特定基因甲基化研究方法建立及LEAFY基因甲基化与早实枳阶段发育关系研究
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摘要
果树大多为多年生木本植物,从种子播种至首次开花结实要经历漫长的童期。童期的最基本特征是实生苗不具备开花能力。多年生木本植物由于个体体积大、童期长、遗传背景复杂等特点,极大地阻碍了木本植物,特别是一些以收获果实、花和种子为主的果树和经济林木的遗传学研究、育种进程和新品种推广。因此,研究木本植物成花分子机理、提早和调控果树成花成为果树学研究中的一个重要课题。早实枳为普通枳的一个实生变异,童期仅1-2年,与普通枳相比大大缩短,为木本植物童期发育研究提供了极好的材料。
果树成花研究,早期主要集中于生理生化基础,环境因子和栽培技术措施对实生苗成花的影响,对于果树开花的分子机制及其遗传控制的研究较少,在果树成花的表观遗传学研究方面也鲜有报道。DNA的甲基化是真核生物的表观遗传调控的重要途径。建立植物基因甲基化研究方法、开展相关基因在阶段发育进程中甲基化与去甲基化的变化与发育进程的关系研究,有助于我们进一步揭示植物发育表观调控的机制,为进一步实现缩短童期和花期调控打下基础。
本研究通过不同浓度的5-AzaC对早实枳种子进行去甲基化处理,观察植株生长状况,分析LFY、TFL等成花相关基因表达量变化,采用生物信息学方法对这些成花相关基因进行了甲基化区域扫描,针对有CpG岛的LFY基因,以亚硫氢酸钠修饰测序的方法对不同发育时期早实枳幼叶中LFY基因甲基化状态进行了比较。主要结果如下:
1.通过0、250、500、1000μM浓度的5-AzaC对培养皿中对早实枳种子连续处理15天后,观察发现随着5-AzaC浓度的加大,种子的生长发育减缓,根的发育显著被抑制。处理15天后的早实枳种子实生苗转入营养土中生长20天以后观察,发现250μM 5-AzaC处理的种子的根和茎的发育均有所减缓,部分叶的发育出现畸形;而500μM 5-AzaC处理后种子的根,茎,叶的生长都出现明显的抑制,并表现畸形;1000μM 5-AzaC处理的种子和20天前相比较几乎没有生长,生长被完全抑制。
2.从5-AzaC处理的早实枳叶片中提取RNA,用Real-time PCR方法分析了5个成花相关基因的表达水平,发现除FT相对表达量是随着5-AzaC处理浓度的增加而逐渐增加外,TFL、LFY、AP1、FLC四个基因的相对表达量都在250μM浓度时达到最大,处理浓度继续升高,基因的相对表达量下降。表明去甲基化处理对基因的转录有影响,大多数基因在低浓度去甲基化处理下,转录水平提高,而高浓度去甲基化会导致基因转录水平下降。
3.使用Methyl Primer Express和EMBOSS CpG Plot Tool软件,依照新旧两种CpG岛的定义,对早实枳中分离得到的5个成花相关基因TFL、LFY、AP1、FLC、FT的基因及启动子序列进行甲基化相关的生物信息学分析,然而仅预测到LFY序列中存在CpG岛。
4.借鉴医学研究中的亚硫氢酸钠修饰测序方法(Bisulfite Sequencing PCR),通过选择设计引物、优化反应条件,并采用巢式PCR方法,建立了柑橘类植物特定基因甲基化研究方法,并成功地应用于LFY基因的甲基化分析。
5.采用改良的亚硫氢酸钠修饰测序方法(Bisulfite Sequencing PCR),对早实枳不同发育阶段LFY基因CpG岛区域和非CpG岛区域的甲基化状态进行了比较。发现LFY 5'-UTR非CpG岛区域的甲基化水平很低,且在不同发育阶段之间没有明显差别;在CpG岛区域,童期时存在一定程度的甲基化并且特定的位点分布规律,在成年期部分位点发生了去甲基化,表明去甲基化参与了LFY在发育进程中的表达调控。
本研究对木本植物特定基因在不同发育进程中甲基化变化进行了分析,研究结果对研究木本植物发育调控分子机理可能有一定的参考意义。
Most fruit trees are woody perennial plants which have a long period of juvenile phase before flowering. A certain characteristic of juvenile phase is the seeding does not have a capability of flowering. Owing to the characteristics such as large individual, long period of juvenile phase and complex genetic backgrounds heavily impede genetic researches and breeding process in woody perennial plants, especially in fruit trees and economic forestry. Consequently, it is an important research area to study molecular mechanism in flowering and regulate the time to flowering in fruit trees. Precocious trifoliate orange is a mutant of trifoliate orange, which flowering time largely shorten to only one or two years. Therefore, precocious trifoliate orange is a suitable material for the molecular and genetic research on juvenile phase of woody perennial plants.
