述的表达载体包括但不限于,如p35S-FAST、PHSE401(中国农业大学陈其军课题组提供)等。
本发明获得的高含油量过表达油菜植株,在其营养生长与生殖生长时期比正常植株发育更好,因此该遗传资源在油菜高油育种中具有潜在的应用价值。
更加详细的技术方案参见具体实施例。
附图说明
图1:BnaC06.NTT1b和BnaA07.NTT1a基因扩增产物的琼脂糖凝胶电泳检测结果。
图2:构建的植物表达载体p35S-FAST-BnaC06.NTT1b(图2a)和p35S-FAST-BnaA07.NTT1(图2b)的质粒图谱。
图3:油菜转化单株的qRT-PCR检测。OE13和OE14是基因BnaC06.NTT1b的超表达株系(图3a),OE22和OE23是基因BnaA07.NTT1a的超表达株系(图3b)。每个株系有3个不同单株,**表示在Student’s t test中P<0.01。
图4:油菜BnNTT1突变体的获得和鉴定。sgRNA位于基因BnaC06.NTT1a、BnaC06.NTT1b、BnaA07.NTT1a和BnaA07.NTT1b的第一个外显子区,获得的2个突变体的靶位点的编辑情况:M55和M56为BnaC06.NTT1b和BnaA07.NTT1a均被编辑的双突。
图5:BnaC06.NTT1b和BnaA07.NTT1a在油菜的表型鉴定。利用GC-MS分析油菜种子的含油量,M55和M56为BnaC06.NTT1b和BnaA07.NTT1a的CRISPR双突变体。OE13和OE14是基因BnaC06.NTT1b的超表达株系,OE22和OE23是基因BnaA07.NTT1a的超表达株系。每个株系有5个不同单株,根据方差分析,不同的字母表示P<0.05的显著差异。
具体实施方式
以下结合具体实施例对本发明作进一步详细说明。应理解,这些实施例仅用于说明本发明而不是用于限制本发明的范围。下列实施例中未详细说明的实验方法,通常按照常规方法或工具书《分子克隆:实验室指南》(New York:Cold Spring Harbor Laboratory,1989)中所述的方法,或者按照生产商提供的操作手册中建议的方法。
实施例1 BnaC06.NTT1b和BnaA07.NTT1a基因的克隆
NTT1基因在油菜C6和A7染色体上各有两个拷贝,分别命名为BnaC06.NTT1a(BnaC06g19090D)、BnaC06.NTT1b(BnaC06g40660D)、BnaA07.NTT1a(BnaA07g38360D)和BnaA07.NTT1b(BnaA07g35740D)。CDS序列分别为序列表SEQ ID NO:1、SEQ ID NO:2、SEQ IDNO:3和SEQ ID NO:4所示的序列,含有完整的ORF阅读框及起始密码子ATG,它们分别编码616、615、616和614个氨基酸(分别见SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ IDNO:8)。在种子发育过程中,我们发现BnaC06.NTT1b和BnaA07.NTT1a的表达水平高于BnaC06.NTT1a和BnaA07.NTT1b。此外,BnaC06.NTT1a和BnaA07.NTT1b具有保守的氨基酸序列和高度相似的蛋白质结构。为便于操作,我们选择基因BnaC06.NTT1b和BnaA07.NTT1a作为候选基因进行克隆。
(1)RNA的提取
总RNA的提取采用全式金公司的TransZol(目录号ET101),提取方案遵从试剂盒使用说明书。需要提前准备的试剂有RNase-free水、氯仿、异丙醇和75%乙醇(由DEPC处理的水配制)。所用试剂和实验用品均经过DEPC灭活RNA酶处理。具体步骤如下:
a.取甘蓝型油菜的叶片于液氮中充分研磨直至粉末状,取100mg研磨好的样品转移到1.5ml离心管中,加入1ml TransZol,之后上下剧烈震荡使之充分混匀,进行匀浆处理,之后室温静置5分钟;
b.向离心管中加入0.2ml氯仿,剧烈振荡15秒,室温孵育3分钟;
c.10,000×g 4℃离心15分钟;
d.将上层无色的水相转移至新的离心管中(体积大约为0.6ml),加入0.5ml经过预冷的异丙醇并颠倒混匀,室温孵育10分钟;
e.10,000×g 4℃离心10分钟,倒去上清,离心管侧壁和底部形成沉淀;向离心管中加入1ml 75%乙醇(DEPC水配制),剧烈涡旋;
f.7,500×g 4℃离心5分钟,去上清,室温晾干沉淀15分钟左右;
g.将沉淀溶于50μl-100μl RNA溶解液中,55-60℃孵育10分钟,将样品保存于-80℃冰箱中备用。
h.取1μl抽提的总RNA在Nanodrop下测定RNA浓度和质量,依据1.8<OD260/OD280<2.0鉴定RNA纯度。同时取2μl进行1%的琼脂糖凝胶电泳,检测完整性和质量。
(2)RNA反转录
以5μg总RNA为模板,依次加入1μl的Anchored Oligo(dT)18Primer,10μl的2×ESReaction Mix,1μl的RT/RI Enzyme Mix,1μl的gDNA Remover,最后用RNase-free Water补充至20μl。将以上反应体系轻轻混匀后置于42℃孵育30min,进行合成第一链cDNA和去除gDNA;之后,85℃加热5秒钟失活RT/RI和gDNA Remover。最后,加入180μl RNase-free Water溶解合成的cDNA。
(3)BnaC06.NTT1b和BnaA07.NTT1a基因的扩增
以上述cDNA为模板,用正向引物5’-GCGGGTACCATGGCAACCGTGATACAAAC-3’和反向引物5’-GCGGGATCCGTATGTTGGTGGGAGCTG-3’扩增得到含有BnaC06.NTT1b全长CDS的片段;用正向引物5’-GCGGGTACCATGGAAGCGGCGATACAAAC-3’和反向引物5’-GCGGGATCCGTATGTTGGTGGGAGCTG-3’扩增得到含有BnaA07.NTT1a全长CDS的片段。采用I-5TM2×High-Fidelity Master Mix(TSINGKE Biologica technology)进行PCR扩增。PCR扩增反应体系为:
按照上述反应体系配置好反应液于PCR管中,在Bio-Rad PCR仪上进行扩增,PCR扩增程序为:
扩增后产物通过1%琼脂糖凝胶电泳检测(图1),扩增获得1848bp的BnaC06.NTT1b全长和1851bp的BnaA07.NTT1a全长,PCR扩增产物的挖胶回收使用天根琼脂凝胶DNA回收试剂盒(DP130227)。
实施例2 BnaC06.NTT1b和BnaA07.NTT1a基因过表达转化载体的构建
(1)对上述获得的BnaC06.NTT1b和BnaA07.NTT1a片段用快速限制性内切酶KpnI和BamHI进行双酶切,本实验所用的限制性内切酶为上海赛默飞世尔科技(中国)有限公司产品。双酶切体系如下:
酶切反应在37℃水浴1.5小时。酶切产物利用北京天根生物工程有限公司的通用型DNA纯化回收试剂盒(DP130227)进行回收。
(2)酶切产物连接到载体p35S-FAST,连接反应体系如下:
连接反应条件:22℃3小时。
(3)转化大肠杆菌DH5α,筛选阳性克隆后提质粒酶切鉴定,选取3个阳性克隆送样测序,分析结果显示,BnaC06.NTT1b和BnaA07.NTT1a基因的CDS序列成功连接上载体,即成功构建并转化植株的植物表达载体p35S-FAST-BnaC06.NTT1b和p35S-FAST-BnaA07.NTT1a(如图2所示)。
(4)将构建正确的重组质粒载体导入农杆菌菌株GV3101,挑选阳性单克隆于-80℃冰箱保存。使用电击法转化农杆菌GV3101感受态,具体实验步骤如下:
a.用纯水、超纯水、无水乙醇分别清洗电转杯,置于超净台晾干;
b.将重组质粒和农杆菌GV3101感受态置于冰上解冻;
c.取构建正确的重组质粒1μl,加入到25μl农杆菌GV3101感受态中,轻轻吸打混匀,避免产生气泡;
d.将上述混合液沿着杯壁迅速转入已经预冷的电转杯中;
e.将电转仪调至1800V,之后用吸水纸擦干电转杯外壁,在1800V电压条件下电击;
f.电击成功后,向电转杯中加入200μl无抗LB液体培养基,轻轻吸打几下,之后将溶液转移至1.5ml无菌离心管中,28℃150r/min活化2小时左右;
g.将复苏的菌株涂到含有抗生素的LB固体培养基上,于28℃培养箱中倒置培养48小时;
h.在平板上挑取单菌落,用载体前引物和目的基因后引物对其进行PCR扩增,1%琼脂糖凝胶电泳检测阳性克隆。
i.取阳性菌液加入抗性的LB液体培养基,于28℃,150r/min摇床中扩大培养至OD值达到0.8,加入等体积的50%(v/v)的甘油混匀,将其保存于-80℃冰箱。
实施例3 BnaNTT1-CRISPR载体的构建
利用中国农业大学陈其军实验室的sgRNA-Cas9系统创建突变体。实验操作步骤如下:
