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春兰(Cymbidium goeringii)是兰科(Orchidaceae)兰属(Cymbidium)中的一个种,具有较高的观赏价值和经济价值。‘宋梅’是中国春兰传统铭品,为梅瓣花的代表品种,被誉为春兰“四大天王”之首,“春兰之王”等,幽香馥郁,浓而不浊,深受广大兰友的喜爱。
镁原卟啉Ⅸ甲基转移酶(Magnesium protoporphyrin Ⅸ methyltransferase,ChlM)是叶绿素合成过程中的关键酶,也是重要的调控酶,其活性受到光照、叶绿体氧化还原状态和叶片中叶酸含量调控。此外,镁原卟啉Ⅸ甲基转移酶不仅影响PSⅠ、PSⅡ和Cytb6f 蛋白复合体内重要蛋白的积累,参与叶绿体到细胞核的反向信号转导、ABA 信号转导,还通过调控ChlH 表达影响镁螯合酶活性[1]。原卟啉Ⅸ甲基转移酶是单基因产物,定位在叶绿体被膜和类囊体膜上,其发挥作用时依赖于腺苷甲硫氨酸( Ado-Met) ,属于S-腺苷-L-甲硫氨酸依赖的甲基转移酶大家族[2-3]。拟南芥At4g25080是编码该甲基转移酶的基因,在烟草[4]、水稻[5]、豌豆[6]、衣藻[7]中均发现ChlM 序列同源物。本研究从‘宋梅’组培苗叶片中克隆了春兰ClChlM1基因,构建pART-ClChlM1植物表达载体,进而在烟草中瞬时表达鉴定了该基因的叶绿素生物合成功能,旨在为春兰叶绿素合成途径的揭示及春兰叶色变异提供理论依据。
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供试材料为春兰‘宋梅’组培苗,保存在四川省农业科学院园艺研究所组培室(见图1)。
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根据天根生化科技有限公司的植物总RNA提取试剂盒说明书,从叶片样品中提取总RNA,利用Nanodrop2000(Thermo Scientific,美国)对RNA的浓度和纯度进行检测,1.5%琼脂糖凝胶电泳检测RNA 完整性。
使用Aidlab公司反转录试剂盒(TUREscript 1st Strand cDNA Synthesis Kit)进行cDNA的合成,反应体系为40 μL:500 ng总RNA,8 μL 5×RT Reaction Mix,2 μL Oligo(dT)引物,2 μL TUREscript H- RTase/RI Mix,采用RNase Free dH2O补足至40 μL。反转录反应程序条件为:42 ℃ 60 min,65 ℃ 10 min。反应结束后,将得到的cDNA放置−20℃保存,用前稀释10倍使用。
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根据表1的引物,从‘宋梅’cDNA中克隆了ClChlM1基因。PCR扩增程序:96 ℃ 10 min;96℃ 30 s;60 ℃ 30 secs,72 ℃ 1 min,30个循环;72 ℃ 10 min。将扩增产物经1.5%琼脂糖凝胶分离后对目的片段进行切胶回收,连入pMT18-T载体并转化大肠杆菌DH10B过夜培养。次日挑选克隆经菌落PCR验证后送至上海美吉生物医药科技有限公司测序。
表 1 引物序列
Table 1. Primers used in the present study
基因名称
Name引物序列
Primer sequence用途
UsageClChlM1-F ATGGCTTCTTTCGCCACCGTTC 基因全长扩增
Gene amplificationClChlM1-R TCAGGCGGCCGGGACGAGGA ClChlM1-XhoI-F CCGCTCGAGATGGCTTCTTTCGCCACC 转基因载体构建 ClChlM1-SalI-R ACGCGTCGACTCAGGCGGCCGGGACG Transgenic vectors Construction Kan-F GATCTGGACGAAGAGCATCAG 转基因检测
Transgenic detectionKan-R CTCGTCAAGAAGGCGATAGAAG ClHemA-F CCAAGATGCTCTGGGTTGATAG qRT-PCR ClHemA-R ATGGGATACGGGCTTCAAATAC ClGsa-qF GTTGATGCCAGGAGGTGTAA qRT-PCR ClGsa-qR GCCCTTCACGGAGTCAATAA ClCHLI-qF CTGAACGTCGATGGATTGAGAG qRT-PCR ClCHLI-qR TATCCTCCGGAGTGACCTTATC ClCHLH-qF GGTATTTCTGCCCTTCCATCTAT qRT-PCR ClCHLH-qR GCAGCTCCACATCTCGTAAA ClLHCB-qF GCATTCGCTGAGTTGAAAGTG qRT-PCR ClLHCB-qR GGTCCCTTTCCTGTGACAATAG ClEF1-α-qF ATTGGTGGAATTGGTACTGTCC Reference gene/qRT-PCR ClEF1-α-qR CCGCAACATTCTTGACATTAAA -
选取铁皮石斛的DcChlM1、马蹄蝴蝶兰的PeChlM1、油棕的EgChlM1、深圳拟兰的AsChlM1、风铃木的HiChlM1、香蕉的MaChlM1、玉米的ZmChlM1、柑橘的CsChlM1、黄麻的CoChlM1,以及春兰的ClChlM1,采用MEGA 5.0构建系统进化树。
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设计酶切引物对ClChlM1基因进行扩增,将扩增后的片段经XhoI和SalI双酶切后连入pBI121-GFP载体,并对拟南芥原生质体进行转化。通过荧光显微镜(Olympus BX51)观察融合蛋白的定位。
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为了获得ClChlM1过量表达载体,将ClChlM1编码区序列插入pART-CAM,获得pART-ClChlM1载体[8]。通过农杆菌介导法将载体导入到本氏烟草中[9]。表1引物检测烟草的阳性转化率。
