棉花诱变种质Gh14-3-3L基因的Eco-TILLING分析
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摘要
【目的】为了验证前期建立的诱变体系和鉴定技术在棉花种质资源创制方面的效果。【方法】本实验利用Eco-TILLING技术和测序技术,对诱变获得的棉花材料纤维相关基因进行分析。【结果】与纤维相关的基因Gh14-3-3L在对照和诱变材料上获得了不同的SNP位点变异,在石引150材料上的扩增产物存在1个SNP位点差异,位于320 bp处;石引200材料的扩增产物存在2个SNP位点,分别位于320和380 bp处;石引250材料扩增产物与对照存在1个SNP位点,位于320 bp处,并且经过酶切和测序结果得到了验证。蛋白比对显示其中1个SNP位点的改变导致开放阅读框(ORF)变短,氨基酸数目减少,蛋白结构发生改变,其结构功能有待于进一步研究;连续3年纤维检测数据显示石引200材料与对照之间存在差异性,推断可能与SNP位点变异有关,研究结果将为深入研究SNP位点变异与绒长改变之间的关联性奠定基础。【结论】建立的诱变体系和鉴定技术能够用于棉花种质资源的创制,所得材料在表型性状和分子水平都发生了变化,为深入研究棉花种质资源的创制提供了新的方法和鉴定技术。
【Objective】The present study aimed to identify the effect of the mutagenesis system and verification technology established in the creation of cotton germplasm resources.【Method】 Eco-TILLING technology and sequencing were used to analyze the gene related to fiber length of cotton mutation materials in this study. 【Result】The result showed that the gene of Gh14-3-3 L had some SNP among standard and mutation materials. There were 1 SNP between ‘Shiyin 150' and CK, and the location was 320 bp; 2 SNP were found between ‘Shinyin 200' and CK, and the location was 320 and 380 bp; 1 SNP was found between ‘Shiyin 250' and CK, the location was 320 bp. These result was verified by digestion and sequencing technology. The protein ratio showed that the open reading frame(ORF) was shortened due to the change of one SNP site, and the number of acid was reduced. The structure of protein was changed and the function would be researched in further. The date of fiber length for three consecutive years showed that there was obvious difference between ‘Shiyin 200'and standard, and related to SNP difference. 【Conclusion】The result would lay a foundation for further study on the relationship between SNP site variation and pile length change in further study.
【Objective】The present study aimed to identify the effect of the mutagenesis system and verification technology established in the creation of cotton germplasm resources.【Method】 Eco-TILLING technology and sequencing were used to analyze the gene related to fiber length of cotton mutation materials in this study. 【Result】The result showed that the gene of Gh14-3-3 L had some SNP among standard and mutation materials. There were 1 SNP between ‘Shiyin 150' and CK, and the location was 320 bp; 2 SNP were found between ‘Shinyin 200' and CK, and the location was 320 and 380 bp; 1 SNP was found between ‘Shiyin 250' and CK, the location was 320 bp. These result was verified by digestion and sequencing technology. The protein ratio showed that the open reading frame(ORF) was shortened due to the change of one SNP site, and the number of acid was reduced. The structure of protein was changed and the function would be researched in further. The date of fiber length for three consecutive years showed that there was obvious difference between ‘Shiyin 200'and standard, and related to SNP difference. 【Conclusion】The result would lay a foundation for further study on the relationship between SNP site variation and pile length change in further study.
引文
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[14]方琴.利用TILLING 技术筛选油菜根毛突变体及根毛发生发育相关基因的分离[D].武汉:华中农业大学硕士学位论文,2009.
[15]汪念.甘蓝型油菜EMS 突变体库的构建及TILLING、EcoTILLING 技术的应用研究[D].武汉:华中农业大学博士学位论文,2009.
[16]Stephen P,Baker D,Girin T,et al.A rich TILLING resource for studying gene function in Brassica rapa[J].BMC Plant Biology,2010,10:62.
[17]Beatriz Martín,Mercedes Ramiro,José M Martínez-Zapateret al.A high-density collection of EMS-induced mutations for TILLING in lands bergerecta genetic background of Arabidopsis[J].BMC Plant Biology,2009(9):147.
[18]Caldwell D G,Nicloa Mc,Shaw Paul,et al.A structured mutation population for forward and reverse genetics in barley(Hordeum vnlgare L.)[J].The Plant Journal,2004,40:143-150.
[19]Liang Chen,Linzhou Huang,Donghong Min.Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)[J].PLOS ONE,2012,7(7):1-10.
