CN104988158B - 甘蓝型油菜核转录因子NF-YA基因BnNF-YA12及其应用 - Google Patents
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Abstract
本发明公开了甘蓝型油菜核转录因子NF‑YA基因BnNF‑YA12及其应用。一种甘蓝型油菜核转录因子NF‑YA基因BnNF‑YA12,具有如SEQ ID NO.1所示的核苷酸序列。其编码的蛋白质,具有如SEQ ID NO.2所示的氨基酸序列。所述的甘蓝型油菜核转录因子NF‑YA基因BnNF‑YA12过表达植株对盐、干旱和ABA处理表现出敏感,说明BnNF‑YA12参与植物的非生物胁迫调控过程。
Description
技术领域
本发明属于基因工程领域,涉及甘蓝型油菜核转录因子NF-YA基因BnNF-YA12及其应用。
背景技术
NF-YA(CBF-B或HAP2)是转录调控因子NF-Y的一个亚基,它与另外两个亚基NF-YB、NF-YC组成异源三聚体,共同结合于下游基因的CCAAT框上,从而激活或抑制基因的表达(Romier等,2003)。CCAAT-box一般位于启动子转录起始位点(TSS)上游-60~-100bp处,是启动子中的基本调控元件之一(Mantovani,1999)。NF-Y是非常保守的一类转录调控因子,每个亚基都有其特定的保守区域。NF-YA的保守区域位于C端,该区域负责与DNA序列结合,引导NF-YB/NF-YC异源二聚体结合于CCAAT-box(Li等,1992)。关于NF-Y异源三聚体的组装,一种可能的组装过程为:首先NF-YB与NF-YC结合形成异源二聚体,接着NF-YA再同异源二聚体结合形成完整的NF-Y,最后转录调控因子NF-Y与下游基因的CCAAT框结合(Ronchi等,1995,Sinha等,1995)。研究发现在拟南芥中,NF-YA与CO的蛋白结构相似。CO同样可以与NF-YB、NF-YC结合形成一个三聚体,该三聚体结合于FT基因的启动子中的CCAAT框上,并激活FT基因的表达。当对NF-YA过表达时,植物会表现出FT基因被抑制的现象。这说明在植物体内NF-YA与CO是相互竞争的关系,大量表达的NF-YA会竞争性地与NF-YB/NF-YC异源二聚体结合,从而造成CO的脱靶,进而抑制了FT基因的表达。
miR169在植物中是一个很大的miRNA基因家族,目前已经在超过40个物种中得到鉴定(Jones-Rhoades等,2006),其在植物的生长和响应环境胁迫上起着重要的作用(Lee等,2010)。在拟南芥中,miR169基因家族有14个成员,经加工最终形成的成熟序列有4种。这四种成熟序列在环境胁迫下拥有独特的表达模式,预示着各自功能的已经特殊化(Licausi等,2011,Singh等,2012,Zhao等,2011)。在植物中,miR169的靶基因包括细胞色素C、蛋白磷酸酶7、CBF、NF-YA(Zhou等,2012)。NF-Y转录调控因子可以结合于下游基因启动子中的CCAAT框,从而激活或抑制下游基因的表达(Mantovani,1999)。Li等研究发现,拟南芥miR69a/c在干旱处理下表达下调,其靶基因AtNF-YA5表达上调;AtNF-YA5的过表达拟南芥植株,在干旱处理下,叶片的失水减少,与WT植株相比抗旱性更高;而ath-miR69a的过表达植株与敲除nf-ya5的突变体植株表型相同,都对干旱高度敏感(Licausi等,2011)。Marco等对拟南芥大的NF-YA基因家族中受miR169调控的AtNF-YA2/3/7/10进行过表达,实验结果显示,过表达AtNF-YA2/3/10会增强植株对多种非生物胁迫的抗性(Zhao等,2011)。在大豆中,通过分析GmNF-YA3在干旱胁迫下的表达模式,发现其对干旱胁迫有反应。利用5’RACE和烟草瞬时表达证明GmNF-YA3是miR169的一个靶基因。GmNF-YA3的过表达植株表现为叶片失水率低,在长期的干旱处理下也能存活,过表达GmNF-YA3增强了植株的抗旱能力。同时,过表达GmNF-YA3的拟南芥对外源施加的ABA很敏感,转基因植株体内与ABA生物合成、ABA信号传导以及胁迫响应相关的基因的转录水平比野生型植株的要高。以上说明GmNF-YA3基因积极参与调节植物的抗旱功能。在杨树(Populus tremuloides)中,ptr-miR169a和靶基因PtrHAP2-5(NF-YA2)参与调控杨树的休眠(Potkar等,2013)。在番茄(Solanumlycopersicum)中,sly-miR169在干旱处理下表达量增加,其靶基因SiNF-YA1/2/3的表达量降低(Zhang等,2011)。综合上所述,miR169/NF-YA调控模块在植物逆境胁迫中起着重要的作用,NF-YA主要是通过miR169调控途径来参与逆境调控网络的。