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按照Alawady and Grimm的方法检测ALA[4]。取烟草叶片,在含有40 mM乙酰丙酸(pH 6.9)的20 mM磷酸盐缓冲液(pH值7.5)中,置于光下培养4小时。取上清液,加入乙酰乙酸煮沸10分钟。加入等量的Ehrlich’s试剂,在553 nm下检测ALA。
检测烟草的叶绿素含量,用95%的乙醇和稀释的丙酮碾磨叶片。用紫外分光光度计检测665 nm, 649 nm 和 470 nm波长[10]。
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引物序列详见表1。ACTIN基因作为内参,通过2−△△Ct法对目的基因表达量进行计算。
Molecular Cloning and Functional Analysis of Magnesium Protoporphyrin Ⅸ Methyltransferase Gene in Tissue Culture Seedlings of Cymbidium goeringii
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摘要: 镁原卟啉IX甲基转移酶(ChlM)是叶绿素合成途径中的关键限速酶之一,在植株生长发育过程中发挥着重要作用。为了了解春兰中ChlM的功能,从春兰‘宋梅’组培苗中克隆了ClChlM1基因。该基因开放阅读框全长945 bp,编码314个氨基酸序列。序列比对显示ClChlM1含有Mg-por_mtran_C结构域,亚细胞定位结果显示该基因定位在叶绿体上。在烟草中过量表达ClChlM1,叶片叶绿素含量及ALA比野生型增加。另外,过量表达烟草叶片面积也大于野生型。在过量表达转基因株系中,编码谷氨酸酯-1-半醛2,1-氨基变位酶(Gsa),叶绿素a/b结合蛋白(LHCB),镁离子螯合酶CHLI和镁离子螯合酶CHLH的基因表达上调。
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关键词:
- 兰属春兰;
- 组培;
- 叶绿素;
- 镁原卟啉IX 甲基转移酶基因
Abstract: The magnesium protoporphyrin IX methyltransferase (ChlM) is one of the key rate-limiting enzymes in chlorophyll synthesis and development of chloroplasts, which plays an important role in plant growth and development. To better understand the role of ChlM in Cymbidium goeringii, ChlM gene was cloned from the tissue culture seedling of Cymbidium goeringii ‘Song Mei’. The full-length open reading frame of ClChlM1 was 945 bp and encoded 314 amino acids. The sequence alignment showed that ClChlM1 contained an Mg-por_mtran_C domain, and subcellular location analysis indicated ClChlM1 was located in chloroplast. Furthermore, the overexpression of ClChlM1 in tobacco leaves could significantly increase the content of chlorophyll and ALA compared with wild type tobacco leaves. In addition, the leaf area of overexpressed tobacco was larger than that of wild type. In the over-expressed transgenic lines, the gene expression of glutamate -1- semialdehyde 2,1- aminomutase (Gsa), chlorophyll a/b binding protein (LHCB), magnesium chelatase CHLI and magnesium chelatase CHLH were up-regulated. -
图 3 ClChlM1同源性比较及系统进化树分析
Fig. 3 Phylogenetic and alignment of ClChlM1 with homologous proteins from other plants
注: DcChlM1 (XP_020691545), PeChlM1 (XP_020570902), EgChlM1 (XP_010909956), AsChlM1 (PKA65777), HiChlM1 (PIN15818), MaChlM1 (XP_009418736), ZmChlM1 (KMZ70281), CsChlM1 (KDO51265), CoChlM1 (OMO52366). Note: DcChlM1 (XP_020691545), PeChlM1 (XP_020570902), EgChlM1 (XP_010909956), AsChlM1 (PKA65777), HiChlM1 (PIN15818), MaChlM1 (XP_009418736), ZmChlM1 (KMZ70281), CsChlM1 (KDO51265), CoChlM1 (OMO52366).