[20]Fatima Dahmani-Mardas,Christelle Troadec,Adnane Boualem.Engineering melon plants with improved fruit shelf life using the TILLING approach [J].PLOS ONE,2010,12(5):1-10.
[21]Joseph E Knoll,M Laura Ramos,Yajuan Zeng.TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)[J].BMC Plant Biology,2011,11:81.
[22]Andrew H.Paterson.Genetic and Genomics of cotton[M].Springer-Verlag New York lnc.,2008.
[23]邓康胜,吕远大,陶涛,等.基于聚丙烯酰胺凝胶电泳的棉花EcoTILLING 技术体系建立[J].南京农业大学学报,2012,35(4):1-7.
[2]徐艳华.小麦EMS 诱变群体的构建及关键性状突变体的筛选[D].郑州:河南农业大学硕士学位论文,2010.
[3]Cristobal Uauy,Francine Paraiso,Pasqualina Colasuonno,et al.A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat [J].BMC Plant Biology,2009(9):115.
[4]Liu X L,Yang ZH,Li G.Biological effects of M1 generation by utilizing EMS mutagen on Maize pollen[J].Journal of Shenyang Agricultural University,2006,37(6):806-810.
[5]张景萍.EMS 诱变玉米花粉突变体生理生化特性研究[D].重庆:西南农业大学硕士学位论文,2004.
[6]殷冬梅,杨秋云,杨海棠,等.花生突变体的EMS 诱变及分子检测[J].中国农学通报,2009,25(5):53-56.
[7]朱保葛,路自县,耿玉轩,等.烷化剂EMS 诱发花生性状变异的效果及高产突变系的选育[J].中国农业科学,1999,30(6):87-89.
[8]孙洁.CEL 1的分离及水稻TILLING 突变库的构建[D].杭州:浙江大学硕士学位论文,2007.
[9]Till B J,Jennifer C,Thomas H T.Discovery of chemically induced mutations in rice by TILLING[J].BMC Plant Biol,2007(7):19.
[10]Wilma Sabetta,Vittorio Alba,Antonio Blanco,et al.Sun-TILL:a TILLING resource for gene function analysis in sunflower[J].Plant Methods,2011( 7):20.
[11]Jennifer L Cooper,Bradley J Till,Robert G Laport,et al.TILLING to detect induced mutations in soybean[J].BMC Plant Biology,2008(8):9.
[12]于秀普.EMS 诱发大豆突变可筛选高蛋白或高脂肪含量的种质资源[J].作物品种资源,1995(1):23-26.
[13]Mireia González,Meihong Xu,Cristina Esteras.Towards a TILLING platform for functional genomics in Piel de Sapo melons[J].BMC Research Notes,2011,4:289.
[14]方琴.利用TILLING 技术筛选油菜根毛突变体及根毛发生发育相关基因的分离[D].武汉:华中农业大学硕士学位论文,2009.
[15]汪念.甘蓝型油菜EMS 突变体库的构建及TILLING、EcoTILLING 技术的应用研究[D].武汉:华中农业大学博士学位论文,2009.
[16]Stephen P,Baker D,Girin T,et al.A rich TILLING resource for studying gene function in Brassica rapa[J].BMC Plant Biology,2010,10:62.
[17]Beatriz Martín,Mercedes Ramiro,José M Martínez-Zapateret al.A high-density collection of EMS-induced mutations for TILLING in lands bergerecta genetic background of Arabidopsis[J].BMC Plant Biology,2009(9):147.
[18]Caldwell D G,Nicloa Mc,Shaw Paul,et al.A structured mutation population for forward and reverse genetics in barley(Hordeum vnlgare L.)[J].The Plant Journal,2004,40:143-150.
[19]Liang Chen,Linzhou Huang,Donghong Min.Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)[J].PLOS ONE,2012,7(7):1-10.
[20]Fatima Dahmani-Mardas,Christelle Troadec,Adnane Boualem.Engineering melon plants with improved fruit shelf life using the TILLING approach [J].PLOS ONE,2010,12(5):1-10.
[21]Joseph E Knoll,M Laura Ramos,Yajuan Zeng.TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)[J].BMC Plant Biology,2011,11:81.
[22]Andrew H.Paterson.Genetic and Genomics of cotton[M].Springer-Verlag New York lnc.,2008.
[23]邓康胜,吕远大,陶涛,等.基于聚丙烯酰胺凝胶电泳的棉花EcoTILLING 技术体系建立[J].南京农业大学学报,2012,35(4):1-7.