发明内容
本发明的目的是提供一种甘蓝型油菜小分子RNA基因bna-miR169o及其靶基因核转录因子NF-YA基因BnNF-YA12。
本发明的另一目的是提供基因BnNF-YA12的应用。
本发明的目的可通过如下技术方案实现:
一种甘蓝型油菜核转录因子NF-YA基因BnNF-YA12,其特征在于具有如SEQ IDNO.1所示的核苷酸序列。
所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12优选以甘蓝型油菜“南盐油1号”cDNA为模板,以BnNF-YA12-F和BnNF-YA12-R为引物,通过PCR扩增得到。
所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12编码的蛋白质,具有如SEQ IDNO.2所示的氨基酸序列。
一种甘蓝型油菜小分子RNA基因bna-miR169o,其靶基因是权利要求1所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12,切割位点位于BnNF-YA12的3‘UTR。
所述的甘蓝型油菜小分子RNA基因bna-miR169o,优选以甘蓝型油菜“南盐油1号”cDNA为模板,以bna-miR169o-F和bna-miR169o-R为引物,通过PCR扩增得到,具有如SEQ IDNO.3所示的核苷酸序列。
一种含有本发明所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12重组载体。
所述的重组载体,优选将甘蓝型油菜核转录因子NF-YA基因BnNF-YA12插入表达载体pCAMBIA1302的NcoI和SpeI酶切位点之间所得。
一种含有所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12重组载体的根癌农杆菌EHA105。
所述的根癌农杆菌,所述的重组载体经电转化转入根癌农杆菌EHA105中。
所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12在构建盐、干旱或外源ABA敏感的转基因植物中的应用。
有益效果:
本发明提供了一株新的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12,该基因与已有的拟南芥、亚麻芥、白菜中的NF-YA9基因的蛋白序列同源度分别为73.8%、71.5%、76.4%。
对bna-miR169o和BnNF-YA12的转基因纯合体进行各项生理实验,结果显示BnNF-YA12的过表达植株对盐、干旱和ABA处理表现出敏感。说明BnNF-YA12参与植物的非生物胁迫调控过程。bna-miR169o的过表达植株与野生型的表型差不多,没有明显的差异。可能的原因是拟南芥中不存在bna-miR169o的靶基因。
附图说明
图1、甘蓝型油菜小分子RNA基因bna-miR169o的前体扩增结果(A01、A08分别代表A染色体组中的01和08染色体);
图2、烟草瞬时表达验证结果
图3、PCR验证转基因植株在转录水平上的表达。A12代表BnNF-YA12
图4、逆境处理下转基因拟南芥株系和野生型的萌发率。
图5、根的伸长率测定。
图6、根系扫描的结果。
具体实施方式
实施例1bna-miR169o基因的克隆及靶基因的预测