(1)登录到网站http://www.cbi.hzau.edu.cn/cgi-bin/CRISPR,筛选靶点。构建两个靶点同时编辑BnaC06.NTT1a(BnaC06g19090D)、BnaC06.NTT1b(BnaC06g40660D)、BnaA07.NTT1a(BnaA07g38360D)和BnaA07.NTT1b(BnaA07g35740D)四个基因的载体,两个靶点均设计在四个基因的第一外显子上,sgRNA1的碱基序列为5’-CCTTACCCGCCAAACCCATCGG-3’,5’端距离起始密码子ATG 34bp,sgRNA2碱基序列为5’-GTGAATATAGTTGCTGAGAGG G-3’,5’端距第一个外显子末端159bp。BnaC06.NTT1b基因中两靶点相距578bp,BnaA07.NTT1a基因的两靶位点间相隔572bp。
(2)设计引物:
DT1-BsF:ATATATGGTCTCGATTGCCTTACCCGCCAAACCCATGTT
DT1-F0:TGCCTTACCCGCCAAACCCATGTTTTAGAGCTAGAAATAGC
DT2-R0:AACTCTCAGCAACTATATTCACCAATCTCTTAGTCGACTCTAC
DT2-BsR:ATTATTGGTCTCGAAACTCTCAGCAACTATATTCACCAA
(3)PCR扩增:以稀释100倍的pCBC-DT1T2为模板进行四引物PCR扩增。DT1-BsF和DT2-BsR为正常引物浓度;DT1-F0和DT2-R0稀释20倍。PCR扩增体系为:
PCR扩增程序为:
(4)纯化回收PCR产物,建立如下酶切-连接体系:
(5)取5μl连接产物转化大肠杆菌感受态,在Kana抗性的LB培养基上筛选阳性克隆。U626-IDF+U629-IDR=726bp菌落PCR鉴定,U626-IDF和U629-IDF测序,测序正确的载体即为构建成功的CRISPR载体。
(6)将构建正确的重组质粒载体导入农杆菌菌株GV3101感受态中,挑选阳性单克隆于-80℃冰箱保存,继续油菜遗传转化工作的开展。
实施例4遗传转化实验
(1)油菜的遗传转化
对构建好的过表达载体和CRISPR载体采用下胚轴暗光培养进行油菜的遗传转化,本发明中用于油菜转化的受体为甘蓝型油菜Westar,具体方法如下:
a.种子的灭菌:
将经过挑选的成熟饱满的甘蓝型油菜‘Westar’种子倒入培养盒中,用75%的酒精浸泡种子1min左右,时间不能超过5分钟;使用无菌水清洗浸泡后的种子1-2次;加入能够淹没种子的0.15%-0.2%升汞溶液(保存于实验室的棕色瓶中,可用实验室5%的母液稀释),消毒15min;用无菌水清洗种子5-6次。
b.播种:
用灭过菌的镊子将种子播种于M0培养基,每个培养皿均匀播种40-50粒种子;播完种后将培养皿放在暗光条件下培养7天左右,温度25℃。
c.农杆菌的培养:
播种五天后,在抗性平板上接种细菌,用经过消毒的接种环蘸取含有目的基因的农杆菌菌液在抗性LB平板上划线;2天后在抗性平板上用牙签或枪头吸取阳性单菌落,置于抗性LB液体培养基中吹打,于28℃,150r/min摇菌。
d.外植体的制备和侵染:
检测菌液在600nm吸收光条件下的浓度(分光光度计),测出菌液的OD值,OD值在0.6-0.8之间为最佳,此时将菌液6000rpm离心10min,弃上清;用与菌液等体积的DM(Dilution Medium)液体重悬,6000rpm离心10min,弃上清;用与菌液相同体积的DM溶液悬浮。之后取2ml菌液于灭菌的培养皿中,20ml DM溶液对其进行稀释(1:10);用无菌解剖刀和镊子将暗光培养7天后的幼苗下胚轴切下,切取长度为0.8-1.0cm,切取外植体时尽量一刀切下,保持其切口整齐;将切取下来的外植体放到已配好浓度的菌液中,侵染30min(时间不能过长),用移液器隔段时间吸打一次,使得切口充分接触菌液。
e.愈伤组织的培养:
用已灭菌的吸水纸吸干外植体上的菌液,再将外植体转移到M1培养基中,每皿50-60个外植体,暗光条件下培养2天左右,温度25℃;两天后,将外植体转移到诱导愈伤的M2培养基中,光照条件下进行培养(白天16h/夜晚8h),温度22℃,之后都在光照条件下进行培养;三周后,将生长正常,两端膨大的外植体转到分化培养基M3中,每2-3周继代一次,长出绿芽为止;切下已经有明显基节的绿芽并将其转入培养基M4中进行生根,需要2-4周,待生根后将其移栽至室外大田里,观察植株田间生长表型(培养基配方见表1)。
表1下胚轴暗光培养的培养基配方
(2)过表达转化单株的鉴定
提取获得的油菜过表达转化单株的基因组DNA,通过PCR检测外源基因片段的插入,本发明中过表达的骨架载体为p35S-FAST,在骨架载体上设计引物FAST-F(5’-AAAGGCCATCGTTGAAGATG-3’)和FAST-R(5’-TCGAACTCAGTAGGATTCTG-3’),通过载体骨架引物与外源片段引物搭配来进行PCR反应(FAST-F和BnaC06.NTT1b-R/BnaA07.NTT1a-R或者BnaC06.NTT1b-F/BnaA07.NTT1a-F和FAST-R),外源片段引物BnaC06.NTT1b-F(5’-GCGggtaccATGGCAACCGTGATACAAAC-3’),BnaC06.NTT1b-R(5’-GCGggatccGTATGTTGGTGGGAGCTG-3’),BnaA07.NTT1a-F(5’-GCGggtaccATGGAAGCGGCGATACAAAC-3’)和BnaA07.NTT1a-R(5’-GCGggatccGTATGTTGGTGGGAGCTG-3’),在PCR水平检测转基因苗。
PCR反应体系为:
PCR反应程序为:
对PCR获得的油菜转基因阳性苗进行qRT-PCR,以检测基因表达量。利用TransZol试剂盒提取转化单株种子的RNA并进行反转录合成cDNA(方法同实施例1),最后用全氏金的PerfectStartTM Green qPCR SuperMix试剂对样品进行荧光定量PCR分析。
定量引物利用Primer 5软件设计得到,产物大小在100bp-200bp之间,设计好后用参考序列进行BLAST比对,确保引物RT-BnaC06.NTT1b-F(5’-TCCTCTTGCTATCCTGAGGA-3’)、RT-BnaC06.NTT1b-R(5’-GAGAAAATGAGCGCAACGTT-3’)、RT-BnaA07.NTT1a-F(5’-GCTTTGGGGTAGTGTGGTTAT-3’)和RT-BnaA07.NTT1a-R(5’-ATTGGCTCCGAGTCCAAACAA-3’)的特异性。BnACTIN7-L(5’-CGCGCCTAGCAGCATGAA-3’)和BnACTIN7-R(5’-GTTGGAAAGTGCTGAGAGATGCA-3’)作为油菜qRT-PCR的内参引物。qRT-PCR反应体系为:
qRT-PCR反应程序为:
qRT-PCR反应在Bio-Rad CFX96 Real-Time System中进行。
根据内参引物进行标准化,不同重复间定量变异用delta-deltathreshold cyclerelative quantification(2-ΔΔCT)的方法计算。最后分析选择油菜过表BnaC06.NTT1b的两个单株OE13和OE14和过表达BnaA07.NTT1a的两个单株OE22和OE23进行进一步分析(图3)。
(3)CRISPR转化单株的鉴定
对获得的油菜CRISPR转化单株进行测序筛选油菜突变体。首先利用引物Cas9-F(5’-CGCACAATCCCACTATCCT-3’)和Cas9-R(5’-CCAGGTCATCGTCGTATGTG-3’)鉴定Cas9蛋白,对于Cas9蛋白阳性单株进行目的基因的特异扩增以及测序鉴定。目的基因的特异扩增的方法为:分别用引物NTT1b-C6-F(5’-ATGGCAACCGTGATACAAAC-3’)和NTT1b-C6-R(5’-GGCTTCATCCACAGTTGTTATC-3’)特异扩增BnaC06.NTT1b;NTT1a-A7-F(5’-ATGGAAGCGGCGATACAAAC-3’)和NTT1a-A7-R(5’-CAAGAGCCTCCGGG-3’)特异扩增BnaA07.NTT1a。扩增方法同实施例4(2)所示。
对扩增的目的片段进行PCR产物测序,分析靶位点的编辑情况。测序结果显示,获得了BnaC06.NTT1b和BnaA07.NTT1a同时被编辑的双突变体株系M55和M56,编辑造成了靶点处不同程度的碱基插入和缺失(图4)。
(4)遗传转化所得转基因植株的含油量分析
采用气相色谱-火焰离子化检测器法测定油菜种子含油量:
a.称取4粒形状大小一致的油菜成熟种子,保证总质量在15mg-18mg范围内,记录下称取的质量,每株材料进行5个重复。
b.将称取好对油菜种子放入干净的提脂管中,加入4.5ml甲醇提取液(95%甲醇,5%浓硫酸,0.01%BHT)。
c.利用玻璃棒研破提脂管中种子的种皮并使种子露出胚。
d.用玻璃针(100μl)量取配好的十七烷酸100μl(C17:0,16.2μmol/ml,分子量270.45)于提脂管中,用力旋紧管盖,上下颠倒混匀。
e.将提脂管置于85℃水浴锅中,水浴2h,让其充分反应。
f.2h后,将提脂管拿出并冷却至室温,之后加入3ml的ddH2O和3ml的正己烷,注意要缓慢加液防止液体溅出,涡旋混匀。
g.1000r/min 25℃离心10min,用胶头滴管吸取1ml左右的上清液于进样瓶中,用于之后的GC-MS分析,暂时不用可放置-20℃冰箱储存。
