CN114438121B - 油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用 - Google Patents
油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用 Download PDFInfo
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Abstract
本发明公开了油菜磷酸烯醇式丙酮酸转运基因BnaPPT1在调控作物含油量中的应用,所述油菜磷酸烯醇式丙酮酸转运基因BnaPPT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示。本发明使用农杆菌介导的遗传转化方法将含有油菜磷酸烯醇式丙酮酸转运基因BnaPPT1的过表达载体转化到作物的基因组中,获得油菜磷酸烯醇式丙酮酸转运基因BnaPPT1超表达的作物品种,测定了过表达和突变体转化材料的含油量、脂质组,分析了转化材料种子的细胞学切片,发现BnaPPT1正调控油菜种子含油量的积累。
Description
技术领域
本发明属于基因工程和作物育种技术领域,涉及一种油菜磷酸烯醇式丙酮酸转运基因BnaPPT1及其编码蛋白的新用途,尤其是在调控作物含油量中的新用途。
背景技术
油菜是我国分布最广、播种面积最大的油料作物,植物油通常以三酰基甘油的形式储存在种子中。植物油用途较广,不仅是食用油的主要来源,而且可用于生产油漆、肥皂、润滑油以及生物柴油。到2030年,全世界对植物油的需求将翻倍。我国是食用油消费大国,2020年我国进口食用油籽1.0614亿吨,同比增加1283万吨,是国内油籽油料总产量6564万吨的1.6倍,对外依赖高。油菜的供油量占国产植物油的47%,因此,提高油菜的含油量有利于保障我国食用油的供给安全。
种子中油的积累是一个复杂的生物学过程,与多种生物学途径密切相关,受如光合作用、种子发育、物质转运、脂质合成、积累和降解。目前,在模式植物拟南芥中已报道超过700个与油脂积累相关的基因或者酶,而油菜的油脂代谢相关基因研究还较少。油菜的含油性状一直是油菜的育种目标之一,创建新型的高油材料质,扩宽了油菜的高油育种的种质资源。
本发明克隆了一个油菜磷酸烯醇式丙酮酸转运基因BnaPPT1,用35S启动子启动BnaPPT1基因的高表达,获得的油菜种子含油量显著升高。同时,利用CRISPR Cas9技术创建BnaPPT1基因的突变体,获得的油菜突变体的种子含油量降低。该结果预示着BnaPPT1基因在调控油菜种子含油量中起着重要作用,它在创建油菜含油量新种质中具有很大的应用前景。
发明内容
本发明的目的在于提供油菜磷酸烯醇式丙酮酸转运基因BnaPPT1、该基因所编码的蛋白、含有该基因的表达载体和重组菌在调控作物含油量中的应用。
本发明中所涉及的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1,该基因编码磷酸烯醇式丙酮酸转运蛋白,在油菜A8和C8染色体上各有一个拷贝,分别是BnaA08.PPT1(BnaA08g07320D)和BnaC08.PPT1(BnaC08g08150D),该基因的核苷酸序列分别如序列表SEQID NO:1和SEQ ID NO:2所示,分别为1218bp和1209bp;该基因编码的蛋白质序列分别如序列表SEQ ID NO:3和SEQ ID NO:4所示,分别编码405和402个氨基酸。可以采用PCR技术从基因组、mRNA和cDNA中扩增得到本发明的目的基因。
申请人通过使用农杆菌介导的遗传转化方法将含有所述的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1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,获得油菜磷酸烯醇式丙酮酸转运基因BnaPPT1超表达的作物品种。同时利用CRISPR/Cas9基因编辑技术获得基因BnaPPT1功能缺失的油菜品种。
接着,测定了过表达和突变体转化材料的含油量、脂质组,分析了转化材料种子的细胞学切片,发现BnaPPT1正调控油菜种子含油量的积累。
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:35S-FAST、PKSE401(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油菜,在其营养生长与生殖生长时期与正常植株没有明显的差异,该遗传资源在油菜高油育种中具有十分重要的应用前景。
附图说明
序列表SEQ ID NO:1和SEQ ID NO:2是分离自甘蓝型油菜Westar的基因BnaA08.PPT1(BnaA08g07320D)和BnaC08.PPT1(BnaC08g08150D)的核苷酸序列,其序列长度分别1218bp、1209bp。