利用生物软件对4个甘蓝型油菜降解组测序数据库(镉处理的根、镉处理的茎叶、对照组的根和对照组的茎叶,本数据来源于周兆胜老师)进行分析(Zhou等,2012),我们得到了bna-miR169基因家族成熟序列以及类似序列在降解组数据库中的表达丰度。从检测结果中发现,bna-miR169m、bna-miR169n、bna-miR169a-b、169Bn_sRNA_51、169Bn_sRNA_1538的成熟序列表达丰度较其他家族成员要高很多。169Bn_sRNA_51尚未被报道,可能是bna-miR169家族的新成员,我们将其命名为bna-miR169o。
将bna-miR169o的成熟序列导入白菜数据库(http://brassicadb.org/brad/)中检索,截取白菜基因组中完全匹配区域以及该区域两端各200bp的序列,用来克隆新成员bna-miR169o的前体序列。根据在白菜数据库中检索并截取的序列,使用引物设计软件Premier5.0,在距离bna-miR169o成熟序列两端各100bp左右的位置设计合适的引物。引物序列如下:
bna-miR169o-F:5'AAGGATGGATGATGATTA 3'(SEQ ID NO.4)
bna-miR169o-R:5'AAGTTGTGATGGTGATAT 3'(SEQ ID NO.5)
以甘蓝型油菜‘南盐油1号’的DNA为模板,用所设计的油菜bna-miR169o的引物进行扩增,其PCR体系与反应程序如下
PCR产物点入1%的琼脂糖凝胶中电泳,得到约200bp左右的特异条带,如图1所示。对目的片段进行切胶回收,连接克隆载体pMD19-T,将阳性菌株送交金维智公司测序。测序获得的序列与数据库检索截取的序列相似度为98.2%,通过在线DNA折叠预测分析,确定新成员bna-miR169o具有miRNA基因的典型结构。
将bna-miR169o的成熟序列和BnNF-YA基因家族的cDNA序列(带3’UTR区域)导入在线靶基因预测网站psRNATarget(http://www.plantgrn.org/psRNATarget/)中,利用网站中在线软件分析,预测结果显示bna-miR169o的潜在靶基因是BnNF-YA12。
实施例2bna-miR169o过表达载体的构建
由于bna-miR169o是非编码基因,其前体序列不存在编码基因的“ATG”,因此只需要在两侧添加XbaⅠ和SacⅠ即可,设计的引物如下,酶切位点如下划线所示:
p35S::bna-miR169o-F:5'GCTCTAGAAAGGATGGATGATGATTATG 3'(SEQ ID NO.6)
p35S::bna-miR169o-R:5'CGAGCTCAAGTTGTGATGGTGATATAAG 3'(SEQ ID NO.7)
以实施例1中的DNA为模板,用高保真酶进行PCR扩增,反应体系:5×PrimerSTARTMBuffer(Mg2+plus)10μL,dNTP Mixture(2.5mM)4μL,p35S::bna-miR169o-F(10μM)1μL,p35S::bna-miR169o-R(10μM)1μL,DNA模板2μL,HS DNA polymerase(2.5U/μL)0.5μL,ddH2O31.5μL,总体积50μL。
反应程序:
将扩增产物跑1%的琼脂糖凝胶电泳,得到长度约为200bp左右的单一条带,与预期的大小相同。将该基因片段克隆至pEASY-Blunt平末端载体上(具体操作参照pEASY-Blunt Cloning Kit说明书进行),即得到克隆载体pEASY-B-bna-miR169o。
pEASY-B-bna-miR169o和空载pBI121分别用XbaⅠ和SacⅠ双酶切,然后将酶切后的pBI121单一片段和克隆载体中的bna-miR169o片段回收,然后用T4连接酶将二者酶连,即得到表达载体pBI121-bna-miR169o,表达载体转化至大肠杆菌感受态细胞DH5α(TAKARAD9057S)中,挑选阳性克隆,送交公司测序。测序结果正确即表明载体构建成功。
实施例3BnNF-YA12过表达载体的构建
在本专利中,我们构建了三种结构的BnNF-YA12过表达载体,分别是BnNF-YA12CDS、BnNF-YA12UTR和BnNF-YA12MUT。BnNF-YA12MUT是通过突变BnNF-YA12UTR的部分碱基而获得的,因此只需要对BnNF-YA12CDS和BnNF-YA12UTR进行引物设计。通过酶切位点选择分析,BnNF-YA12CDS和BnNF-YA12UTR都选用NcoⅠ、SpeⅠ,由于BnNF-YA12的起始密码子“ATG”后面的碱基是“G”,只需在“ATG”前面加上“CC”就可以构成NcoⅠ,再在前面添加保护碱基“CATG”,5’端可以直接添加SpeⅠ(5’-A↓CTAGT-3’)。BnNF-YA12CDS过表达载体的构建需去掉终止密码子。设计的引物如下,酶切位点如下划线所示:
p35S::BnNF-YA12CDS-F:5'CATGCCATGGGAATGGAAACTGAAGACA 3'(SEQ ID NO.8)
p35S::BnNF-YA12CDS-R:5'GACTAGTCTTGATAGCTAAAAGAGGTT 3'(SEQ ID NO.9)
p35S::BnNF-YA12UTR-F:5'CATGCCATGGGAATGGAAACTGAAGACA 3'(SEQ ID NO.10)
p35S::BnNF-YA12UTR-R:5'GACTAGTCTCAATCACATAGTTTCGGA 3'(SEQ ID NO.11)
以甘蓝型油菜‘南盐油1号’的cDNA为模板,用高保真酶进行PCR扩增,反应体系和反应程序同实施例2,将扩增产物跑1%的琼脂糖凝胶电泳,分别得到长度约为900bp和1100bp左右的单一条带,与预期的大小相同。将该基因片段克隆至pEASY-Blunt平末端载体上(具体操作参照pEASY-Blunt Cloning Kit说明书进行),即得到克隆载体pEASY-B-BnNF-YA12CDS和pEASY-B-BnNF-YA12UTR。
使用全式金公司的Fast Mutagenesis System点突变试剂盒,对BnNF-YA12UTR上的miR169切割位点进行多碱基的突变。详细步骤见试剂盒说明书。针对BnNF-YA12UTR切割位点的序列设计突变引物(F:5'CAAGTAACCTCTACGCTAACTTATCATTCTTG3'(SEQ ID NO.12);R:5'AAGTTAGCGTAGAGGTTACTTGATAGCTAAAA3'(SEQ ID NO.13)),以上面测序成功的pEASY::BnNF-YA12UTR质粒作为模板,进行点突变PCR扩增,反应体系和反应程序如下:
取10μLPCR产物,1%琼脂糖凝胶电泳检测。观察到目的条带后,加1μLDMT酶与PCR产物中,混匀,37℃孵育1h。取2-5μL用于转化DMT感受态细胞,挑取阳性克隆送交公司测序。
pEASY-B-BnNF-YA12CDS/UTR/MUT和空载pCAMBIA1302分别用NcoⅠ和SpeⅠ双酶切,然后将酶切后的pCAMBIA1302单一片段和克隆载体中的BnNF-YA12CDS/UTR/MUT片段回收,然后用T4连接酶将二者酶连,即得到表达载体pCAMBIA1302-BnNF-YA12CDS/UTR/MUT,表达载体转化至大肠杆菌感受态细胞DH5α(TAKARA D9057S)中,挑选阳性克隆,送交公司测序。测序结果正确即表明载体构建成功。
实施例4bna-miR169o基因的靶基因验证
分别将实施例2和3中构建的表达载体pBI121-bna-miR169o和pCAMBIA1302-BnNF-YA12CDS/UTR/MUT转化到根癌农杆菌EHA105中,通过菌液PCR筛选转化成功的阳性转化子。将携带pBI121-bna-miR169o的农杆菌分别与携带pCAMBIA1302-BnNF-YA12CDS/UTR/MUT的农杆菌两两混合注射入烟草(Nicotiana benthamiana)叶片细胞中。烟草生长两天后,分别采样并提取烟草叶片的总RNA。利用反转录试剂盒将RNA反转录成cDNA,根据定量引物设计要求,在BnNF-YA12的CDS区域设计一对定量引物,扩增产物大小为80-150bp,定量引物如下:
BnNF-YA12qPCR-F:5'CAAACCATCCTTCTCAACC 3'(SEQ ID NO.14)
BnNF-YA12qPCR-R:5'ATAAGCCAACAAAGATAGAGCC 3'(SEQ ID NO.15)
实时定量结果如图2所示,从柱形图中可以看出,bna-miR169o对无切割识别位点的BnNF-YA12CDS以及切割识别位点突变的BnNF-YA12MUT的表达量没有影响。而具有切割识别位点的BnNF-YA12UTR,在共转bna-miR169o的过表达载体的情况下,其表达量显著地下降。实验证明BnNF-YA12是bna-miR169o的靶基因。