(以上提取脂肪酸过程用的有机试剂均是色谱纯级别。)
h.设置高效气相色谱气质联用分析仪(GC-MS)参数:将分流比设成10:1,选择Rtx-Wax(0.25mm×30m)色谱柱,设置GC进样口温度为230℃,柱箱初始温度为170℃并保持2min,之后温度以5℃/min的速率上升至230℃保持5min。MS进样口温度设置为230℃,调谐电压为0.2kV。之后GC-MS自动进样进行分析。
i.利用GC-MS的峰图结果对物质进行定性分析。利用GC-MS的峰图结果对物质进行定量分析:结合物质的分子量和质量,根据内标计算脂肪酸组成和含油率。
含油量结果显示,与WT(39.25±0.51)相比,BnaC06.NTT1b基因的过表达材料含油量分别为OE13(42.97±0.33)和OE14(41.77±0.26),显著上升2.5-3.7个百分点(图5a);BnaA07.NTT1a基因的过表达材料含油量分别为OE22(41.96±0.37)和OE23(42.80±0.85),显著上升2.7-3.5个百分点(图5a);而突变体材料的含油量分别为M55(36.44±0.33)和M56(37.56±0.55),显著下降1.7-2.8个百分点(图5a)。另外,脂肪酸组成结果显示,与WT相比,过表达材料的C16:0、C18:0、C18:1、C18:2和C18:3含量均显著增加(图5b)。
这些结果表明,BnaC06.NTT1b和BnaA07.NTT1a在调控甘蓝型油菜种子含油量和脂肪酸组成中确实发挥着重要作用。
序列表
<110> 华中农业大学
<120> 油菜三磷酸核苷酸转运蛋白基因 BnNTT1在调控作物含油量中的应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1851
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 1
atggaagcgg cgatacaaac cagaggaatc ctctccttac ccgccaaacc catcggagcg 60
agaagaagcc ttctccaccc atcccacggc ttaaagcata gacttttctc ctccaagcca 120
agaactccac ccgccttgtc tctctccttc aagaaagctc aatcctttga cccaaagatt 180
tccatttccc acaaggagag aaccaccgag ttcatatgca aggcggaggc agacggagga 240
gctctgctca acgaaggcga cacggcagcg attgtaccat caccaaagat cttcggcgtg 300
gaggtcacaa cgttgaaaaa gattatacct ttaggtttaa tgttcttttg cattctattc 360
aactacacga tcctgaggga cacgaaggac gtcttggtcg taacggctaa aggaagttcg 420
gcagagatta tacctttctt gaagacgtgg gtcaatctcc ctatggctat tgggttcatg 480
ctcctctaca ccaagctctc caatgttctc tccaagaagg ctctctttta caccgttatt 540
atccctttca tcatctactt tggggccttt ggtttcgtta tgtaccctct cagcaactat 600
attcacccgg atgctcttgc tgataagctc cttgctgctc tcggtccaag gttcatgggt 660
cctcttgcta tcttgaggat ttggagtttc tgtttgtttt atgtcatggc tgagctttgg 720
ggtagtgtgg ttatttcagt tctcttctgg ggctttgcta atcagatcac aactgtggat 780
gaagccaaga aattctatcc cttgttcgga cttggagcca atgttgcgct gattttctcc 840
ggaagaacgg tgaaatactt ttctaacttg agaaagaatc ttggtcctgg agttgatggc 900
tgggcagttt cattgaaagc catgatgagc attgtggtgg gaatggggct tgccatctgt 960
ttcctctatt ggtgggttaa tagatacgtt cctcttccaa ctcgtagcct gaagaagaag 1020
aacaaaccaa agatgggaac gatggagagc ttgaagttct tggtgtcatc accatacatt 1080
agggatcttg ctactttagt ggtagcgtat ggtattagta tcaaccttgt ggaagtcaca 1140
tggaaatcaa agcttaaagc tcagtttcct agcccgaacg agtactcggc gtttatgggt 1200
gacttctcaa cctgcactgg tattgcaaca ttcacaatga tgcttctcag tcaatacgta 1260
tttgataagt atggttgggg agtagctgca aagatcactc caactgttct cttattgact 1320
ggcgttgcct tcttctctct gatactgttt ggtggcccat tcgcaccact tgttgccaag 1380
cttggtatga ctccgctgct tgcagctgtt tatgttggtg ctctccagaa tatcttcagc 1440
aagagtgcca agtacagctt gttcgatcct tgtaaagaaa tggcttacat ccccttggat 1500
gaggatacca aggttaaagg gaaagctgca attgatgtgg tttgcaaccc gttagggaaa 1560
tcagggggtg ctctaataca gcagttcatg atcttatcct ttggatcact agccaattca 1620
acaccatacc taggaatgat actgttggtc attgtcactg catggttagc tgcagctaag 1680
tcactggagg gacagttcaa tgccttgagg tctgaagaag agctggagaa ggaaatggag 1740
agagcttcat cggtgaagat acctgttgtg tctcaagagg aaggtggaaa cggttccctt 1800
ggagaatcta caagcagttg gcctgagaaa tcagctccca ccaacatata a 1851
<210> 2
<211> 1848
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 2
atggcaaccg tgatacaaac cagaggactt ctctccttac ccgccaaacc catcggagtg 60
aggagccttc tccagccatc ccacggctta aagcacagac ttttcgcctc caaaccgaga 120
aatccacctg ccttgtctct atcctctaac ggccaccaga aatttcaatc ctttgagcca 180
acccttcccg ggatttccat ttcccacaag gcgagaagca ccgagttcat atgcaaagcg 240
gaggcagcgt ccgccggcga aggcgacacg gcggcgattt cagcgtcgcc gaagatcttc 300
ggcgtggagg tcacgacctt gaaaaagata atacctttag ggttgatgtt cttttgcatt 360
ctgttcaact acacaatcct gagggacacg aaggacgtct tggtggtgac tgcgaaaggg 420
agctcagctg agattatacc tttcttgaag acgtgggtca atctccctat ggccattggc 480
ttcatgctcc tctacaccaa gctctccaat gttctctcca aaagggctct cttttacacc 540
gttatcatcc ctttcatcct ctactttggg gccttcggtt tcgtcatgta ccctctcagc 600
aactatattc accccgaagc tcttgctgat aagctcctcg ccactctcgg cccaaggttc 660
atgggtcctc ttgctatcct gaggatttgg agtttctgtt tgttttatgt catggctgag 720