Most researches on the mechanism of fruit tree flowering mainly focus on physiological, environmental factors or cultivation techniques at the initial stages, however, the molecular and genetic mechanisms are still not understand clearly, even few researches reported the epigenetic mechanisms of fruit tree flowering. DNA methylation is an important aspect of epigenetic regulation in eukaryotes. Establish a protocol for analyzing DNA methylation status in plant and study on the relationship between related genes methylation stauts and developmental phase are both better for us to explore the epigenetic mechanism in plant and lies a foundation for shortening juvenile phase.
According to the phenotypic characteristics of precocious trifoliate orange mutant seedlings Treated by different 5-AzaC concentrations, we analyzed LFY, TEL etc. related flowering genes' relative expression levels in precocious trifoliate orange which treated by different 5-AzaC concentration. Then, we employed bioimformatic soft wares to analyze these genes in order to find whether these genes contain a CpG island. Fortunately, there is a CpG island found in LFY gene sequence. Therefore, we analyses cytosine methylation status in LFY CpG island DNA sequence from precocious trifoliate orange mutant in juvenile and adult phase by using bisulfate sequencing PCR method. The main results are as follows:
1. Precocious trifoliate orange seeds continuous treated by 0, 250, 500, 1000μM 5-AzaC for 15 days in the dark. The results showed that the seeds developed slower with increasing 5-AzaC concentrations, especially for the root development. Then the seeds transplanted in the soil after 15 days treatment. After the seedlings grew in the soil for 20 days in the describe condition, the results showed that the root of seedlings treated by 250μM 5-AzaC grew slowly compared to the control, and the seedlings showed several aberrant leaves; the root and stem development of seedlings were obvious inhibited by :500μM 5-AzaC treatment, and most of leaves exhibit aberrant phenotype; the growth of 1000μM 5-AzaC treated seeding nearly standstill compared with 20 days ago.
2. Extracted RNA from the leaves of different 5-AzaC concentrations treatment, and then analyzed five flowering genes' relative expression levels by using Real-time PCR. The results showed that FT relative expression level increased with the 5-AzaC concentrations. While, TFL, LFY, API, FLC showed highest relative expression levels in 250μM treatment, and these genes relative expression levels sharply decreased with higher concentrations. This means that demethylation treatment influences these genes expression, and most genes show high expression level in low demethylation treatment. However, the expression level would be decreased with higher concentration.
3. According to Methyl Primer Express and EMBOSS CpG Plot Tool soft wares analysis, we adopted both the old and new CpG island criteria to search and analyze flowering genes TFL, LFY, API, FLC, FT that isolated from precocious trifoliate orange. The results showed that only LFY sense chain DNA sequence contained a CpG island.
4. According to the references which successful analyzed gene sequence methylation status in mammalian, we optimized the modification protocol and adopted nested PCR by selecting proper primers to establish the bisulfite sequencing PCR protocol for analyzing DNA sequence methylation in citrus. This protocol has been successfully used for analyzing IFF DNA sequence methylation status.