实施例5BnNF-YA12拟南芥转基因
1、蘸花法(农杆菌介导)转化拟南芥
农杆菌侵染重悬液的配制:蔗糖(5%),silwet L-77(0.02%),农杆菌菌体,无菌水;将保藏的含目的基因表达载体的农杆菌划线活化(有抗生素的固体YEP培养基),挑取单克隆接种于50mL加抗生素的YEP液体培养基(Kan(50mg·L-1)和Str(50mg·L-1)),28℃恒温摇床,200rpm,培养2d,至OD600=0.8~1.5;收集菌体:取上述菌液10mL分装与2mL的离心管中,4000~6000rpm离心3~4min,弃上层的培养基,用等体积的重悬液重悬菌体;用一次性胶头滴管吸取侵染菌液,小心滴入拟南芥的花蕾上,确保整个花絮都被浸润(第一次侵染时需将已经开花的花朵剪掉);侵染后的拟南芥套上塑料袋,暗培养12~24h;去掉塑料袋后正常培养5~7d,再进行第二次侵染,同一植株可连续侵染2~3次,以增加转化的效率;转化2~3次的拟南芥恢复正常培养,及时剪除新长出的侧芽。在拟南芥生长后期可少量浇水,以加速拟南芥成熟;待少量角果开始变黄时,用信封将整株拟南芥包住,加速种子成熟;收取种子:去除杂质,将干净的种子装入1.5mL离心管中,存放于4℃冰箱中。
2、拟南芥转基因阳性苗的筛选。
实验准备:70%酒精溶液、30%次氯酸钠溶液,无菌水,灭菌的枪头,含有抗生素的MS固体培养基(Hyg浓度为25mg·L-1,Kan浓度为50mg·L-1);取适量待筛选的转基因拟南芥种子于10mL离心管中,加入去离子水浸泡至少30min;在无菌超净台中,70%的酒精浸泡拟南芥种子1min,倒去上层溶液后用无菌水冲洗2~3次;再用30%次氯酸钠浸泡种子5min,然后用无菌水冲洗6~7次;用无菌枪头吸取灭菌的种子涂布于MS平板上,再加入适量无菌水,轻轻摇晃平板使种子尽量铺展开来,小心吸取多余的无菌水;将铺有种子的平板开盖放置于超净台中,吹干培养基表面的水分,然后封好平板,4℃放置2d;冷处理后的种子放置于长日照(16-h light/8-h dark),22~24℃环境中培养7~10d,挑选长势良好的拟南芥(阳性苗株型较大且根系较长),移栽到基质中生长;待拟南芥有一定的生物量时,采集叶片提取基因组DNA,并进行阳性苗验证。
3、拟南芥转基因阳性苗的鉴定
通过PCR扩增验证转基因阳性苗。
设计至少两对引物用以PCR验证。一对引物分别是:Forward primer在35S启动子区域里,Reverse Primer在目的基因的CDS区域里。另一对引物分别是:Forward primer在目的基因的CDS区域里,Reverse Primer在标记基因GFP区域里。目的基因的验证引物如下:
1302-35S-F:5'CGTTGAAGATGCCTCTGCCG 3'(SEQ ID NO.16)
BnNF-YA12-R:5'CTTGATAGCTAAAAGAGGTT 3'(SEQ ID NO.17)
为了更准确地验证转基因阳性苗,可以对PCR验证扩增的片段进行测序分析。
4、拟南芥转基因纯合体的筛选
分别取40粒左右单株收取(F3代)的种子,按照5.1.2.4中筛选转基因阳性苗的方法处理种子,并将种子放置于带有抗性的MS固体培养基上。冷处理后的种子放置于长日照(16-h light/8-h dark),22~24℃环境中培养7~10d。所有幼苗都长势良好的株系即为纯合体株系,将确定为纯合体株系的幼苗移栽到机制中继续生长,等待收取下一代的种子。
已经筛选出35S::bna-miR169o和35S::BnNF-YA12的纯合体,bna-miR169o转基因纯合体株系的个数为4个,BnNF-YA12的转基因纯合体株系的个数为1个。分别提取35S::bna-miR169o和35S::BnNF-YA12纯合体的总RNA,反转录成cDNA,通过PCR验证转基因纯合体在转录水平上的表达。PCR验证结果如图3所示。
实施例5BnNF-YA12拟南芥转基因株系的发芽率测定
