杨树不定根发育的基因表达调控研究
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摘要
生根性状在杨树遗传改良中占有重要地位,研究杨树硬枝插穗不定根发育的分子机理不仅有理论意义,而且有潜在应用价值。插穗不定根发育是一个复杂的生物学过程,本研究以南林895杨(Populus×euramericana cv.‘Nanlin895’)硬枝插穗为材料,利用杨树全基因组芯片剖析插穗不定根发育的分子生物学事件,挖掘和鉴定不定根发育相关基因及其功能。同时,运用“遗传基因组学”策略,进行杨树不定根发育的eQTLs分析,探索基因表达调控与表型之间的相关关系。本论文主要结果如下:
利用15张杨树全基因组芯片检测了杨树硬枝插穗生根过程中5个发育阶段的基因表达变化,基于SAM软件中的Mulclass算法共筛选到14630个基因在不定根发育过程中显著差异表达。
运用实时定量RT-PCR技术对芯片检测结果进行了验证,33个验证基因在两种方法中的表达结果具有极高的吻合度,表明了杨树不定根基因组芯片实验及其数据分析具有较高的准确性和可靠性。
整合多个公共数据库中的杨树分子生物学资源对筛选到的差异表达基因进行相关的生物学功能注释,并列举和分析了部分可能对不定根发育研究有重要意义的基因,这为杨树不定根发育相关基因的克隆提供依据,也为进一步开展插穗不定根分子发育生物学研究奠定了基础。
基于杨树全基因组表达谱芯片分析,分离克隆了10个杨树不定根发育相关基因的全长cDNA序列,以及1个Aux/IAA基因启动子序列,并对这些基因或启动子的结构及其进化关系进行了生物信息学分析。
构建了PeAux/IAA1、PeAux/IAA2、PeAux/IAA3、PeAux/IAA4、PeABP1、PeRHD3_1、PeRHD3_2、PeRHD3_3等8个基因的过量表达载体,以及1个启动子的GUS表达载体,采用农杆菌介导法进行表达载体在拟南芥(Arabidopsis thaliana)、南林895杨和欧洲杂种山杨T89(Populus tremula×Populus tremuloides Michx. clone T89)等受体材料中的遗传转化,并对获得的转基因植株进行了初步检测和表型观察。
在已完成的杨树插穗生根性状QTLs定位图谱基础上,结合不定根发育的全基因组芯片检测数据,进行了杨树插穗不定根发育的eQTLs分析,探讨插穗生根性状相关QTLs的分子生物学基础。
Rooting ability was one of the target traits in Populus genetic improvement. Therefore, it has important significance in both theory and practice to research on molecular mechanism of adventitious root development for Populus. As adventitious root development in cuttings is a highly complex biological progress, in this study, Affymetrix poplar whole-genome array was used to elucidate molecular biological events of adventitious root development of hardwood cuttings from a hybrid Populus clone of Populus×euramericana cv.’Nanlin895’, and some related genes were cloned and identified. In addition, expression QTLs analysis based on genetical genomics explored the relationship between gene expression regulation and rooting traits of Populus hardwood cuttings. The main results were as follows:
Fifteen GeneChip? poplar whole-genome arrays were used to monitor gene expression profiles during adventitious development stages of hardwood cuttings, and then 14630 significant differently expressed genes were identified at five different developmental stages according to Mulclass SAM comparison.
To confirm gene expression data of poplar arrays, thirty-three of 14630 differential expressed genes were verified by quantitative real-time RT-PCR. The results suggested that the poplar array experimental method for adventitious root development was feasible and credible.
Integrating molecular biology resources to annotate poplar genome arrays in various biological contexts, furthermore, parts of these differential expressed genes were enumerated. Undoubtedly, the outcome would contribute new ideas on the study of molecular mechanism for adventitious root development in Populus hardwood cuttings.
Based on the results above, ten full-length cDNA of genes possibly related to adventitious root development were cloned from emerging roots of cuttings, and the corresponding promoter of PeAux/IAA2 gene was obtained. Moreover, sequence analysis indicted their potential biological function in rooting of cuttings.
Plant expression vectors for eight genes of PeAux/IAA1、PeAux/IAA2、PeAux/IAA3、PeAux/IAA4、PeABP1、PeRHD3_1、PeRHD3_2、PeRHD3_3 and one promoter PeAux/IAA2 were constructed and used for Agrobacterium-mediate transformation of Arabidopsis thaliana、Populus×euramericana cv-‘Nanlin895 and Populus tremula×Populus tremuloides Michx-clone T89). Some transgenic plants were obtained and further phenotypic identified.