序列表SEQ ID NO:3和SEQ ID NO:4是分离自甘蓝型油菜基因Westar的基因BnaA08.PPT1(BnaA08g07320D)和BnaC08.PPT1(BnaC08g08150D)编码的蛋白质序列,分别编码405、402个氨基酸。
图1:BnaC08.PPT1克隆。
图2:构建的BnaC08.PPT1过表达载体的示意图。
图3:转化单株的qRT-PCR检测。*表示在Student’s t test中P<0.05,**表示在Student’s t test中P<0.01。
图4:油菜BnaPPT1突变体的获得和鉴定。sgRNA1和sgRNA2分别位于基因BnaPPT1的第一和第二个外显子区,获得的4个突变体的靶位点的编辑情况:CR28和CR54为BnaA08.PPT1和BnaC08.PPT1均被编辑的双突,CR5为BnaA08.PPT1被编辑的单突,CR105为BnaC08.PPT1被编辑的单突。
图5:BnaPPT1在油菜的表型鉴定。(a)利用近红外分析油菜种子含油量。CR28和CR54是BnaA08.PPT1和BnaC08.PPT1的CRISPR双突变体,CR5和CR105分别是BnaA08.PPT1和BnaC08.PPT1被编辑的单突,OE1和OE11是超表达株系。每个株系有6-8个不同单株,**表示在Student’s t test中P<0.05。(b)种子脂质TAG和DAG测定。每个株系有6个不同单株,**表示在Student’s t test中P<0.05。
图6:种子透射电镜观察。
具体实施方式
以下结合具体实施例,进一步定义本发明。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。下列实施例中未注明具体条件的实验方法,通常按照常规条件如工具书《分子克隆:实验室指南》(New York:Cold Spring Harbor Laboratory,1989)中所述的条件,或者按照生产商提供的操作手册中建议的方法。
实施例1 BnaC08.PPT1基因的克隆
PPT1编码一个磷酸烯醇式丙酮酸转运蛋白,在油菜A8和C8染色体上各有一个拷贝,分别是BnaA08.PPT1(BnaA08g07320D)和BnaC08.PPT1(BnaC08g08150D),CDS序列分别为序列表SEQ ID NO:1和SEQ ID NO:2所示的序列,包括完整的ORF阅读框及起始密码子ATG,分别编码405和402个氨基酸(分别见序列表SEQ ID NO:3和SEQ ID NO:4所示),通过BLAST比对发现BnaA08.PPT1和BnaC08.PPT1同源性很高,在95%以上,随机选取一个拷贝BnaC08.PPT1进行基因克隆。
(1)RNA的提取
总RNA的提取采用全式金公司的TansZol(目录号ET101),取甘蓝型油菜Westar叶片于液氮中研磨粉碎,取100mg研磨好的样品转移到1.5mL离心管中,加入1mL的TransZol,上下剧烈颠倒数次使之充分混匀,室温静置5分钟;加0.2mL氯仿,剧烈振荡15秒,室温孵育3分钟;10000xg 4℃离心15分钟,此时样品分为三层,无色的水相(上层),中间层,粉红色有机相(下层);转移无色的水相于新的离心管中,加入0.5mL异丙醇,颠倒混匀,室温孵育10分钟;10000xg 4℃离心10分钟,去上清,在管侧和管底形成胶状沉淀;加1mL75%乙醇(DEPC处理的水配制),剧烈涡旋;7500xg 4℃离心5分钟;弃上清,室温晾干沉淀;沉淀溶于50-100μlRNA溶解液中;55℃孵育10分钟。取lμl抽提的总RNA在Nanodrop下测定RNA浓度,依据1.8<OD260/OD280<2.0鉴定RNA纯度。同时取lμl进行1%agrose电泳,检测完整性。
(2)cDNA的合成
反转录使用的是全式金One-Step gDNA Removal and cDNASynthesis SuperMix(目录号AE311)。以2μg总RNA为模板,依次加入Anchored Oligo(dT)18Primer 1μl,2×ES Reaction Mix 10μl,/>RT/RI Enzyme Mix 1μl,gDNARemover 1μl,RNase-free Water补充至20μl。将以上体系轻轻混匀后置于42℃ 30min,此步是合成第一链cDNA和去除gDNA。85℃加热5秒钟失活/>RT/RI和gDNA Remover。加入180μl RNase-free Water溶解合成的cDNA,待用。
(3)BnaC08.PPT1基因的扩增
根据上述分析所得的CDS序列设计引物,以甘蓝型油菜Westar的cDNA为模板,正向引物BnPPT-XmaⅠ-F序列为5’-GCGcccgggACCATGGCCCAGAGCTCCGCCGTCTT-3’,反向引物BnPPT-BamHⅠ-R序列为5’-GCGggatccCGGCAGTCTTAGGCTTTG-3’,扩增得到含有BnaC08.PPT1全长CDS的片段。采用I-5TM2×High-Fidelity Master Mix(TSINGKE Biologicatechnology)进行PCR扩增。PCR扩增体系如下:
