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开花是高等植物进入生殖生长的标志,是植物完成生活史的必要环节。在拟南芥Arabidopsis thaliana中的研究发现,开花过程受多种内源因素和外源环境因素的影响,由复杂基因网络严格调控[1]。光周期途径中植物开花受季节性昼长变化影响[2];赤霉素(GA)途径是在非诱导光周期途径的条件下,在开花过程中发挥作用[3];春化途径中植物开花要受到低温积累的影响[4];自主途径中通过感知植物自身内部发育状态而调控植物开花[5],多种途径的成花调节信号最终都集中于2个整合因子:开花促进因子FLOERING LOCUS T(FT)和SUPPRESSPR OF OVEREXPRESION OF CONSTANS 1(SOC1)[6-8]。在拟南芥中,过表达FT基因会明显促进SOC1基因的表达量;同时在ft突变体中,SOC1的表达量明显下降[9-11],但是对ft和soc1双突变体与ft单突变体植株的开花时间进行统计,结果显示没明显差异,可知除FT蛋白调控SOC1的表达外,还存在另外的途径影响SOC1基因的表达[10-13]。其他开花控制因子可以通过FT和SOC1参与开花途径的调控,例如开花抑制因子SHORT VEGETATIVE PHASE(SVP)可以结合SOC1和FT的调控序列,对FT和SOC1产生抑制作用[14],进而下调它们的表达量。近年,发现拟南芥中属于J蛋白家族一员的J3蛋白,可以与SVP蛋白相互作用调控SOC1和FT基因的表达量,而实现对成花信号的调节,在调控开花途径中发挥着十分重要的作用[15]。J蛋白(J-domain protein)是广泛存在于动植物中的一个庞大的分子伴侣家族,该类蛋白N末端包含1个约由70个氨基酸构成的保守序列——J结构域而被命名为J蛋白[16-17]。J蛋白的J结构域由4个α-螺旋组成,第2和第3个螺旋之间有一个极为保守的由组氨酸、脯氨酸、天冬氨酸组成的HPD三肽,这是J结构域的重要特征[18];邻近J结构域的是G/F结构域(富含甘氨酸和苯丙氨酸);之后是锌指结构域。根据结构域的不同J蛋白可分为4类[19]:Ⅰ类J蛋白包含所有常见的结构;Ⅱ类J蛋白缺少锌指结构;Ⅲ类J蛋白只有J结构域;Ⅳ类J蛋白也被称作J-like蛋白,这类蛋白的J结构域没有HPD模块[20-21]。目前发现拟南芥中共有120个J蛋白,其中Ⅰ类J蛋白8个,Ⅱ类J蛋白16个,Ⅲ类J蛋白92个,Ⅳ类J蛋白4个[22]。在植物中,有关J蛋白的研究主要集中在拟南芥中,现有的结果表明,J蛋白可以参与多种生物学过程,包括叶绿体的发育[23-25]、植物的抗逆性[26-27]、信号转导[28-29]和成花途径[15]等。在拟南芥开花调控中,J3蛋白通过与SVP等其他开花关键因子相互作用而影响植物成花过程,SVP的突变体svp-41表现为早花现象,J3的突变j3-1表现为晚花现象,同时在j3-1和svp-41的双突变体中,拟南芥表现出早花现象,与svp-41的单突变体的表型相似,这一结果表明J3基因通过与SVP的相互作用实现对开花时间的调控[30-31],进一步研究发现在细胞核中J3基因通过与营养生长短期(SVP)蛋白直接相互作用,衰减SVP蛋白与SOC1和FT调控序列的结合从而上调SOC1和FT基因的表达量[15]。竹类植物开花周期较长并具有时间上的不确定性,而且很多竹子开花后会成片死亡,雷竹Phyllostachys violascens是一种优良的笋用竹种,近年来,雷竹普遍开花,开花后部分雷竹会死亡,导致雷竹笋产量大幅度下降,带来严重的经济损失。有关竹类植物开花的研究已经很多,但是竹子成花机制和花期调控模式至今尚未完全清楚[32]。目前,竹子中还未见有关J3基因生物学功能的研究,本研究以雷竹为研究对象,通过同源克隆技术获得1个J3同源基因,经过亚细胞定位、表达模式和功能分析,以期能为深入研究J3同源基因在竹类植物开花调控机制中的作用提供科学依据。
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雷竹材料来源于浙江农林大学智能温室大棚,选取位于同一竹鞭上的笋、花芽、花,开花和不开花雷竹的幼叶、成叶、茎秆,标记分装后放入液氮中速冻,保存在-80 ℃超低温冰箱。
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用全RNA提取试剂盒(鼎国)分别提取采集的雷竹材料的RNA,以提取的RNA(少于0.5 g·L-1)为模板,用Prime ScriptTM RT Master Mix(Perfect Real Time)试剂盒(Clontech),反转录获得雷竹各组织的cDNA,检测合格后,于-20 ℃保存备用。
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通过本地Blast,从毛竹Phyllostachys edulis基因组文库中找到一个候选基因序列号(bphylf027h21),根据该候选基因的基因序列设计引物(J3,表 1),雷竹cDNA为模板,进行聚合酶链式反应(PCR)扩增,琼脂糖凝胶电泳检测PCR扩增条带,用胶回收试剂盒(上海生工生物公司)回收纯化电泳条带,将获得的目的片段与pMD19(simple)-T载体置于连接仪上16 ℃连接1.0~3.0 h,之后转化大肠埃希菌Escherichia coli DH5α感受态细胞(热转化法),37 ℃恒温培养箱培养6.0~8.0 h,挑取白色圆形单菌落,菌落PCR鉴定出阳性单克隆,送测序,得到碱基序列。