对转基因拟南芥和野生型(WT)植物种子进行消毒,用灭菌过牙签随机挑取每个转基因株系的40粒种子,分别点播到含有不同浓度NaCl(50mM、100mM和150mM),甘露醇(200mM、300mM和400mM)和ABA(0.5μM和1μM)的MS平板中,观察种子萌发对NaCl、干旱和ABA的敏感性。点播后种子4℃冰箱中春化3d,平板正置,温室(22℃)正常培养。当种子幼根已经穿出种皮被认为萌发,每天统计种子的萌发率。BnNF-YA12的拟南芥转基因纯合体在100mMNaCl、300mM甘露醇和0.5μM ABA处理下的萌发率如图4所示,结果显示BnNF-YA12的拟南芥转基因株系在这三种处理下的萌发率都低于野生型,对逆境处理表现出敏感。
实施例5BnNF-YA12拟南芥转基因株系的根伸长率测定
灭菌后的转基因和野生型种子,点播到MS平板中萌发。种子萌发后,用牙签小心将野生型和转基因株系3棵萌发的幼苗分别转移到加NaCl、甘露醇、ABA的MS固体平板中,每个处理做3组生物重复。NaCl的浓度分别设定为120mM和150mM,甘露醇的浓度分别设定为250mM和350mM,ABA的浓度分别设定为0.1μM和0.25μM。每隔两天测量一次根生长的长度,总共计数5次,每次测量的时间点需保持一致(如14:00)。对多次测量结果取平均,算出根的生长率。根的伸长率统计结果如图5所示,从图中可以发现,在三种非生物胁迫处理下(NaCl、甘露醇、ABA),BnNF-YA12的拟南芥转基因株系的根伸长率要低于野生型。
实施例6BnNF-YA12拟南芥转基因株系的根系扫描
分别将实施例5中完成根的伸长率测定的转基因和野生型拟南芥植株移出,放入装有清水的透明方形塑料平板中。用牙签小心将每株拟南芥的根系分开,并放入根系扫描仪中进行根系扫描,最终获得并记录总根长(Length)、表面积(SurfArea)、平均直径(AvgDiam)、根体积(RootVolume)、根尖数(Tips)等五项数据。统计分析结果如图6所示,在0.25μM ABA处理下,过表达BnNF-YA12的拟南芥的总根长、根总面积、根体积、根尖数都要比野生型的低,但是二者的根平均直径没有明显的差异。在250mM甘露醇处理下,过表达BnNF-YA12的拟南芥的五项指标则都明显低于野生型。类似地,在120mM的NaCl处理下,对于过表达BnNF-YA12的拟南芥而言,在盐处理下,其根平均直径与野生型的一致,其余四项指标都明显低于野生型。
Claims (1)
1.甘蓝型油菜核转录因子NF-YA基因BnNF-YA12在构建盐、干旱或外源ABA敏感的转基因植物中的应用; 所述的甘蓝型油菜核转录因子NF-YA基因BnNF-YA12核苷酸序列如SEQID NO.1所示。
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Non-Patent Citations (4)
Title |
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Cloning and characterization of microRNAs from Brassica napus;Lei Wang et al.;《FEBS Letters》;20070716;第581卷;第3848–3856页 * |
Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium;Zhao Sheng Zhou et al.;《Journal of Experimental Botany》;20121231;第63卷(第12期);第4597-4613页 * |
Mingxiang Liang et al..Identification and characterization of NF‑Y transcription factor families in Canola (Brassica napus L.).《Planta》.2013,第239卷第14页右栏第1段,第22页第4行, 第23页. * |
Multiple NUCLEAR FACTOR Y Transcription Factors Respond to Abiotic Stress in Brassica napus L.;Li Xu et al.;《PLOS ONE》;20141030;第9卷(第10期);摘要,第4页图1 * |
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