PCR扩增程序:98℃总变性1min;98℃变性15sec,55℃退火15sec,72℃延伸30sec,34circles;72℃总延伸5min。
扩增后产物通过琼脂糖凝胶电泳检测(图1),扩增获得1209bp的BnaC08.PPT1全长,产物挖胶回收使用天根琼脂糖凝胶回收试剂盒(http://www.tiangen.com/)。
实施例2 BnaC08.PPT1基因过表达转化载体的构建
(1)对上述获得的BnaC08.PPT1片段用快速限制性内切酶XmaI和BamHI进行双酶切,双酶切体系如下:
酶切反应在37℃水浴锅进行,时间1小时。酶切产物用天根的DNA纯化试剂盒回收。
(2)酶切产物连接到载体35S-FAST,该载体含有组成型表达启动子和抗生素标记物。
连接方法如下:
连接反应条件:22℃3小时。
(3)转化大肠杆菌DH5α,转化方法如下:
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,BnaC08.PPT1基因的CDS序列成功连接上载体,即成功构建了转化植株的植物表达载体35S-FAST-BnaC08.PPT1(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。导入方法如下:
a.清洗电转杯:先用纯水洗,再用超纯水洗,倒掉,再用无水乙醇清洗(用1ml枪头吹打),倒掉无水乙醇,置于超净台晾干;
b.取农杆菌感受态GV3101 20μl;
c.取构建正确的重组质粒0.8μl,加入到20μl感受态中,轻轻吸打混匀,避免产生气泡;
d.将洗好吹干的电转杯置于冰中预冷,再将上述混合液靠杯壁打入;
e.将电转仪调至1800V;
f.将电转杯从冰中取出,用吸水纸擦干净电转杯外壁;
g.将电转杯放入仪器,连续按两下“push”键,几秒钟之后听到“滴”的一声则成功;
h.电击成功后,向电转杯中加入400μl无抗LB,吸打几下,转移到无菌离心管中;
i.28℃活化2h左右,取100μl涂到含有双抗,用封口膜封好,倒置于28度培养箱培养2天,挑斑检测。
(5)农杆菌菌落检测
挑选菌落于双抗LB中,28℃培养2小时,取适量菌液进行PCR检测,保存阳性农杆菌菌液。
实施例3 BnaPPT1-CRISPR载体的构建
利用中国农业大学生物学院陈其军团队的sgRNA-Cas9系统创建油菜BnaPPT1突变体。实验操作步骤如下:
(1)登录到网站http://www.genome.arizona.edu/crispr/CRISPRsearch.html,筛选靶点CAGGCGCGAAGCGGCGAGTCGG和CAGGCGCGAAGCGGCGAGTAGG,分别位于基因的第一和第二个外显子区,可以同时靶向BnaA08.PPT1和BnaC08.PPT1.
(2)设计引物
DT1-BsF:ATATATGGTCTCGATTGCAGGCGCGAAGCGGCGAGTGTT
DT1-F0:TGCAGGCGCGAAGCGGCGAGTGTTTTAGAGCTAGAAATAGC
DT2-R0:AACTTCTTCTCCGTTGTCTTGTCAATCTCTTAGTCGACTCTAC
DT2-BsR:ATTATTGGTCTCGAAACTTCTTCTCCGTTGTCTTGTCAA
(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。扩增体系为:
PCR扩增程序:98℃总变性1min;98℃变性15sec,56℃退火25sec,72℃延伸25sec,34circles;72℃总延伸5min。
(4)纯化回收PCR产物,建立如下酶切-连接体系(restriction-ligation):
反应条件:5hours at 37℃,5min at 50℃,10min at 80℃
(5)转化转化大肠杆菌DH5α:取5μL转化大肠杆菌感受态,Kan板筛选。阳性克隆筛选,方法如下:
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BnPMT6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,挑选阳性单克隆于-80℃冰箱保存。转化方法同实施例2中所示:
实施例4遗传转化实验
(1)油菜的遗传转化
对构建好的BnaC08.PPT1过表达载体和CRISPR载体进行油菜的遗传转化,使用农杆菌介导的遗传转化方式,本发明中用于油菜转化的受体为甘蓝型油菜Westar,具体操作流程详见参考文献:An efficient Agrobacterium-mediated transformation methodusing hypocotyl as explants for Brassica napus.