表 1 本研究所用的引物
Table 1. PCR primers used in this study
引物名称 序列(5'→3') 用途 J3-F CTCTCCGTCTCACTCCGCTC 基因开放阅读框(ORF)扩增 J3-R GTCAAATTGCCTTTATAGAA PvJ3-F ATGTTCGGGCGCGCGCCG ORF扩增 PvJ3-R TTACTGTTGTGCGCACTGCAC PvJ3-1-F CCGGAATTCTGGAGAGAGCATTAACG 荧光定量PCR PvJ3-1-R CCGGAATTCCTTCTGCTGGAGGAC PeNTB-F TCTTGTTTGACACCGAAGAGGAG 荧光定量PCR内参引物 PeNTB-R AATAGCTGTCCCTGGAGGAGTTT PvJ3-2-F TCCCCCGGGATGTTCGGGCGCGCG 亚细胞定位 PvJ3-2-R CGCGGATCCCTGTTGTGCGCACTGCACTC PvJ3-3-F tccATCGATATGTTCGGGCGCGCG 过表达载体构建 PvJ3-3-R cgcGGATcCttaCTGTTGTGCGCACTGCACTC 用Vector NTI预测基因序列的开放阅读框(ORF)区,据此设计引物(PvJ3,表 1),重复上述实验过程,最终测序得到雷竹PvJ3基因序列。运用TMHMM和ProtScale分析PvJ3蛋白的结构特征和理化性质;Smart在线分析其蛋白的结构域;通过Clustal X2比对雷竹PvJ3与其他物种中的J3同源基因的氨基酸序列的相似性;并用Mega 5.0构建系统发育树,选择邻近法(NJ),bootstrap回复次数为1 000次。
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用雷竹各组织部位的cDNA为模板,通过实时定量聚合酶链式反应(qRT-PCR),分析雷竹PvJ3基因的表达模式。根据实验要求设计目的基因的定量引物(PvJ3-1,表 1),毛竹的PeNTB基因作为内参基因[33]。各种雷竹组织做3次重复,避光条件下操作,参照SYBR Premix Ex TaqTMⅡ(Takara)试剂盒要求加样。PCR扩增程序(两步法):预变性95 ℃,5 min;95 ℃,30 s;60 ℃,30 s,循环数为40。反应结束后,分析确定数据的可靠性,选择合适的数据,用2-△△CT方法[34]对数据进行分析。
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根据PvJ3基因ORF区序列(不含终止密码子)和pCaM35S-sGFP载体序列上的酶切位点,在目的基因ORF区的上下游分别引入酶切位点SmaI和BamHI,设计引物(PvJ3-2,表 1),提取pMD19-PvJ3质粒做为模板,进行PCR反应,胶回收纯化得到带有酶切位点的基因片段与pMD19(simple)-T载体连接转化后,阳性单克隆送测序。将测序正确的单克隆进行扩大培养,用质粒提取试剂盒(上海生工生物公司)提取质粒,用限制性内切酶SmaI和BamHI分别对pMD19-PvJ3和pCaM35S-sGFP载体质粒进行双酶切实验,之后用T4 DNA连接酶在16 ℃条件下过夜连接胶回收的酶切产物,连接产物转化大肠埃希菌DH5α感受态细胞,经测序正确,瞬时表达载体PvJ3-GFP构建成功。通过基因枪轰击洋葱Allium cepa表皮,25 ℃下避光暗培养1~2 d后,用荧光显微镜观察绿色荧光蛋白信号,金粉的准备和样品的制备参照文献[35]。
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根据PvJ3基因ORF区序列和pC1301载体(含有35 S启动子)的序列设计引物(PvJ3-3,表 1),在目的基因ORF区的上下游同样分别引入酶切位点SmaI和BamHI,构建过表达载体pC1301-PvJ3,实验方法参照1.5节。
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通过热转化法将构建成功的pC1301-PvJ3过表达载体转入农杆菌Agrobacterium tumefaciens GV3101感受态细胞中,提取阳性单克隆的质粒,重新转化大肠埃希菌DH5α感受态细胞,再次测序检验转入重组质粒是否正确。用已正确转入pC1301-PvJ3重组载体的农杆菌配制侵染液,侵染进入花期的野生型拟南芥花序[36],侵染3次,间隔约1周·次-1,侵染后的拟南芥即T0代植株,将消毒处理后的T0代种子均匀撒在加入潮霉素的1/2 MS(Murashige and Skoog)固体培养基上,潮霉素质量浓度为100.00 mg·L-1,按照V(潮霉素溶液):V(1/2MS培养基)=1.50:1 000.00比例加入培养基中,筛选抗性植株,以采用十六烷基三甲基溴化铵(CTAB)法[37]提取的抗性植株的基因组DNA为模板,PCR鉴定出T1代阳性植株,并对植株表型进行初步统计。
Cloning and functional analysis of the PvJ3 gene from Phyllostachys violascens