5. To compare the methylation status of LFY CpG region with 5'-UTR at both juvenile and adult phase of precocious trifoliate orange by using bisulfite sequencing PCR, we found that LFY 5'-UTR which not in CpG island region showed unmethylation and no differences in different developmental phase. While, there was a low degree methylation status in CpG island region at juvenile phase and the lower degree methylation happened in the same region at adult phase, which meant demethylation might be regulate LFY gene expression from juvenile transition to adult phase.
The work is the first time to study on a certain gene's methylation status in the different developmental stages in woody perennial plants; the results might provide some evidence for further study.
果树成花研究,早期主要集中于生理生化基础,环境因子和栽培技术措施对实生苗成花的影响,对于果树开花的分子机制及其遗传控制的研究较少,在果树成花的表观遗传学研究方面也鲜有报道。DNA的甲基化是真核生物的表观遗传调控的重要途径。建立植物基因甲基化研究方法、开展相关基因在阶段发育进程中甲基化与去甲基化的变化与发育进程的关系研究,有助于我们进一步揭示植物发育表观调控的机制,为进一步实现缩短童期和花期调控打下基础。
本研究通过不同浓度的5-AzaC对早实枳种子进行去甲基化处理,观察植株生长状况,分析LFY、TFL等成花相关基因表达量变化,采用生物信息学方法对这些成花相关基因进行了甲基化区域扫描,针对有CpG岛的LFY基因,以亚硫氢酸钠修饰测序的方法对不同发育时期早实枳幼叶中LFY基因甲基化状态进行了比较。主要结果如下:
1.通过0、250、500、1000μM浓度的5-AzaC对培养皿中对早实枳种子连续处理15天后,观察发现随着5-AzaC浓度的加大,种子的生长发育减缓,根的发育显著被抑制。处理15天后的早实枳种子实生苗转入营养土中生长20天以后观察,发现250μM 5-AzaC处理的种子的根和茎的发育均有所减缓,部分叶的发育出现畸形;而500μM 5-AzaC处理后种子的根,茎,叶的生长都出现明显的抑制,并表现畸形;1000μM 5-AzaC处理的种子和20天前相比较几乎没有生长,生长被完全抑制。
2.从5-AzaC处理的早实枳叶片中提取RNA,用Real-time PCR方法分析了5个成花相关基因的表达水平,发现除FT相对表达量是随着5-AzaC处理浓度的增加而逐渐增加外,TFL、LFY、AP1、FLC四个基因的相对表达量都在250μM浓度时达到最大,处理浓度继续升高,基因的相对表达量下降。表明去甲基化处理对基因的转录有影响,大多数基因在低浓度去甲基化处理下,转录水平提高,而高浓度去甲基化会导致基因转录水平下降。
3.使用Methyl Primer Express和EMBOSS CpG Plot Tool软件,依照新旧两种CpG岛的定义,对早实枳中分离得到的5个成花相关基因TFL、LFY、AP1、FLC、FT的基因及启动子序列进行甲基化相关的生物信息学分析,然而仅预测到LFY序列中存在CpG岛。
4.借鉴医学研究中的亚硫氢酸钠修饰测序方法(Bisulfite Sequencing PCR),通过选择设计引物、优化反应条件,并采用巢式PCR方法,建立了柑橘类植物特定基因甲基化研究方法,并成功地应用于LFY基因的甲基化分析。
5.采用改良的亚硫氢酸钠修饰测序方法(Bisulfite Sequencing PCR),对早实枳不同发育阶段LFY基因CpG岛区域和非CpG岛区域的甲基化状态进行了比较。发现LFY 5'-UTR非CpG岛区域的甲基化水平很低,且在不同发育阶段之间没有明显差别;在CpG岛区域,童期时存在一定程度的甲基化并且特定的位点分布规律,在成年期部分位点发生了去甲基化,表明去甲基化参与了LFY在发育进程中的表达调控。
本研究对木本植物特定基因在不同发育进程中甲基化变化进行了分析,研究结果对研究木本植物发育调控分子机理可能有一定的参考意义。
Most fruit trees are woody perennial plants which have a long period of juvenile phase before flowering. A certain characteristic of juvenile phase is the seeding does not have a capability of flowering. Owing to the characteristics such as large individual, long period of juvenile phase and complex genetic backgrounds heavily impede genetic researches and breeding process in woody perennial plants, especially in fruit trees and economic forestry. Consequently, it is an important research area to study molecular mechanism in flowering and regulate the time to flowering in fruit trees. Precocious trifoliate orange is a mutant of trifoliate orange, which flowering time largely shorten to only one or two years. Therefore, precocious trifoliate orange is a suitable material for the molecular and genetic research on juvenile phase of woody perennial plants.