(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35S-FAST,在骨架载体上设计引物FAST Primer-F3(5’-AAAGGCCATCGTTGAAGATG-3’),通过载体骨架引物与外源片段引物搭配来进行PCR(FASTPrimer-F3和BnPPT-BamHⅠ-R,BnPPT-BamHⅠ-R序列如实施例1所示),在PCR水平检测转基因苗。PCR体系为:Easy taq polymerase 0.15μL,10mM dNTP 0.4μL,10×buffer 2μL,DNA模板2μL,引物FAST Primer-F3 2μL,引物BnPPT-BamHⅠ-R 2μL,补ddH2O至20μL。PCR条件:94℃总变性5min;94℃变性30sec,55℃退火30sec,72℃延伸30sec,34circles;72℃总延伸5min。
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。提取转化单株种子的RNA并进行cDNA的合成(方法同实施例1),定量引物利用Primer 3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-PPT1-F(5’-CAATCAGCTGGTGTGAATGTT-3’)和RT-PPT1-R(CTTCACTCTGGAG-TATAAGAA-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(详见Zhou et al 2012:BnMs3 is required fortapetal differentiation and degradation,microspore separation,and pollen-wallbiosynthesis in Brassica napus)。反应体系为:
反应程序:94℃30s;94℃10s,60℃15s,72℃30s,45个循环;绘制溶解曲线。qRT-PCR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。最后分析获得油菜过表达转化单株OE1和OE11(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Cas9-570-F(5’-AGACCGTGAAGGTTGTGGAC-3’)和Cas9-570-R(5’-TAGTGATCTGCCGTGTCTC-G-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PPT1-A8-F(5’-gtctctcctccgaatgtcgt-3’)和PPT1-A8-R(5’-ctttgttagggactggtaac-3’)特异扩增BnaA08.PPT1;PPT1-C8-F(5’-ccgcctcctcgaatctcagcg-3’)和PPT1-C8-R(5’-attgacaattcccggaaaagc-3’)特异扩增BnaC08.PPT1。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,测序结果用DSDecode在线网站(http://skl.scau.edu.cn/dsdecode/)分析靶位点的编辑情况。测序结果显示,获得了BnaPPT1被编辑的多个突变体独立株系(CR5、CR28、CR54和CR105)。其中CR28和CR54为BnaA08.PPT1和BnaC08.PPT1均被编辑的双突,CR5为BnaA08.PPT1被编辑的单突,CR105为BnaC08.PPT1被编辑的单突(图4)。
(4)转化所得植株的含油量相关表型分析
①采用近红外分析仪测定油菜种子含油量
利用近红外分析仪,对成熟期收获的油菜种子进行品质分析,获得种子含油量数据,测定仪器由华中农业大学国家油菜工程技术研究中心提供。
含油量结果显示,受体背景材料Westar的含油量是41.73±1.29,2个超表达株系OE1和OE11的含油量分别是43.80±0.56和45.02±0.51,显著升高了5.0和7.9个百分点。突变体材料CR5、CR28、CR54和CR105的含油量分别是41.22±0.83、32.57±0.50、39.50±1.17和42.48±0.52,不难看出,双突变体CR28和CR54的含油量显著下降22和5.3个百分点,但是单突CR5和CR105的含油量没有显著改变(图5a)。
②采用LC-MS/MS方法测定油菜种子脂质含量
利用6500 plus Qtrap,对成熟期收获的油菜种子进行脂质提取与分析,结果显示,成熟种子中95%以上的脂质为TAG(三酰基甘油,Triacylglyceride),DAG(二酰基甘油,Diacylglycerol)含量较少。与WT相比,OE1中TAG和DAG含量分别增加了24.6%和34.0%,CR28中TAG和DAG含量分别减少了20.5%和44.6%(图5b)。
③透射电镜观察油菜种子油体
取种子子叶切片,在透射电镜下分析油体。结果显示OE中油体总面积和每个油体的面积较大,而突变体中则较小,并且突变体的油体密度明显低于OE和WT。相比之下,在一个细胞中蛋白体等其他组织的比例在突变体中比OE和WT中更大(图6)。
综上所述,基因BnaPPT1在调控油菜含油量中发挥重要的作用。
序列表
<110> 华中农业大学
<120> 油菜BnaPPT1基因及其编码蛋白在调控作物含油量中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1218
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 1
atgcagagct ccgccgtctt ctccctctct ccctccctcc ctctcctgaa accacgccgc 60
ctctcccttc gccatcccgt caccgcctcc tcgaacctca acgtctctcc tccgaatgtc 120
gtctctgttc ctcctctacc tcgtcgatct tggcgtctgg cctcttccga ctcgccgctt 180
cgcgcctggt ccggtctccc ttccgtctcc tccccttccc tcgacactaa ccgcttcaaa 240
actgcagcta cggcagttcc ggagaacgct gaggaaggag aaggaagtgg aaagatgact 300