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摘要: 拟南芥Arabidopsis thaliana中J3基因参与花期的调节,调控开花时间。为了探究竹类植物中J3基因的功能,根据植物中J同源基因的高度保守性,采用同源克隆技术从雷竹Phyllostachys vilascens中克隆出1个J3基因,命名为PvJ3。该基因的开放阅读框(ORF)区长为1 260 bp,编码419个氨基酸。通过同源比对和系统进化树分析,可知雷竹PvJ3蛋白和其他物种中J3同源蛋白的氨基酸序列同源性很高。通过实时定量聚合酶链式反应(qRT-PCR),发现PvJ3基因在同一竹鞭的笋、花芽、花,开花与不开花雷竹的茎秆、成叶、幼叶中都有表达,且开花雷竹茎秆中表达量最高,表明PvJ3基因在雷竹各组织部位都有表达。亚细胞定位分析结果显示,PvJ3蛋白定位在细胞质和细胞核。通过转化拟南芥对其功能进行了初步分析,表型统计结果显示,PvJ3转基因拟南芥与野生型相比,开花时间明显提早,而且出现多主茎现象,表明PvJ3可以促使植物提前开花并且参与植物茎秆的发育。Abstract: The J3 gene mediates the integration of flowering signals in Arabidopsis thaliana. To explore the functions of the J3 gene in bamboo plants, a J3 homolog named PvJ3 was isolated from Phyllostachys violascens, and a preliminary analysis of its function was conducted with Ph. violascens. Analyses included a sequencing alignment and phylogenetic analysis, real-time quantitative PCR, and protein subcellular localization. Results showed that the phenotype of overexpression of PvJ3 in Ph. violascens predated flowering and had more main stems. The full-length ORF of the PvJ3 in Ph. violascens was 1 260 bp which encoded 419 amino acids. The sequencing alignment and phylogenetic analysis of PvJ3 showed a high homology with other species. Real-time quantitative PCR results showed that the PvJ3 was expressed in all organs both in flowering and non-flowering bamboo plants and was highest in the stem of flowering bamboo plants. Protein subcellular localization showed that PvJ3 was located in the cytoplasm and cell nucleus. These results suggest that PvJ3 may promote flowering, may participate in stem development, and may provide a scientific basis for the regulation of bamboo flowering.
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Key words:
- forest tree breeding /
- Phyllostachys violascens /
- PvJ3 /
- subcellular localization /
- functional analysis
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表 1 本研究所用的引物
Table 1. PCR primers used in this study
引物名称 序列(5'→3') 用途 J3-F CTCTCCGTCTCACTCCGCTC 基因开放阅读框(ORF)扩增 J3-R GTCAAATTGCCTTTATAGAA PvJ3-F ATGTTCGGGCGCGCGCCG ORF扩增 PvJ3-R TTACTGTTGTGCGCACTGCAC PvJ3-1-F CCGGAATTCTGGAGAGAGCATTAACG 荧光定量PCR PvJ3-1-R CCGGAATTCCTTCTGCTGGAGGAC PeNTB-F TCTTGTTTGACACCGAAGAGGAG 荧光定量PCR内参引物 PeNTB-R AATAGCTGTCCCTGGAGGAGTTT PvJ3-2-F TCCCCCGGGATGTTCGGGCGCGCG 亚细胞定位 PvJ3-2-R CGCGGATCCCTGTTGTGCGCACTGCACTC PvJ3-3-F tccATCGATATGTTCGGGCGCGCG 过表达载体构建 PvJ3-3-R cgcGGATcCttaCTGTTGTGCGCACTGCACTC -
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