草莓CHS基因分离、克隆和病毒诱导基因沉默（VIGS）载体构建
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摘要
查尔酮合成酶（chalcone synthase，CHS）基因是植物体内合成类黄酮物质的重要基因，在植物的生长、发育和抗逆境反应中起着至关重要的作用。本研究分别以拟南芥AtCHS（AT5G13930）及FaCHS（AY997297）序列于NCBI Fragariavesca WGS数据库做blast分析，搜索到2条草莓CHS同源序列分别为FvCHS1-A和FvCHS2-A。此外，在现有的草莓EST数据库（NCBI Fragaria EST，GDR octoploid strawberry ESTs，GDR diploid strawberry ESTs）对这两条基因进行blast分析，结果表明，FvCHS1在草莓果实有表达，并有一些对应的EST序列，而没有与FvCHS2对应的EST序列。
为了从八倍体草莓中分离出CHS基因以供VIGS研究，用软件Primer4.0进行如下特异引物设计：
FaCHS1-U：5’-TGCAAGACAAGACATGGTGGT-3’，
FaCHS1-L：5’-GGAACATCTTTGAGGAGGTGAA-3’；
FaCHS2-U：5’-TACGGATTGCCAAGGACATA-3’，
FaCHS2-L：5’-CAACAGTCTCCACAGTAAGTC-3’。接着利用RT-PCR分离这两个基因。结果从成熟草莓果实的cDNA克隆出FaCHS1特异的基因片段，长为540bp；从花和花芽的混合cDNA克隆出FaCHS2特异的基因片段，长为639bp。利用生物信息学的方法预测分析了草莓CHS基因的同源性和理化性质。
VIGS是研究基因功能的有效途径。它是将目的基因片段导入到病毒载体中，并用该病毒载体去侵染植物，由于植物自身免疫系统的原因，使得目的基因发生沉默。本研究通过反方向插入，将从久香草莓中获得的FaCHS1和FaCHS2基因片段分别嵌入到pTRV2病毒载体中。通过菌落PCR和质粒酶切鉴，已成功构建出含有FaCHS1和FaCHS2基因片段的病毒载体。并将构建好的病毒载体成功的转化到农杆菌GV3101中。总之，本研究将为接下来的通过农杆菌侵染研究草莓CHS基因功能奠定了良好的基础。
Chalcone synthase (CHS) is important in flavonoids compounds synthesis inplants, and play a crucial role in plant development and diverse resistant reactions. Inthis work, two woodland strawberry CHS homologs FvCHS1and FvCHS2were firstidentified via BLAST analysis againt NCBI Fragaria vesca WGS database usingarabidopsis AtCHS (AT5G13930) and FaCHS (AY997297) EST sequences. Further,these two genes were BLAST analyzied againt all three available databases forstrawberry ESTs (NCBI Fragaria EST，GDR octoploid strawberry ESTs，GDR diploidstrawberry ESTs). It was found that FvCHS1is expressed and has several ESTsequences, while no EST sequence is available for FvCHS2.
To isolate CHS genes from octoploid strawberry for VIGS study, specificprimersets were designed using Primer4.0software as following:
FaCHS1-U：5’-TGCAAGACAAGACATGGTGGT-3’;
FaCHS1-L：5’-GGAACATCTTTGAGGAGGTGAA-3’；
FaCHS2-U：5’-TACGGATTGCCAAGGACATA-3’;
FaCHS2-L：5’-CAACAGTCTCCACAGTAAGTC-3’.RT-PCR was then performed to isolate these two genes. The540bp long fragmentspecific to FaCHS1gene was cloned from ripe fruit cDNAs, while the639bp longfragment specific to FaCHS2gene was cloned from the mixed cDNAs from flowersand floral buds. Sequence homology and physical and chemical properties for thededuced strawberry CHS proteins were analyzied using corresponding bioinformaticsmethods.
Virus-induced gene silencing (VIGS) is an effective method for gene functionalstudy. The target gene fragment was first introduced into the virus carrier. When thevirus carrier was used to infect plant, target gene will be silenced similar like that theplant's own immune system being activated. In this work, we introduced FaCHS1andFaCHS2fragments into pTRV2virus carrier, respectively.The colony PCR andrestriction enzymes digestion uniformly confirmed that FaCHS1and FaCHS2genefragments were successfully inserted into the virus carrier with reverse orientation. Later, these virus carriers were successfully transformed into agrobacterium strainGV3101. In sum, this work laid a good foundation for functional study of strawberryCHS genes via agrobacterium-mediated infection experiments in the near future.
