甘蓝型油菜花序长发夹lhRNAi文库的构建
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摘要
异源四倍体油菜含有大量多拷贝基因及冗余基因,难以通过T-DNA插入、物理及化学诱变等常规突变体库创制方法发掘功能基因。为开发适应于油菜简单高效的突变体库创制方法,本研究通过滚环复制方法构建了甘蓝型油菜的花序lhRNAi文库,并对这种新型的干扰文库进行质量评估;然后将该花序lhRNAi文库对甘蓝型油菜进行遗传转化,获得763株T0植株,从中鉴定分离出74株具有可见表型变异的花发育相关突变体,包括花瓣减少、形状异常,柱头卷曲,雄蕊退化萎缩,雄性不育、死蕾或花蕾闭合等。说明通过滚环复制介导的lhRNAi文库的构建方法可以成功应用到油菜中,这将为油菜花序发育相关基因的功能研究提供重要的研究平台。
Allopolyploid Brassica napus contains a large number of homologous and redundant genes.It is difficulty to identify novel genes related to important agronomic traits by traditional T-DNA insertional,physical and chemical mutagenesis methods.In this study,a genome-wide inflorescence lhRNAi library of B.napus was constructed by the rolling circle amplification(RCA)-mediated lhRNAi construction technology.Subsequently,the inflorescence lhRNAi library was transformed into B.napus to produce 763 T0 transgenic plants.A total of 74 transgenic lines exhibited visible mutant phenotypes,including petal reduction in the number,stamen degeneration shrinkage,stigma distortion and exposure,dead or closed buds and male sterility.These results demonstrated the construction method of lhRNAi library which were successfully applied to rapeseed and provided an important research platform for gene functions related to rape flower inflorescence development.
Allopolyploid Brassica napus contains a large number of homologous and redundant genes.It is difficulty to identify novel genes related to important agronomic traits by traditional T-DNA insertional,physical and chemical mutagenesis methods.In this study,a genome-wide inflorescence lhRNAi library of B.napus was constructed by the rolling circle amplification(RCA)-mediated lhRNAi construction technology.Subsequently,the inflorescence lhRNAi library was transformed into B.napus to produce 763 T0 transgenic plants.A total of 74 transgenic lines exhibited visible mutant phenotypes,including petal reduction in the number,stamen degeneration shrinkage,stigma distortion and exposure,dead or closed buds and male sterility.These results demonstrated the construction method of lhRNAi library which were successfully applied to rapeseed and provided an important research platform for gene functions related to rape flower inflorescence development.
引文
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[2] Wang X,Wang H,Wang J,et al. The genome of the mesopolyploid crop species Brassica rapa[J]. Nat Genet,2011,43(10):1035-1039.
[3] Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[J].Nature,2000,408(6814):796-815.
[4] Liu S,Liu Y,Yang X,et al. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes[J]. Nat Commun,2014,5:3930.
[5] Bolle C,Schneider A,Leister D. Perspectives on systematic analyses of gene function in Arabidopsis thaliana:new tools,topics and trends[J]. Curr Genomics,2011,12(1):1-14.
[6] Chang Y,Long T,Wu C. Effort and contribution of TDNA insertion mutant library for rice functional genomics research in China:review and perspective[J]. J Plant Ecology,2012,54(12):953-966.
[7] Wang N,Long T,Yao W,et al. Mutant resources for the functional analysis of the rice genome[J]. Mol Plant,2013,6(3):596-604.
[8] Kolesnik T,Szeverenyi I,Bachmann D,et al. Establishing an efficient Ac/Ds tagging system in rice:largescale analysis of Ds flanking sequences[J]. Plant J,2004,37(2):301-314.
[9] Piffanelli P,Droc G,Mieulet D,et al. Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library[J]. Plant Mol Biol,2007,65(5):587-601.
[10] Till B J,Cooper J,Tai T H,et al. Discovery of chemically induced mutations in rice by TILLING[J]. BMC Plant Biol,2007,7:19.