ctttggggta gtgtcgtcat ttctgttctc ttctggggct ttgctaatca gataacaact 780
gtggatgaag ccaagaaatt ctatccttta ttcggacttg gagccaacgt tgcgctcatt 840
ttctctggaa gaacggtgaa atacttctct aacttgagaa agaatcttgg tcctggagtt 900
gacggatggg cagtttcact gaaagccatg atgagcattg tggttgggat gggacttgcc 960
atctgtttcc tctattggtg ggttaataca tatgttccgc ttcctgcccg tagccagaag 1020
aagaagaaca aaccgaagat gggaacgata gagagcttga agttcttggt gtcatcacca 1080
tacataaggg atcttgctac tttagtggtg gcgtatggta ttagtatcaa ccttgtggaa 1140
gtcacttgga aatcaaagct taaagctcag tttcctagcc cgaacgagta ctcagcattt 1200
atgggggact tctcaacctg caccggtatt gcaacattca cgatgatgct tctcagccaa 1260
tacgtatttg ataaatatgg ctggggagta gcagcaaaga tcaccccaac tgttctgcta 1320
ttgactggcg ttgccttctt ctccctgata ctgtttggcg gcccattcgc accacttgtt 1380
gccaagcttg gtatgactcc gctacttgca gctgtctatg ttggtgccct tcagaatata 1440
ttcagcaaga gtgccaagta cagcttgttc gatccctgta aagagatggc ctatatcccc 1500
ttggatgagg ataccaaggt taaagggaaa gctgcgattg acgtggtatg caacccgttg 1560
gggaaatcag ggggtgctct aatacagcag ttcatgatct tatcatttgg atcactagcc 1620
aattcaacgc cctacctagg aatgatattg ttggtcattg tcactgcctg gttagctgca 1680
gctaagtcgc tggagggaca gttcaacgct ttgaggtctg aggaagagct ggagaaggaa 1740
atggagagag cttcgtcggt gaagatccct gttgtgtctc aagatgaagg ttcccttgga 1800
gaatcttcaa gcagttcacc tgagaaatca gctcccacca acatatag 1848
<210> 3
<211> 1851
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 3
atggaagcgg cgatacaaac cagaggaatc ctctccttac ccgccaaacc catcggagcg 60
agaagaagcc ttctccaccc atcccacggc ttaaagcata gacttttctc ctccaagcca 120
agaactccac ccgccttgtc cctctccttc aagaaagctc aatcctttga gccaacgatt 180
tccatttccc acaaggagag aagccacgag ttcatatgca aggcggaggc agacggagga 240
gctctcctca acgaaggcga caccgccgca gtcgtaccat caccaaagat cttcggcgtg 300
gaagtcacaa cgttgaaaaa gatcatacct ttaggcttaa tgttcttttg cattctgttc 360
aactacacga tcctgaggga cacgaaggat gttttggtgg tgacggcgaa aggaagttca 420
gctgagatta ttcccttttt gaagacgtgg gtcaatctcc ctatggctat tgggttcatg 480
ctcctctaca ccaagctctc caacgttctg tccaaaaagg ctctctttta caccgttatt 540
atccctttca tcatctactt tggggccttt ggtttcgtta tgtaccctct cagcaactat 600
attcacccgg aggctcttgc tgataagctc cttgctgctc tcggtccaag gttcatgggt 660
cctcttgcta tcttgaggat ttggagtttc tgtttgtttt atgtcatggc tgagctttgg 720
ggtagtgtgg ttatttcagt tctcttctgg ggctttgcta atcagatcac aactgtggat 780
gaagccaaga aattctaccc cttgtttgga ctcggagcca atgttgcgct gattttctct 840
ggaagaacgg tgaagtattt ttctaacttg agaaaaaatc ttggtcctgg agttgacggc 900
tgggcagttt cattgaaagc tatgatgagc attgttgtgg gaatgggact tgccatttgt 960
tttctatatt ggtgggttaa tagatacgtt cctcttccaa ctcgtagcct gaagaagaag 1020
aacaaaccta agatgggaac gatggagagc ttgaagttct tggtgtcatc accatacatt 1080
agagatcttg ctactttagt tgtggcgtat ggtattagta tcaaccttgt ggaagtcaca 1140
tggaaatcaa agcttaaagc tcagtttcct agcccaaacg actactcagc cttcatgggt 1200
gacttctcaa catgcactgg tattgcaaca ttcacaatga tgcttctcag tcaatacgta 1260
tttgataagt atggctgggg agtagctgcg aagatcactc caactgttct gttattgact 1320
ggtgtcgcct tcttctcttt gatattgttt ggtggcccat ttgcaccact tgttgccaag 1380
cttggtatga ctccgctact ggcagctgtt tatgttggtg ctctccagaa tatcttcagc 1440
aagagtgcca agtacagctt gttcgatcct tgtaaagaaa tggcttacat ccccttggat 1500
gaggatacca aggtcaaagg gaaagctgca attgacgtgg tttgcaaccc gttggggaaa 1560
tcagggggtg ctctaatcca acagttcatg atcttatcat ttggatcact agccaattca 1620
acaccatacc taggaatgat actgttggtc attgtcactg catggttagc tgcggctaag 1680
tcactggagg gacagttcaa tgcgttgagg tctgaagaag agctagagaa ggaaatggag 1740
agagcttcgt ctgtgaagat acctgttgtg tctcaagagg aaggtggaaa cggttctctt 1800
ggagaatcaa ctagcagttg gcctgagaaa tcagctccca ccaacatata a 1851
<210> 4
<211> 1845
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 4
atggcaaccg cgattcaaac cagaggactt ctctccttac ccgccaaacc catcggagtg 60
aggagccttc tccagccctc ccacggctta aagcaccgcc ttttcgcctc caagccgaga 120
aatccacctg ccttgtctct atcctctaag aaatttcaat cctttgagcc aacccttccc 180
gggatttcca tttcccacaa gctgaggagc accgagttca tatgcaaggc ggagggagca 240
