基于基因芯片分析芜菁雌蕊退化突变体（tpa）与野生型的转录组差异
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摘要
近年来,雌蕊发育是植物发育理论研究的一个热点,国内外学者做了大量与雌蕊发育相关的理论和实验的探讨,应用很多新的技术和方法,取得了明显的研究进展,极大地丰富了相关领域的研究基础。但是,雌蕊发育的遗传调控机制仍有许多问题需要深入研究。以本实验室2002年从多代自交的芜菁021-03株系中发现的雌蕊退化突变体tpa (turnip pistil abortion)为材料,采用晶芯(?)29K拟南芥基因组寡核苷酸芯片和拟南芥基因组ATH1芯片等技术对芜菁雌蕊发育的转录组差异进行了研究,取得以下研究结果：
(1)通过田间调查以及显微观察发现tpa具有独特的突变表型。形态学观察结果表明,雌蕊退化突变体的萼片、花瓣、雄蕊正常发育,与可育植株比较无明显差异,但是突变体的部分雌蕊在发育早期开始萎缩,最后只能发育成细丝状的不育雌蕊,部分雌蕊甚至完全缺失。目前对tpa突变体及野生型的测交结果的统计分析结果显示,可育植株单株自交的分离比例有以下几种类型：0,1：4.3,1：5.3,1：11,1 : 15和1：28。不育植株交可育植株后代的分离比例有：0,1：1：1：1.3,1：1.4,1：1.5,1：2.3。不符合单基因隐性遗传规律,很有可能是双基因隐性遗传控制的。
(2)通过晶芯(?)29 K拟南芥全基因组寡核苷酸芯片分析tpa及其野生型的花蕾转录组差异,发现了一些可能与雌蕊退化突变相关的候选基因。tpa及其野生型植株的花蕾转录组中表达有较大差异(fold change绝对值大于2)的基因有94个,其中在tpa花蕾转录组中上调表达的基因有63个,在野生型植株的花蕾转录组中上调表达的基因有31个,没有在tpa或野生型植株花蕾转录组中特异表达的基因。我们选取了其中26个差异基因进行RT-PCR验证,结果显示,这些基因在不同的组织中具有不同的表达特性,在tpa与野生型植株中上述基因的表达差异并不是十分显著。有14个基因是在花蕾中特异表达的,其它12个基因在花蕾、茎、叶片中都有表达。
(3)利用tpa及其野生型植株的开放花制备的mRNA反转录成cDNA与拟南芥ATH1芯片进行杂交,筛选出在tpa及其野生型植株中表达有差异的基因,我们获得了152个在野生型(W1)和完全退化株(M3)转录组中差异表达的基因,61个在W1和部分退化株(I2)转录组中差异表达的基因,以及24个在I2和M3转录组中差异表达的基因,上述差异基因分别属于细胞壁合成与调控、防卫与胁迫反应、转录调控、蛋白质代谢以及普通新陈代谢等9个不同的功能类别。通过对41个基因的RT-PCR验证,获得了At2g42840、At1g57750、At5g20630、At2g03090、At3g08030、At5g08000、At2g28790、At5g63310和At2g24270等9个在tpa及野生型植株中具显著不同的时空表达特性的基因。目前,对芜菁雌蕊退化突变体的遗传特性分析,细胞学观察以及相关基因在芜菁中的表达分析正在继续之中。
Recently, research on gynoecium development has been one of the highlights in plant developmental biology for last decade, and there are a lot of progress made in the genetic pathways underlying the initiation and development of the gynoecium to date. However, more efforts are needed to unravel the puzzles of gynoecium development. Our laboratory obtained a tpa (turnip pistil abortion) mutant which was employed to explore the genetic mechanism of gynoecium development in Brassica crops. Some interesting results have been obtained as follow:
(1) After morphological investigation and microscope observation, we found the phenotpye of tpa is unique in the turnip. In this tpa mutant, sepal, petal and stamen are not affected. However, most of the gynoecium in the tpa plant began to shrink during the early development, finally the shape of pistil turn into filiform, moreover, lots of flowers even lack of pistil completely. Using the pollen of tpa plant cross with wild type plant, the tpa mutant character can be inherited successfully. The segregation ratios of the self cross for wild type plant were the following forms:0,1:4.3,1:1.3,1:11,1:15,1:28. And the segregation ratios of the offspring for tpa plant crossed with wild type plan were the following forms:0,1:1,1:1.3,1:1.4,1:1.5, 1:2.3. Our data suggests the tpa mutant is not controlled by single recessive gene,but most likely controlled by two recessive genes.
(2) Global analysis the genes expression difference during the gynoecium development in turnip. Through the Jinxin (?) 29K microarray (The Arabidopsis thaliana Genome Oligo Set Version3.0) analysis, we found some potential gene related to gynoecium development. According to the data came from microarray analysis, there were 94 differential genes,63 genes were up-regulated and 31 genes were down-regulated in the floral buds of tpa plants. We choosed 26 genes for RT-PCR analysis, the results show there were a few difference of these genes expression between the floral buds of tpa and wild type plants. Fourteen genes expressed specifically in the floral buds, and the other 12 genes all expressed among the floral buds, stems and leaves with-no remarkable difference.
(3) The Arabidopsis ATH1 genome array was employed to analyze the transcriptome differences between the tpa mutants and their wild type flowers. Data analysis results indicated there were differences in 152 genes between the wild type (W1) and the pistil aborted completely (M3), likewise,61 genes with transcriptional difference between the W1 and the pistil aborted partially (12), and 24 genes with transcriptional difference between the 12 and M3. All of the previous genes were classed in functional category as follow:cell wall biosynthesis and regulation, defence and stressing reaction, transcriptional regulation, protein metabolism and normal metabolism, respectively. Out of 41 genes,9 genes with significant temporal and spatial transcription difference:(At2g42840, At1g57750, At5g20630, At2g03090, At3g08030, At5g08000, At2g28790, At5g63310, At2g24270), have beeen obtained through the RT-PCR screening and confirmation.