Most researches on the mechanism of fruit tree flowering mainly focus on physiological, environmental factors or cultivation techniques at the initial stages, however, the molecular and genetic mechanisms are still not understand clearly, even few researches reported the epigenetic mechanisms of fruit tree flowering. DNA methylation is an important aspect of epigenetic regulation in eukaryotes. Establish a protocol for analyzing DNA methylation status in plant and study on the relationship between related genes methylation stauts and developmental phase are both better for us to explore the epigenetic mechanism in plant and lies a foundation for shortening juvenile phase.
According to the phenotypic characteristics of precocious trifoliate orange mutant seedlings Treated by different 5-AzaC concentrations, we analyzed LFY, TEL etc. related flowering genes' relative expression levels in precocious trifoliate orange which treated by different 5-AzaC concentration. Then, we employed bioimformatic soft wares to analyze these genes in order to find whether these genes contain a CpG island. Fortunately, there is a CpG island found in LFY gene sequence. Therefore, we analyses cytosine methylation status in LFY CpG island DNA sequence from precocious trifoliate orange mutant in juvenile and adult phase by using bisulfate sequencing PCR method. The main results are as follows:
1. Precocious trifoliate orange seeds continuous treated by 0, 250, 500, 1000μM 5-AzaC for 15 days in the dark. The results showed that the seeds developed slower with increasing 5-AzaC concentrations, especially for the root development. Then the seeds transplanted in the soil after 15 days treatment. After the seedlings grew in the soil for 20 days in the describe condition, the results showed that the root of seedlings treated by 250μM 5-AzaC grew slowly compared to the control, and the seedlings showed several aberrant leaves; the root and stem development of seedlings were obvious inhibited by :500μM 5-AzaC treatment, and most of leaves exhibit aberrant phenotype; the growth of 1000μM 5-AzaC treated seeding nearly standstill compared with 20 days ago.
2. Extracted RNA from the leaves of different 5-AzaC concentrations treatment, and then analyzed five flowering genes' relative expression levels by using Real-time PCR. The results showed that FT relative expression level increased with the 5-AzaC concentrations. While, TFL, LFY, API, FLC showed highest relative expression levels in 250μM treatment, and these genes relative expression levels sharply decreased with higher concentrations. This means that demethylation treatment influences these genes expression, and most genes show high expression level in low demethylation treatment. However, the expression level would be decreased with higher concentration.
3. According to Methyl Primer Express and EMBOSS CpG Plot Tool soft wares analysis, we adopted both the old and new CpG island criteria to search and analyze flowering genes TFL, LFY, API, FLC, FT that isolated from precocious trifoliate orange. The results showed that only LFY sense chain DNA sequence contained a CpG island.
4. According to the references which successful analyzed gene sequence methylation status in mammalian, we optimized the modification protocol and adopted nested PCR by selecting proper primers to establish the bisulfite sequencing PCR protocol for analyzing DNA sequence methylation in citrus. This protocol has been successfully used for analyzing IFF DNA sequence methylation status.
5. To compare the methylation status of LFY CpG region with 5'-UTR at both juvenile and adult phase of precocious trifoliate orange by using bisulfite sequencing PCR, we found that LFY 5'-UTR which not in CpG island region showed unmethylation and no differences in different developmental phase. While, there was a low degree methylation status in CpG island region at juvenile phase and the lower degree methylation happened in the same region at adult phase, which meant demethylation might be regulate LFY gene expression from juvenile transition to adult phase.
The work is the first time to study on a certain gene's methylation status in the different developmental stages in woody perennial plants; the results might provide some evidence for further study.
引文
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