gcgtcctccg gcgacggaga cacggcggcg attgcagcgt cgccgaagat cttcggcgtg 300
gaggtcacga ccttgaaaaa gattatacct ttagggttga tgttcttttg cattctgttc 360
aattacacaa tcctgaggga cacgaaggac gtcttggtgg tgactgcgaa agggagctca 420
gctgagatta tacctttcct gaagacgtgg gtcaatctcc ctatggccat tggcttcatg 480
ctcctctaca ccaagctctc caatgttctc tccaaaaagg ctctctttta caccgttatc 540
atccctttca tcctctactt tggggccttc ggtttcgtca tgtaccctct cagcaactat 600
attcaccccg aagctcttgc tgataagctc cttgccactc tcggcccaag gttcatgggt 660
cctcttgcta tcctgaggat ttggagtttc tgtttgtttt atgtcatggc tgagctttgg 720
ggtagtgtgg tcatttctgt tctcttctgg ggctttgcta atcagatcac aactgtggat 780
gaagccaaga aattctatcc tttattcgga cttggagcca atgttgcgct cattttctct 840
ggaagaacgg tgaaatactt ctctaacttg agaaagaatc ttggtcctgg agttgacgga 900
tgggcagttt cactgaaagc catgatgagc attgtggttg ggatgggact tgccatctgt 960
ttcctctatt ggtgggtcaa tacatacgtt ccgcttccag cacgtagcca gaagaagaag 1020
aacaaaccga agatgggaac gatggagagc ttgaagttct tggtgtcatc accatacatt 1080
agagatcttg ctactttagt ggtggcgtat ggtattagta tcaacctcgt ggaagtcact 1140
tggaaatcaa agctcaaagc tcagttccct agcccgaacg agtactcagc atttatgggg 1200
gacttctcaa cctgcactgg tattgcaaca ttcacgatga tgcttctcag ccaatacgta 1260
tttgataagt atggttgggg agtagcagca aagatcaccc caactgttct gctattgact 1320
ggcgttgcct tcttctctct gatactgttt ggcggcccat tcgcaccact tgttgccaag 1380
cttggtatga ctccgttact cgcagctgtc tatgttggtg cccttcagaa tatattcagc 1440
aagagtgcca agtacagctt gttcgatcct tgtaaagaaa tggcctatat ccctttggat 1500
gaggacacca aggtcaaagg gaaagctgcg attgacgtgg tatgcaaccc gttggggaaa 1560
tcagggggtg ctctgataca gcagttcatg atcttatcat ttggatcact agccaattca 1620
acgccctacc taggaatgat cttgttggtc attgtcactg cgtggttagc tgcagctaag 1680
tcgctggagg gacagttcaa cgcgttgagg tctgaggaag agctggagaa ggaaatggag 1740
agagcttcgt cggtgaagat ccctgttgtg tctcaagatg aagaaggttc cctgggagaa 1800
tcttcaagca gttcacctga gaaatcagct cccaccaaca tttag 1845
<210> 5
<211> 616
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 5
Met Glu Ala Ala Ile Gln Thr Arg Gly Ile Leu Ser Leu Pro Ala Lys
1 5 10 15
Pro Ile Gly Ala Arg Arg Ser Leu Leu His Pro Ser His Gly Leu Lys
20 25 30
His Arg Leu Phe Ser Ser Lys Pro Arg Thr Pro Pro Ala Leu Ser Leu
35 40 45
Ser Phe Lys Lys Ala Gln Ser Phe Asp Pro Lys Ile Ser Ile Ser His
50 55 60
Lys Glu Arg Thr Thr Glu Phe Ile Cys Lys Ala Glu Ala Asp Gly Gly
65 70 75 80
Ala Leu Leu Asn Glu Gly Asp Thr Ala Ala Ile Val Pro Ser Pro Lys
85 90 95
Ile Phe Gly Val Glu Val Thr Thr Leu Lys Lys Ile Ile Pro Leu Gly
100 105 110
Leu Met Phe Phe Cys Ile Leu Phe Asn Tyr Thr Ile Leu Arg Asp Thr
115 120 125
Lys Asp Val Leu Val Val Thr Ala Lys Gly Ser Ser Ala Glu Ile Ile
130 135 140
Pro Phe Leu Lys Thr Trp Val Asn Leu Pro Met Ala Ile Gly Phe Met
145 150 155 160
Leu Leu Tyr Thr Lys Leu Ser Asn Val Leu Ser Lys Lys Ala Leu Phe
165 170 175
Tyr Thr Val Ile Ile Pro Phe Ile Ile Tyr Phe Gly Ala Phe Gly Phe
180 185 190
Val Met Tyr Pro Leu Ser Asn Tyr Ile His Pro Asp Ala Leu Ala Asp
195 200 205
Lys Leu Leu Ala Ala Leu Gly Pro Arg Phe Met Gly Pro Leu Ala Ile
210 215 220
Leu Arg Ile Trp Ser Phe Cys Leu Phe Tyr Val Met Ala Glu Leu Trp
225 230 235 240
Gly Ser Val Val Ile Ser Val Leu Phe Trp Gly Phe Ala Asn Gln Ile
245 250 255
Thr Thr Val Asp Glu Ala Lys Lys Phe Tyr Pro Leu Phe Gly Leu Gly
260 265 270
Ala Asn Val Ala Leu Ile Phe Ser Gly Arg Thr Val Lys Tyr Phe Ser
275 280 285
Asn Leu Arg Lys Asn Leu Gly Pro Gly Val Asp Gly Trp Ala Val Ser
290 295 300
Leu Lys Ala Met Met Ser Ile Val Val Gly Met Gly Leu Ala Ile Cys
305 310 315 320
Phe Leu Tyr Trp Trp Val Asn Arg Tyr Val Pro Leu Pro Thr Arg Ser
325 330 335
Leu Lys Lys Lys Asn Lys Pro Lys Met Gly Thr Met Glu Ser Leu Lys
340 345 350
Phe Leu Val Ser Ser Pro Tyr Ile Arg Asp Leu Ala Thr Leu Val Val
355 360 365
Ala Tyr Gly Ile Ser Ile Asn Leu Val Glu Val Thr Trp Lys Ser Lys
370 375 380
Leu Lys Ala Gln Phe Pro Ser Pro Asn Glu Tyr Ser Ala Phe Met Gly
385 390 395 400
Asp Phe Ser Thr Cys Thr Gly Ile Ala Thr Phe Thr Met Met Leu Leu
405 410 415