Research of genetic characteristics and genes which involve in the gynoecium development in tpa mutant are still in progress.
(1)通过田间调查以及显微观察发现tpa具有独特的突变表型。形态学观察结果表明,雌蕊退化突变体的萼片、花瓣、雄蕊正常发育,与可育植株比较无明显差异,但是突变体的部分雌蕊在发育早期开始萎缩,最后只能发育成细丝状的不育雌蕊,部分雌蕊甚至完全缺失。目前对tpa突变体及野生型的测交结果的统计分析结果显示,可育植株单株自交的分离比例有以下几种类型：0,1：4.3,1：5.3,1：11,1 : 15和1：28。不育植株交可育植株后代的分离比例有：0,1：1：1：1.3,1：1.4,1：1.5,1：2.3。不符合单基因隐性遗传规律,很有可能是双基因隐性遗传控制的。
(2)通过晶芯(?)29 K拟南芥全基因组寡核苷酸芯片分析tpa及其野生型的花蕾转录组差异,发现了一些可能与雌蕊退化突变相关的候选基因。tpa及其野生型植株的花蕾转录组中表达有较大差异(fold change绝对值大于2)的基因有94个,其中在tpa花蕾转录组中上调表达的基因有63个,在野生型植株的花蕾转录组中上调表达的基因有31个,没有在tpa或野生型植株花蕾转录组中特异表达的基因。我们选取了其中26个差异基因进行RT-PCR验证,结果显示,这些基因在不同的组织中具有不同的表达特性,在tpa与野生型植株中上述基因的表达差异并不是十分显著。有14个基因是在花蕾中特异表达的,其它12个基因在花蕾、茎、叶片中都有表达。
(3)利用tpa及其野生型植株的开放花制备的mRNA反转录成cDNA与拟南芥ATH1芯片进行杂交,筛选出在tpa及其野生型植株中表达有差异的基因,我们获得了152个在野生型(W1)和完全退化株(M3)转录组中差异表达的基因,61个在W1和部分退化株(I2)转录组中差异表达的基因,以及24个在I2和M3转录组中差异表达的基因,上述差异基因分别属于细胞壁合成与调控、防卫与胁迫反应、转录调控、蛋白质代谢以及普通新陈代谢等9个不同的功能类别。通过对41个基因的RT-PCR验证,获得了At2g42840、At1g57750、At5g20630、At2g03090、At3g08030、At5g08000、At2g28790、At5g63310和At2g24270等9个在tpa及野生型植株中具显著不同的时空表达特性的基因。目前,对芜菁雌蕊退化突变体的遗传特性分析,细胞学观察以及相关基因在芜菁中的表达分析正在继续之中。
Recently, research on gynoecium development has been one of the highlights in plant developmental biology for last decade, and there are a lot of progress made in the genetic pathways underlying the initiation and development of the gynoecium to date. However, more efforts are needed to unravel the puzzles of gynoecium development. Our laboratory obtained a tpa (turnip pistil abortion) mutant which was employed to explore the genetic mechanism of gynoecium development in Brassica crops. Some interesting results have been obtained as follow:
(1) After morphological investigation and microscope observation, we found the phenotpye of tpa is unique in the turnip. In this tpa mutant, sepal, petal and stamen are not affected. However, most of the gynoecium in the tpa plant began to shrink during the early development, finally the shape of pistil turn into filiform, moreover, lots of flowers even lack of pistil completely. Using the pollen of tpa plant cross with wild type plant, the tpa mutant character can be inherited successfully. The segregation ratios of the self cross for wild type plant were the following forms:0,1:4.3,1:1.3,1:11,1:15,1:28. And the segregation ratios of the offspring for tpa plant crossed with wild type plan were the following forms:0,1:1,1:1.3,1:1.4,1:1.5, 1:2.3. Our data suggests the tpa mutant is not controlled by single recessive gene,but most likely controlled by two recessive genes.
(2) Global analysis the genes expression difference during the gynoecium development in turnip. Through the Jinxin (?) 29K microarray (The Arabidopsis thaliana Genome Oligo Set Version3.0) analysis, we found some potential gene related to gynoecium development. According to the data came from microarray analysis, there were 94 differential genes,63 genes were up-regulated and 31 genes were down-regulated in the floral buds of tpa plants. We choosed 26 genes for RT-PCR analysis, the results show there were a few difference of these genes expression between the floral buds of tpa and wild type plants. Fourteen genes expressed specifically in the floral buds, and the other 12 genes all expressed among the floral buds, stems and leaves with-no remarkable difference.
(3) The Arabidopsis ATH1 genome array was employed to analyze the transcriptome differences between the tpa mutants and their wild type flowers. Data analysis results indicated there were differences in 152 genes between the wild type (W1) and the pistil aborted completely (M3), likewise,61 genes with transcriptional difference between the W1 and the pistil aborted partially (12), and 24 genes with transcriptional difference between the 12 and M3. All of the previous genes were classed in functional category as follow:cell wall biosynthesis and regulation, defence and stressing reaction, transcriptional regulation, protein metabolism and normal metabolism, respectively. Out of 41 genes,9 genes with significant temporal and spatial transcription difference:(At2g42840, At1g57750, At5g20630, At2g03090, At3g08030, At5g08000, At2g28790, At5g63310, At2g24270), have beeen obtained through the RT-PCR screening and confirmation.
Research of genetic characteristics and genes which involve in the gynoecium development in tpa mutant are still in progress.
引文
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