图 4 ClChlM1亚细胞定位。
不同的数字代表不同的荧光场。485 nm用于绿色荧光观察,587 nm用于荧光探针观察,DIC用于明亮视野观察,Merge代表485 nm, 587 nm 和DIC的重叠区域。叶绿体定位基因At5g19750作为标记。
Fig. 4 Subcellular location of ClChlM1 in Arabidopsis protoplast
Note: Different figures represent different fluorescence fields. 485 nm is used for green fluorescence observation, 587 nm is used for fluorescence probe observation and DIC is used for bright field observation. Merge represents the overlapping area of 485 nm, 587 nm and DIC. The Chloroplast localization gene At5g19750 is used as a marker.
图 5 ClChlM1烟草转基因
A, 共培养; B, 在分化培养基上形成的kanamyc抗性体细胞胚; C, 在分化培养基上形成的kanamyc抗性植株; D, 生根培养; E, PCR鉴定, 1, 2, 3为空载体转基因株系,命名为CK1, CK2, CK, 4, 5, 6为空载体转基因株系,命名为T1,T2, T3; F, 转基因株系表型
Fig. 5 Tobacco ClChlM1 transgenic
A, Co-culture; B, Kanamycin-resistant somatic embryos formed on differentiation medium; C, Kanamycin-resistant plantlet formed on differentiation medium; D, Rooting culture; E, PCR identification. 1, 2, 3 are empty vector transgnic line, named CK1, CK2, CK, and 4, 5, 6 are empty vector transgnic line, named T1,T2, T3; F, Phenotype of transgenic strain.
表 1 引物序列
Tab. 1 Primers used in the present study
基因名称
Name引物序列
Primer sequence用途
UsageClChlM1-F ATGGCTTCTTTCGCCACCGTTC 基因全长扩增
Gene amplificationClChlM1-R TCAGGCGGCCGGGACGAGGA ClChlM1-XhoI-F CCGCTCGAGATGGCTTCTTTCGCCACC 转基因载体构建 ClChlM1-SalI-R ACGCGTCGACTCAGGCGGCCGGGACG Transgenic vectors Construction Kan-F GATCTGGACGAAGAGCATCAG 转基因检测
Transgenic detectionKan-R CTCGTCAAGAAGGCGATAGAAG ClHemA-F CCAAGATGCTCTGGGTTGATAG qRT-PCR ClHemA-R ATGGGATACGGGCTTCAAATAC ClGsa-qF GTTGATGCCAGGAGGTGTAA qRT-PCR ClGsa-qR GCCCTTCACGGAGTCAATAA ClCHLI-qF CTGAACGTCGATGGATTGAGAG qRT-PCR ClCHLI-qR TATCCTCCGGAGTGACCTTATC ClCHLH-qF GGTATTTCTGCCCTTCCATCTAT qRT-PCR ClCHLH-qR GCAGCTCCACATCTCGTAAA ClLHCB-qF GCATTCGCTGAGTTGAAAGTG qRT-PCR ClLHCB-qR GGTCCCTTTCCTGTGACAATAG ClEF1-α-qF ATTGGTGGAATTGGTACTGTCC Reference gene/qRT-PCR ClEF1-α-qR CCGCAACATTCTTGACATTAAA -
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