Ser Gln Tyr Val Phe Asp Lys Tyr Gly Trp Gly Val Ala Ala Lys Ile
420 425 430
Thr Pro Thr Val Leu Leu Leu Thr Gly Val Ala Phe Phe Ser Leu Ile
435 440 445
Leu Phe Gly Gly Pro Phe Ala Pro Leu Val Ala Lys Leu Gly Met Thr
450 455 460
Pro Leu Leu Ala Ala Val Tyr Val Gly Ala Leu Gln Asn Ile Phe Ser
465 470 475 480
Lys Ser Ala Lys Tyr Ser Leu Phe Asp Pro Cys Lys Glu Met Ala Tyr
485 490 495
Ile Pro Leu Asp Glu Asp Thr Lys Val Lys Gly Lys Ala Ala Ile Asp
500 505 510
Val Val Cys Asn Pro Leu Gly Lys Ser Gly Gly Ala Leu Ile Gln Gln
515 520 525
Phe Met Ile Leu Ser Phe Gly Ser Leu Ala Asn Ser Thr Pro Tyr Leu
530 535 540
Gly Met Ile Leu Leu Val Ile Val Thr Ala Trp Leu Ala Ala Ala Lys
545 550 555 560
Ser Leu Glu Gly Gln Phe Asn Ala Leu Arg Ser Glu Glu Glu Leu Glu
565 570 575
Lys Glu Met Glu Arg Ala Ser Ser Val Lys Ile Pro Val Val Ser Gln
580 585 590
Glu Glu Gly Gly Asn Gly Ser Leu Gly Glu Ser Thr Ser Ser Trp Pro
595 600 605
Glu Lys Ser Ala Pro Thr Asn Ile
610 615
<210> 6
<211> 615
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 6
Met Ala Thr Val Ile Gln Thr Arg Gly Leu Leu Ser Leu Pro Ala Lys
1 5 10 15
Pro Ile Gly Val Arg Ser Leu Leu Gln Pro Ser His Gly Leu Lys His
20 25 30
Arg Leu Phe Ala Ser Lys Pro Arg Asn Pro Pro Ala Leu Ser Leu Ser
35 40 45
Ser Asn Gly His Gln Lys Phe Gln Ser Phe Glu Pro Thr Leu Pro Gly
50 55 60
Ile Ser Ile Ser His Lys Ala Arg Ser Thr Glu Phe Ile Cys Lys Ala
65 70 75 80
Glu Ala Ala Ser Ala Gly Glu Gly Asp Thr Ala Ala Ile Ser Ala Ser
85 90 95
Pro Lys Ile Phe Gly Val Glu Val Thr Thr Leu Lys Lys Ile Ile Pro
100 105 110
Leu Gly Leu Met Phe Phe Cys Ile Leu Phe Asn Tyr Thr Ile Leu Arg
115 120 125
Asp Thr Lys Asp Val Leu Val Val Thr Ala Lys Gly Ser Ser Ala Glu
130 135 140
Ile Ile Pro Phe Leu Lys Thr Trp Val Asn Leu Pro Met Ala Ile Gly
145 150 155 160
Phe Met Leu Leu Tyr Thr Lys Leu Ser Asn Val Leu Ser Lys Arg Ala
165 170 175
Leu Phe Tyr Thr Val Ile Ile Pro Phe Ile Leu Tyr Phe Gly Ala Phe
180 185 190
Gly Phe Val Met Tyr Pro Leu Ser Asn Tyr Ile His Pro Glu Ala Leu
195 200 205
Ala Asp Lys Leu Leu Ala Thr Leu Gly Pro Arg Phe Met Gly Pro Leu
210 215 220
Ala Ile Leu Arg Ile Trp Ser Phe Cys Leu Phe Tyr Val Met Ala Glu
225 230 235 240
Leu Trp Gly Ser Val Val Ile Ser Val Leu Phe Trp Gly Phe Ala Asn
245 250 255
Gln Ile Thr Thr Val Asp Glu Ala Lys Lys Phe Tyr Pro Leu Phe Gly
260 265 270
Leu Gly Ala Asn Val Ala Leu Ile Phe Ser Gly Arg Thr Val Lys Tyr
275 280 285
Phe Ser Asn Leu Arg Lys Asn Leu Gly Pro Gly Val Asp Gly Trp Ala
290 295 300
Val Ser Leu Lys Ala Met Met Ser Ile Val Val Gly Met Gly Leu Ala
305 310 315 320
Ile Cys Phe Leu Tyr Trp Trp Val Asn Thr Tyr Val Pro Leu Pro Ala
325 330 335
Arg Ser Gln Lys Lys Lys Asn Lys Pro Lys Met Gly Thr Ile Glu Ser
340 345 350
Leu Lys Phe Leu Val Ser Ser Pro Tyr Ile Arg Asp Leu Ala Thr Leu
355 360 365
Val Val Ala Tyr Gly Ile Ser Ile Asn Leu Val Glu Val Thr Trp Lys
370 375 380
Ser Lys Leu Lys Ala Gln Phe Pro Ser Pro Asn Glu Tyr Ser Ala Phe
385 390 395 400
Met Gly Asp Phe Ser Thr Cys Thr Gly Ile Ala Thr Phe Thr Met Met
405 410 415
Leu Leu Ser Gln Tyr Val Phe Asp Lys Tyr Gly Trp Gly Val Ala Ala
420 425 430
Lys Ile Thr Pro Thr Val Leu Leu Leu Thr Gly Val Ala Phe Phe Ser
435 440 445
Leu Ile Leu Phe Gly Gly Pro Phe Ala Pro Leu Val Ala Lys Leu Gly
450 455 460
Met Thr Pro Leu Leu Ala Ala Val Tyr Val Gly Ala Leu Gln Asn Ile
465 470 475 480
Phe Ser Lys Ser Ala Lys Tyr Ser Leu Phe Asp Pro Cys Lys Glu Met
485 490 495
Ala Tyr Ile Pro Leu Asp Glu Asp Thr Lys Val Lys Gly Lys Ala Ala
500 505 510
Ile Asp Val Val Cys Asn Pro Leu Gly Lys Ser Gly Gly Ala Leu Ile
515 520 525
Gln Gln Phe Met Ile Leu Ser Phe Gly Ser Leu Ala Asn Ser Thr Pro
530 535 540
Tyr Leu Gly Met Ile Leu Leu Val Ile Val Thr Ala Trp Leu Ala Ala
545 550 555 560
Ala Lys Ser Leu Glu Gly Gln Phe Asn Ala Leu Arg Ser Glu Glu Glu
565 570 575
Leu Glu Lys Glu Met Glu Arg Ala Ser Ser Val Lys Ile Pro Val Val
580 585 590
Ser Gln Asp Glu Gly Ser Leu Gly Glu Ser Ser Ser Ser Ser Pro Glu
595 600 605