为了从八倍体草莓中分离出CHS基因以供VIGS研究，用软件Primer4.0进行如下特异引物设计：
FaCHS1-U：5’-TGCAAGACAAGACATGGTGGT-3’，
FaCHS1-L：5’-GGAACATCTTTGAGGAGGTGAA-3’；
FaCHS2-U：5’-TACGGATTGCCAAGGACATA-3’，
FaCHS2-L：5’-CAACAGTCTCCACAGTAAGTC-3’。接着利用RT-PCR分离这两个基因。结果从成熟草莓果实的cDNA克隆出FaCHS1特异的基因片段，长为540bp；从花和花芽的混合cDNA克隆出FaCHS2特异的基因片段，长为639bp。利用生物信息学的方法预测分析了草莓CHS基因的同源性和理化性质。
VIGS是研究基因功能的有效途径。它是将目的基因片段导入到病毒载体中，并用该病毒载体去侵染植物，由于植物自身免疫系统的原因，使得目的基因发生沉默。本研究通过反方向插入，将从久香草莓中获得的FaCHS1和FaCHS2基因片段分别嵌入到pTRV2病毒载体中。通过菌落PCR和质粒酶切鉴，已成功构建出含有FaCHS1和FaCHS2基因片段的病毒载体。并将构建好的病毒载体成功的转化到农杆菌GV3101中。总之，本研究将为接下来的通过农杆菌侵染研究草莓CHS基因功能奠定了良好的基础。
Chalcone synthase (CHS) is important in flavonoids compounds synthesis inplants, and play a crucial role in plant development and diverse resistant reactions. Inthis work, two woodland strawberry CHS homologs FvCHS1and FvCHS2were firstidentified via BLAST analysis againt NCBI Fragaria vesca WGS database usingarabidopsis AtCHS (AT5G13930) and FaCHS (AY997297) EST sequences. Further,these two genes were BLAST analyzied againt all three available databases forstrawberry ESTs (NCBI Fragaria EST，GDR octoploid strawberry ESTs，GDR diploidstrawberry ESTs). It was found that FvCHS1is expressed and has several ESTsequences, while no EST sequence is available for FvCHS2.
To isolate CHS genes from octoploid strawberry for VIGS study, specificprimersets were designed using Primer4.0software as following:
FaCHS1-U：5’-TGCAAGACAAGACATGGTGGT-3’;
FaCHS1-L：5’-GGAACATCTTTGAGGAGGTGAA-3’；
FaCHS2-U：5’-TACGGATTGCCAAGGACATA-3’;
FaCHS2-L：5’-CAACAGTCTCCACAGTAAGTC-3’.RT-PCR was then performed to isolate these two genes. The540bp long fragmentspecific to FaCHS1gene was cloned from ripe fruit cDNAs, while the639bp longfragment specific to FaCHS2gene was cloned from the mixed cDNAs from flowersand floral buds. Sequence homology and physical and chemical properties for thededuced strawberry CHS proteins were analyzied using corresponding bioinformaticsmethods.
Virus-induced gene silencing (VIGS) is an effective method for gene functionalstudy. The target gene fragment was first introduced into the virus carrier. When thevirus carrier was used to infect plant, target gene will be silenced similar like that theplant's own immune system being activated. In this work, we introduced FaCHS1andFaCHS2fragments into pTRV2virus carrier, respectively.The colony PCR andrestriction enzymes digestion uniformly confirmed that FaCHS1and FaCHS2genefragments were successfully inserted into the virus carrier with reverse orientation. Later, these virus carriers were successfully transformed into agrobacterium strainGV3101. In sum, this work laid a good foundation for functional study of strawberryCHS genes via agrobacterium-mediated infection experiments in the near future.
引文
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[3]李莉,杨雷,杨莉,等.草莓果实生长发育及主要营养成分变化规律研究[J].江西农业报,2006,18(20):67-70．
[4]Maas J.L, Galletta G.J, Ellagic Acid.Anticareinogen in Fruits[J]. Espeeiauyt in Straw--berry.Hortseience.1991,26(1):10.
[5]罗学冰,贺良明.草莓的营养价值与保健功能[J].中国食物与营养,2011,17(4):74-76.
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[7]张玉娟.草莓高产栽培技术[J].现代园艺,2008,10:68-70.
[8]佟盛芳.草莓关键栽培技术[J].北方园艺,2007,9:51-52.
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[10]Peter M Waterhouse, Christopher A Helliwell. Exploring genomes by RNA-induced genesilencing[J]. Nature Reviews Genetics,2003,4:29-38.
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[14] Liu Y, Schiff M, Dinesh-Kumar S P. Involvement of MEK1MAPKK, NTF6MAPK,WRKY/MYB transcription factors. Col1and CRT1in N-mediated resistance to tobaccomosaicvirus[J]. Plant J,2004,38:800-809.
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[16] Burton R A, Gibeaut D M, Baeie A, et a1.Virus-induced silencing of a plant cellulosesynthase gene[J]. Plant Cel1,2000,12:69-706.
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