[11] Ahn J H,Kim J,Yoo S J,et al. Isolation of 151 mutants that have developmental defects from T-DNA tagging[J]. Plant Cell Physiol,2007,48(1):169-178.
[12] Wang M B,Masuta C,Smith N A,et al. RNA silencing and plant viral diseases[J]. Mol Plant Microbe Interac,2012,25(10):1275-1285.
[13] Axtell M J. Classification and comparison of small RNAs from plants[J]. Annu Rev Plant Biol,2013,64:137-159.
[14] Jones-Rhoades M W,Bartel D P,Bartel B. MicroRNAS and their regulatory roles in plants[J]. Annu Rev Plant Biol,2006,57:19-53.
[15] Chen S,Hofius D,Sonnewald U,et al. Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA[J].Plant J,2003,36(5):731-740.
[16] Guo H S,Fei J F,Xie Q,et al. A chemical-regulated inducible RNAi system in plants[J]. Plant J,2003,34(3):383-392.
[17] Mohr S E,Perrimon N. RNAi screening:new approaches,understandings,and organisms[J]. Wiley Interdiscip Rev RNA,2012,3(2):145-158.
[18] Shirane D,Sugao K,Namiki S,et al. Enzymatic production of RNAi libraries from c DNAs[J]. Nat Genet,2004,36(2):190-196.
[19] Mao Y B,Cai W J,Wang J W,et al. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol[J]. Nat Biotechnol,2007,25(11):1307-1313.
[20] Wang M B,Waterhouse P M. Application of gene silencing in plants[J]. Curr Opin Plant Biol,2002,5(2):146-150.
[21] Smith N A,Singh S P,Wang M B,et al. Total silencing by intron-spliced hairpin RNAs[J]. Nature,2000,407(6802):319-320.
[22] Wang L,Zheng J,Luo Y,et al. Construction of a genomewide RNAi mutant library in rice[J]. Plant Biotechnol J,2013,11(8):997-1005.
[23] Wang L,Luo Y Z,Zhang L,et al. Rolling circle amplification-mediated hairpin RNA(RMHR)library construction in plants[J]. Nucleic Acids Res,2008,36(22):149.
[24] Zhang F,Sun Y,Pei W,et al. Involvement of Os Pht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice[J]. Plant J,2015,82(4):556-569.
[25] Chalhoub B,Denoeud F,Liu S,et al. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome[J]. Science,2014,345(6199):950-953.
[26] De Block M,De Brouwer D,Tenning P. Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants[J]. Plant Physiol,1989,91(2):694-701.
[27] Barnes W M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates[J]. Proc Natl Acad Sci U S A,1994,91(6):2216-2220.
[28] Zeng X,Yan X,Yuan R,et al. Identification and analysis of MS5d:a gene that affects double-strand break(DSB)repair during meiosis I in Brassica napus microsporocytes[J]. Front Plant Sci,2016,7:1966.
[29] Austin R S,Vidaurre D,Stamatiou G,et al. Nextgeneration mapping of Arabidopsis genes[J]. Plant J,2011,67(4):715-725.
[30] Hirochika H,Guiderdoni E,An G,et al. Rice mutant resources for gene discovery[J]. Plant Mol Biol,2004,54(3):325-334.
[31] Lizardi P M,Huang X,Zhu Z,et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification[J]. Nat Genet,1998,19(3):225-232.
[32] Dean F B,Nelson J R,Giesler T L,et al. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification[J]. Genome Res,2001,11(6):1095-1099.
[33] Wang L,Fan Y L. Rolling circle amplification-mediated hairpin RNA(RMHR)library construction in plants[J]. Nucl Acid Res,2008,36(22):e149.
[34] Wesley S V,Helliwell C A,Smith N A,et al. Construct design for efficient,effective and high-throughput gene silencing in plants[J]. Plant J,2001,27(6):581-590.
[35] Frser A G,Kamath R S,Zipperlen P,et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference[J]. Nature,2000,408(6810):325-330.
[36] Kamath R S,Fraser A G,Dong Y,et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi[J]. Nature,2003,421(6920):231-237.