Lys Ser Ala Pro Thr Asn Ile
610 615
<210> 7
<211> 616
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 7
Met Glu Ala Ala Ile Gln Thr Arg Gly Ile Leu Ser Leu Pro Ala Lys
1 5 10 15
Pro Ile Gly Ala Arg Arg Ser Leu Leu His Pro Ser His Gly Leu Lys
20 25 30
His Arg Leu Phe Ser Ser Lys Pro Arg Thr Pro Pro Ala Leu Ser Leu
35 40 45
Ser Phe Lys Lys Ala Gln Ser Phe Glu Pro Thr Ile Ser Ile Ser His
50 55 60
Lys Glu Arg Ser His Glu Phe Ile Cys Lys Ala Glu Ala Asp Gly Gly
65 70 75 80
Ala Leu Leu Asn Glu Gly Asp Thr Ala Ala Val Val Pro Ser Pro Lys
85 90 95
Ile Phe Gly Val Glu Val Thr Thr Leu Lys Lys Ile Ile Pro Leu Gly
100 105 110
Leu Met Phe Phe Cys Ile Leu Phe Asn Tyr Thr Ile Leu Arg Asp Thr
115 120 125
Lys Asp Val Leu Val Val Thr Ala Lys Gly Ser Ser Ala Glu Ile Ile
130 135 140
Pro Phe Leu Lys Thr Trp Val Asn Leu Pro Met Ala Ile Gly Phe Met
145 150 155 160
Leu Leu Tyr Thr Lys Leu Ser Asn Val Leu Ser Lys Lys Ala Leu Phe
165 170 175
Tyr Thr Val Ile Ile Pro Phe Ile Ile Tyr Phe Gly Ala Phe Gly Phe
180 185 190
Val Met Tyr Pro Leu Ser Asn Tyr Ile His Pro Glu Ala Leu Ala Asp
195 200 205
Lys Leu Leu Ala Asp Leu Gly Pro Arg Phe Met Gly Pro Leu Ala Ile
210 215 220
Leu Arg Ile Trp Ser Phe Cys Leu Phe Tyr Val Met Ala Glu Leu Trp
225 230 235 240
Gly Ser Val Val Ile Ser Val Leu Phe Trp Gly Phe Ala Asn Gln Ile
245 250 255
Thr Thr Val Asp Glu Ala Lys Lys Phe Tyr Pro Leu Phe Gly Leu Gly
260 265 270
Ala Asn Val Ala Leu Ile Phe Ser Gly Arg Thr Val Lys Tyr Phe Ser
275 280 285
Asn Leu Arg Lys Asn Leu Gly Pro Gly Val Asp Gly Trp Ala Val Ser
290 295 300
Leu Lys Ala Met Met Ser Ile Val Val Gly Met Gly Leu Ala Ile Cys
305 310 315 320
Phe Leu Tyr Trp Trp Val Asn Arg Tyr Val Pro Leu Pro Thr Arg Ser
325 330 335
Leu Lys Lys Lys Asn Lys Pro Lys Met Gly Thr Met Glu Ser Leu Lys
340 345 350
Phe Leu Val Ser Ser Pro Tyr Ile Arg Asp Leu Ala Thr Leu Val Val
355 360 365
Ala Tyr Gly Ile Ser Ile Asn Leu Val Glu Val Thr Trp Lys Ser Lys
370 375 380
Leu Lys Ala Gln Phe Pro Ser Pro Asn Glu Tyr Ser Ala Phe Met Gly
385 390 395 400
Asp Phe Ser Thr Cys Thr Gly Ile Ala Thr Phe Thr Met Met Leu Leu
405 410 415
Ser Gln Tyr Val Phe Asp Lys Tyr Gly Trp Gly Val Ala Ala Lys Ile
420 425 430
Thr Pro Thr Val Leu Leu Leu Thr Gly Val Ala Phe Phe Ser Leu Ile
435 440 445
Leu Phe Gly Gly Pro Phe Ala Pro Leu Val Ala Lys Leu Gly Met Thr
450 455 460
Pro Leu Leu Ala Ala Val Tyr Val Gly Ala Leu Gln Asn Ile Phe Ser
465 470 475 480
Lys Ser Ala Lys Tyr Ser Leu Phe Asp Pro Cys Lys Glu Met Ala Tyr
485 490 495
Ile Pro Leu Asp Glu Asp Thr Lys Val Lys Gly Lys Ala Ala Ile Asp
500 505 510
Val Val Cys Asn Pro Leu Gly Lys Ser Gly Gly Ala Leu Ile Gln Gln
515 520 525
Phe Met Ile Leu Ser Phe Gly Ser Leu Ala Asn Ser Thr Pro Tyr Leu
530 535 540
Gly Met Ile Leu Leu Val Ile Val Thr Ala Trp Leu Ala Ala Ala Lys
545 550 555 560
Ser Leu Glu Gly Gln Phe Asn Ala Leu Arg Ser Glu Glu Glu Leu Glu
565 570 575
Lys Glu Met Glu Arg Ala Ser Ser Val Lys Ile Pro Val Val Ser Gln
580 585 590
Glu Glu Gly Gly Asn Gly Ser Leu Gly Glu Ser Thr Ser Ser Trp Pro
595 600 605
Glu Lys Ser Ala Pro Thr Asn Ile
610 615
<210> 8
<211> 614
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 8
Met Ala Thr Ala Ile Gln Thr Arg Gly Leu Leu Ser Leu Pro Ala Lys
1 5 10 15
Pro Ile Gly Val Arg Ser Leu Leu Gln Pro Ser His Gly Leu Lys His
20 25 30
Arg Leu Phe Ala Ser Lys Pro Arg Asn Pro Pro Ala Leu Ser Leu Ser
35 40 45
Ser Lys Lys Phe Gln Ser Phe Glu Pro Thr Leu Pro Gly Ile Ser Ile
50 55 60
Ser His Lys Leu Arg Ser Thr Glu Phe Ile Cys Lys Ala Glu Gly Ala
65 70 75 80
Ala Ser Ser Gly Asp Gly Asp Thr Ala Ala Ile Ala Ala Ser Pro Lys
85 90 95
Ile Phe Gly Val Glu Val Thr Thr Leu Lys Lys Ile Ile Pro Leu Gly
100 105 110
Leu Met Phe Phe Cys Ile Leu Phe Asn Tyr Thr Ile Leu Arg Asp Thr
115 120 125
Lys Asp Val Leu Val Val Thr Ala Lys Gly Ser Ser Ala Glu Ile Ile
130 135 140
Pro Phe Leu Lys Thr Trp Val Asn Leu Pro Met Ala Ile Gly Phe Met
145 150 155 160
Leu Leu Tyr Thr Lys Leu Ser Asn Val Leu Ser Lys Lys Ala Leu Phe