aaggttctgg agctcggctt gctgtttgcg atgtggtacc ttttcaatat ctacttcaac 360
atctacaaca agcaggtttt gaaagctctt cacgccccaa tgactgtcac tttagttcag 420
tttgccgtcg gtagtgtcct cattaccttc atgtgggctc ttaaccttta caagaggccc 480
aagatctctg ctgctcagct tgcggccatc ttgccacttg cagttgtgca cactcttggt 540
aatctcttta ctaacatgag cctcgggaaa gtctctgttt cctttactca caccattaaa 600
gccatggagc ctttcttctc cgttgtcttg tctgccatgt ttctcggcga ggtgcctact 660
ccatgggtca tcggttccat tattccaatc gttggtggag ttgcacttgc ttcagtgaca 720
gaggtctcat tcaactgggc tggctttttg agtgcaatgg cttcaaactt gactaaccaa 780
tcccgtaacg tgcttagcaa aaaagtcatg gtcaagaaag atgattcttt ggacaacatc 840
accctcttct ctataataac gttaatgtct ctctttctga tggcccctgt gactttcttc 900
tccgaaggaa tcaagttcac tccgtcatac atccaatcag ctggtgtgaa tgttcaacaa 960
atctacacca agtctcttat tgctgcactc tgcttccacg cataccagca ggtgtcgtac 1020
atgatattgg cgagagtatc acccgtaaca cattctgtag gaaactgtgt gaagcgtgtg 1080
gtggtcattg tgagctctgt catcttcttc aagacaccgg tctcgcctgt taatgctttt 1140
ggaaccggaa tcgccctagc gggagttttc ttatactcca gagtgaagcg tatcaagcca 1200
aagcctaaga ctgcttaa 1218
<210> 2
<211> 1209
<212> DNA
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 2
atgcagagct ccgccgtctt ctccgcctct ccctccctcc ctctcctgaa accaggccgc 60
ctctcccttc gccatcccgt caccgcctcc tcgaatctca gcgtctctcc tccgaatgtc 120
gtctctgttc ctcctctacc tcgccgatct tggcgtctcg cctcttccga ctcgccgctt 180
cgcgcctggt ccggtctccc ttccgtatcc tccccttccc tcgacactaa ccgcttcaaa 240
actgcagcta cggcagttcc ggaggaagga gaaggaagtg gaaagatgac taaggttctg 300
gagctcggct tgctgtttgc gatgtggtac cttttcaata tctacttcaa catctacaac 360
aagcaggttt tgaaagctct tcacgcccca atgactgtca ctttagttca gtttgccgtc 420
ggtagtgtcc tcattacctt catgtgggct cttaaccttt acaagaggcc caagatctct 480
gctgctcagc ttgcggccat cttgccactt gcagttgtgc acactcttgg taatctcttt 540
actaacatga gcctcgggaa agtctctgtt tcctttactc acaccattaa agccatggag 600
cctttcttct ccgttgtctt gtctgccatg tttctcggcg aggtgcctac tccatgggtc 660
atcggttcca ttattccaat cgttggtgga gttgcacttg cttcagtgac agaggtctca 720
ttcaactggg ctggcttttt gagtgcaatg gcttcaaact tgactaacca atcccgtaac 780
gtgcttagca aaaaagtcat ggtcaagaaa gatgattctt tggacaacat caccctcttc 840
tctataataa cgttgatgtc tctctttctg atggcccctg tgactttctt ctccgaagga 900
atcaagttca ctccgtcata catccaatca gctggtgtga atgttcaaca aatctacacg 960
aagtctctta ttgctgcact ctgcttccac gcataccagc aggtgtcgta catgatattg 1020
gcgagagtat cacccgtaac acattctgta ggaaactgtg tgaagcgtgt ggtggtcatc 1080
gtgagctctg tcatcttctt caagacaccg gtctcgcctg ttaatgcttt tggaaccgga 1140
atcgccctag cgggagtttt cttatactcc agagtgaagc gtatcaagcc aaagcctaag 1200
actgcctaa 1209
<210> 3
<211> 405
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 3
Met Gln Ser Ser Ala Val Phe Ser Leu Ser Pro Ser Leu Pro Leu Leu
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<210> 4
<211> 402
<212> PRT
<213> 甘蓝型油菜 (Brassica napus L.)