Finally, the molecular mechanism on adventitious root development of Populus hardwood cuttings was outlined base on the results of functional mapping and eQTLs analysis of these associated QTLs.
利用15张杨树全基因组芯片检测了杨树硬枝插穗生根过程中5个发育阶段的基因表达变化,基于SAM软件中的Mulclass算法共筛选到14630个基因在不定根发育过程中显著差异表达。
运用实时定量RT-PCR技术对芯片检测结果进行了验证,33个验证基因在两种方法中的表达结果具有极高的吻合度,表明了杨树不定根基因组芯片实验及其数据分析具有较高的准确性和可靠性。
整合多个公共数据库中的杨树分子生物学资源对筛选到的差异表达基因进行相关的生物学功能注释,并列举和分析了部分可能对不定根发育研究有重要意义的基因,这为杨树不定根发育相关基因的克隆提供依据,也为进一步开展插穗不定根分子发育生物学研究奠定了基础。
基于杨树全基因组表达谱芯片分析,分离克隆了10个杨树不定根发育相关基因的全长cDNA序列,以及1个Aux/IAA基因启动子序列,并对这些基因或启动子的结构及其进化关系进行了生物信息学分析。
构建了PeAux/IAA1、PeAux/IAA2、PeAux/IAA3、PeAux/IAA4、PeABP1、PeRHD3_1、PeRHD3_2、PeRHD3_3等8个基因的过量表达载体,以及1个启动子的GUS表达载体,采用农杆菌介导法进行表达载体在拟南芥(Arabidopsis thaliana)、南林895杨和欧洲杂种山杨T89(Populus tremula×Populus tremuloides Michx. clone T89)等受体材料中的遗传转化,并对获得的转基因植株进行了初步检测和表型观察。
在已完成的杨树插穗生根性状QTLs定位图谱基础上,结合不定根发育的全基因组芯片检测数据,进行了杨树插穗不定根发育的eQTLs分析,探讨插穗生根性状相关QTLs的分子生物学基础。
Rooting ability was one of the target traits in Populus genetic improvement. Therefore, it has important significance in both theory and practice to research on molecular mechanism of adventitious root development for Populus. As adventitious root development in cuttings is a highly complex biological progress, in this study, Affymetrix poplar whole-genome array was used to elucidate molecular biological events of adventitious root development of hardwood cuttings from a hybrid Populus clone of Populus×euramericana cv.’Nanlin895’, and some related genes were cloned and identified. In addition, expression QTLs analysis based on genetical genomics explored the relationship between gene expression regulation and rooting traits of Populus hardwood cuttings. The main results were as follows:
Fifteen GeneChip? poplar whole-genome arrays were used to monitor gene expression profiles during adventitious development stages of hardwood cuttings, and then 14630 significant differently expressed genes were identified at five different developmental stages according to Mulclass SAM comparison.
To confirm gene expression data of poplar arrays, thirty-three of 14630 differential expressed genes were verified by quantitative real-time RT-PCR. The results suggested that the poplar array experimental method for adventitious root development was feasible and credible.
Integrating molecular biology resources to annotate poplar genome arrays in various biological contexts, furthermore, parts of these differential expressed genes were enumerated. Undoubtedly, the outcome would contribute new ideas on the study of molecular mechanism for adventitious root development in Populus hardwood cuttings.
Based on the results above, ten full-length cDNA of genes possibly related to adventitious root development were cloned from emerging roots of cuttings, and the corresponding promoter of PeAux/IAA2 gene was obtained. Moreover, sequence analysis indicted their potential biological function in rooting of cuttings.
Plant expression vectors for eight genes of PeAux/IAA1、PeAux/IAA2、PeAux/IAA3、PeAux/IAA4、PeABP1、PeRHD3_1、PeRHD3_2、PeRHD3_3 and one promoter PeAux/IAA2 were constructed and used for Agrobacterium-mediate transformation of Arabidopsis thaliana、Populus×euramericana cv-‘Nanlin895 and Populus tremula×Populus tremuloides Michx-clone T89). Some transgenic plants were obtained and further phenotypic identified.
Finally, the molecular mechanism on adventitious root development of Populus hardwood cuttings was outlined base on the results of functional mapping and eQTLs analysis of these associated QTLs.
引文
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