165 170 175
Tyr Thr Val Ile Ile Pro Phe Ile Leu Tyr Phe Gly Ala Phe Gly Phe
180 185 190
Val Met Tyr Pro Leu Ser Asn Tyr Ile His Pro Glu Ala Leu Ala Asp
195 200 205
Lys Leu Leu Ala Thr Leu Gly Pro Arg Phe Met Gly Pro Leu Ala Ile
210 215 220
Leu Arg Ile Trp Ser Phe Cys Leu Phe Tyr Val Met Ala Glu Leu Trp
225 230 235 240
Gly Ser Val Val Ile Ser Val Leu Phe Trp Gly Phe Ala Asn Gln Ile
245 250 255
Thr Thr Val Asp Glu Ala Lys Lys Phe Tyr Pro Leu Phe Gly Leu Gly
260 265 270
Ala Asn Val Ala Leu Ile Phe Ser Gly Arg Thr Val Lys Tyr Phe Ser
275 280 285
Asn Leu Arg Lys Asn Leu Gly Pro Gly Val Asp Gly Trp Ala Val Ser
290 295 300
Leu Lys Ala Met Met Ser Ile Val Val Gly Met Gly Leu Ala Ile Cys
305 310 315 320
Phe Leu Tyr Trp Trp Val Asn Thr Tyr Val Pro Leu Pro Ala Arg Ser
325 330 335
Gln Lys Lys Lys Asn Lys Pro Lys Met Gly Thr Met Glu Ser Leu Lys
340 345 350
Phe Leu Val Ser Ser Pro Tyr Ile Arg Asp Leu Ala Thr Leu Val Val
355 360 365
Ala Tyr Gly Ile Ser Ile Asn Leu Val Glu Val Thr Trp Lys Ser Lys
370 375 380
Leu Lys Ala Gln Phe Pro Ser Pro Asn Glu Tyr Ser Ala Phe Met Gly
385 390 395 400
Asp Phe Ser Thr Cys Thr Gly Ile Ala Thr Phe Thr Met Met Leu Leu
405 410 415
Ser Gln Tyr Val Phe Asp Lys Tyr Gly Trp Gly Val Ala Ala Lys Ile
420 425 430
Thr Pro Thr Val Leu Leu Leu Thr Gly Val Ala Phe Phe Ser Leu Ile
435 440 445
Leu Phe Gly Gly Pro Phe Ala Pro Leu Val Ala Lys Leu Gly Met Thr
450 455 460
Pro Leu Leu Ala Ala Val Tyr Val Gly Ala Leu Gln Asn Ile Phe Ser
465 470 475 480
Lys Ser Ala Lys Tyr Ser Leu Phe Asp Pro Cys Lys Glu Met Ala Tyr
485 490 495
Ile Pro Leu Asp Glu Asp Thr Lys Val Lys Gly Lys Ala Ala Ile Asp
500 505 510
Val Val Cys Asn Pro Leu Gly Lys Ser Gly Gly Ala Leu Ile Gln Gln
515 520 525
Phe Met Ile Leu Ser Phe Gly Ser Leu Ala Asn Ser Thr Pro Tyr Leu
530 535 540
Gly Met Ile Leu Leu Val Ile Val Thr Ala Trp Leu Ala Ala Ala Lys
545 550 555 560
Ser Leu Glu Gly Gln Phe Asn Ala Leu Arg Ser Glu Glu Glu Leu Glu
565 570 575
Lys Glu Met Glu Arg Ala Ser Ser Val Lys Ile Pro Val Val Ser Gln
580 585 590
Asp Glu Glu Gly Ser Leu Gly Glu Ser Ser Ser Ser Ser Pro Glu Lys
595 600 605
Ser Ala Pro Thr Asn Ile
610
Claims (8)
1.油菜三磷酸核苷酸转运蛋白基因BnNTT1在调控作物含油量和脂肪酸组成中的应用,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4所示。
2.油菜三磷酸核苷酸转运蛋白基因BnNTT1所编码的蛋白在调控作物含油量和脂肪酸组成中的应用,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4所示,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1所编码的蛋白的氨基酸序列如SEQ ID NO:5或SEQ ID NO:6或SEQ ID NO:7或SEQ IDNO:8所示。
3.含有油菜三磷酸核苷酸转运蛋白基因BnNTT1的表达载体在调控作物含油量和脂肪酸组成中的应用,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4所示。
4.含有油菜三磷酸核苷酸转运蛋白基因BnNTT1的重组菌在调控作物含油量和脂肪酸组成中的应用,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ ID NO:3或SEQ ID NO:4所示。
5.如权利要求4所述的应用,其特征在于:所述重组菌为重组农杆菌。
6.一种提高作物含油量的方法,其特征在于:使用农杆菌介导的遗传转化方法将含有油菜三磷酸核苷酸转运蛋白基因BnNTT1且由组成型启动子启动的过表达载体转化到作物的基因组中,获得油菜三磷酸核苷酸转运蛋白基因BnNTT1超表达的作物品种,所述油菜三磷酸核苷酸转运蛋白基因BnNTT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2或SEQ IDNO:3或SEQ ID NO:4所示。
7.如权利要求6所述提高作物含油量的方法,其特征在于:所述组成型启动子为CaMV35S。
8.如权利要求6所述提高作物含油量的方法,其特征在于:所述作物为油菜。
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CN114438121A (zh) * | 2022-01-12 | 2022-05-06 | 华中农业大学 | 油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用 |
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CN115927237B (zh) * | 2022-10-21 | 2024-03-29 | 华中农业大学 | 一种油菜海藻糖-6-磷酸合成酶基因在调控含油量与脂肪酸组成中的用途 |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114438121A (zh) * | 2022-01-12 | 2022-05-06 | 华中农业大学 | 油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用 |
CN114438121B (zh) * | 2022-01-12 | 2023-12-22 | 华中农业大学 | 油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用 |
CN114990120A (zh) * | 2022-06-27 | 2022-09-02 | 华中农业大学 | 一种油菜长链非编码rna基因及其在提高油菜含油量及种子粒重中的应用 |
CN114990120B (zh) * | 2022-06-27 | 2024-03-12 | 华中农业大学 | 一种油菜长链非编码rna基因及其在提高油菜含油量及种子粒重中的应用 |
CN115927237B (zh) * | 2022-10-21 | 2024-03-29 | 华中农业大学 | 一种油菜海藻糖-6-磷酸合成酶基因在调控含油量与脂肪酸组成中的用途 |
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