<400> 4
Met Gln Ser Ser Ala Val Phe Ser Ala Ser Pro Ser Leu Pro Leu Leu
1 5 10 15
Lys Pro Gly Arg Leu Ser Leu Arg His Pro Val Thr Ala Ser Ser Asn
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35 40 45
Arg Ser Trp Arg Leu Ala Ser Ser Asp Ser Pro Leu Arg Ala Trp Ser
50 55 60
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65 70 75 80
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Ala Ala Gln Leu Ala Ala Ile Leu Pro Leu Ala Val Val His Thr Leu
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Gly Asn Leu Phe Thr Asn Met Ser Leu Gly Lys Val Ser Val Ser Phe
180 185 190
Thr His Thr Ile Lys Ala Met Glu Pro Phe Phe Ser Val Val Leu Ser
195 200 205
Ala Met Phe Leu Gly Glu Val Pro Thr Pro Trp Val Ile Gly Ser Ile
210 215 220
Ile Pro Ile Val Gly Gly Val Ala Leu Ala Ser Val Thr Glu Val Ser
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Phe Asn Trp Ala Gly Phe Leu Ser Ala Met Ala Ser Asn Leu Thr Asn
245 250 255
Gln Ser Arg Asn Val Leu Ser Lys Lys Val Met Val Lys Lys Asp Asp
260 265 270
Ser Leu Asp Asn Ile Thr Leu Phe Ser Ile Ile Thr Leu Met Ser Leu
275 280 285
Phe Leu Met Ala Pro Val Thr Phe Phe Ser Glu Gly Ile Lys Phe Thr
290 295 300
Pro Ser Tyr Ile Gln Ser Ala Gly Val Asn Val Gln Gln Ile Tyr Thr
305 310 315 320
Lys Ser Leu Ile Ala Ala Leu Cys Phe His Ala Tyr Gln Gln Val Ser
325 330 335
Tyr Met Ile Leu Ala Arg Val Ser Pro Val Thr His Ser Val Gly Asn
340 345 350
Cys Val Lys Arg Val Val Val Ile Val Ser Ser Val Ile Phe Phe Lys
355 360 365
Thr Pro Val Ser Pro Val Asn Ala Phe Gly Thr Gly Ile Ala Leu Ala
370 375 380
Gly Val Phe Leu Tyr Ser Arg Val Lys Arg Ile Lys Pro Lys Pro Lys
385 390 395 400
Thr Ala
Claims (7)
1.油菜磷酸烯醇式丙酮酸转运基因BnaPPT1在调控作物含油量中的应用,所述油菜磷酸烯醇式丙酮酸转运基因BnaPPT1的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.权利要求1中所述的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1所编码的蛋白在调控作物含油量中的应用,所述蛋白的氨基酸序列如SEQ ID NO:3或SEQ ID NO:4所示。
3.含有权利要求1中所述的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1的表达载体在调控作物含油量中的应用。
4.含有权利要求1中所述的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1的重组菌在调控作物含油量中的应用。
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权利要求1中所述的油菜磷酸烯醇式丙酮酸转运基因BnaPPT1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,获得油菜磷酸烯醇式丙酮酸转运基因BnaPPT1超表达的作物品种。
7.如权利要求6所述提高作物含油量的方法,其特征在于:所述作物为油菜。
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