番茄转录辅激活子LeMBF1在转基因材料中的功能分析
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摘要
转录辅激活因子在真核基因表达中通过桥连转录因子/其他调控元件和基础转录装置起着十分重要的作用。根据转录辅激活因子的作用可将其分为两类:一类是促进或保持修饰染色质结构的酶的活性;一类是通过与转录因子结合促进一般转录装置与启动子的结合,多蛋白桥梁因子(multiprotein bridging factor 1, MBF1)就属于这一类。MBF1是一个高度保守的关于内皮细胞分化、激素调节、脂类代谢、中枢神经系统发育和组氨酸新陈代谢等不同过程的转录辅激活蛋白,不同有机体的MBF1蛋白通过与C-Jun、GCN4、ATF1或其他核受体的相互作用连接TATA盒蛋白激活基因转录。植物MBF1s参与植物的生长发育和多种胁迫反应,超表达MBF1可以调节多种信号转导途径和激活多种防御因子的表达,提高植物对多种胁迫的抵抗力,如高温,干旱,病原菌侵染等,为培育高抗性的转基因作物提供了新的路径。目前在番茄中关于此基因还没有相关的报道,本论文拟通过在番茄中超表达MBF1基因研究其对逆境胁迫的抗性能力,从而明确MBF1基因的分子作用机制,主要研究内容及结果如下:
1、再生番茄植株的获得
将LeMBF1基因与经Pst I酶切的pDH51载体连接形成中间载体,再经EcoR I酶切获得了包含35S启动子、MBF1基因、35S终止子的片段,此片段与经EcoR I酶切的pBIN19载体连接,构成双元表达载体质粒pBIN19-MBF1。以AC++番茄子叶为外植体,通过农杆菌LBA4404介导,将CaMV 35S启动子调控下的MBF1基因转入番茄,经过50 mg/L Kan及500 mg/L的Sm筛选,获得一部分生根的再生植株。
2、Southern分析
选择11个转化株系进行Southern分析,结果表明在这些转化株系中有8个是多拷贝,有3个是单拷贝。
3、Northern分析
选择04、05、08和09进行Northern分析,检测结果显示这4个株系中LeMBF1均获得表达。
4、下游基因检验
克隆番茄Actin、PR1、PR2、PR4、PR6、Pti4、HSP90、Apx2、CHS和Zat基因的片段,以Actin为内参,在AC++植株和转基因植株中进行半定量RT-PCR分析。结果显示:1) HSP90、Apx2、Zat、PR1、PR4和PR6基因的表达水平在LeMBF1超表达植株中均明显增强,LeMBF1正调控这些基因;2) CHS,PR2和Pti4基因的表达水平在AC++植株和转基因植株中没有明显变化,表达量基本一致。
5、生物胁迫实验
用芽孢杆菌侵染AC++和转基因植株的叶片,利用平板菌落计数法检测叶片上的菌落总数。结果显示:转基因植株对细菌的抵抗力显然比AC++植株强。用赤霉菌侵染AC++和转基因植株的叶片,以Actin为内参进行半定量RT-PCR分析下游防卫反应基因的表达。结果显示:1)在AC++植株中,LeMBF1、PR1、PR2、PR4、PR6和Pti4基因的表达水平明显增强。2)在转基因植株中,LeMBF1、PR2、PR4、PR6和Pti4基因的表达水平明显增强,但PR1基因的表达水平轻微减弱。3)与AC++植株相比,转基因植株中LeMBF1、PR2、PR4和Pti4基因的表达水平明显较高,而是PR1和PR6基因的表达水平反而低。LeMBF1可能通过激活部分PRs和Pti4基因的表达提高植物的抗病性。
6、高温实验
将AC++植株和转基因植株放置于39°C高温,统计植株在高温下的存活率。结果显示:转基因植株的耐热性比AC++植株强。利用半定量RT-PCR分析高温条件下防卫反应相关基因在AC++和转基因植株中的表达。结果显示:1)在AC++和转基因植株中,LeMBF1、PR1、HSP90、Apx2、CHS和Zat基因的表达水平均明显升高。2)与AC++植株相比,转基因植株中LeMBF1、PR1、HSP90、Apx2和Zat基因的表达水平明显较强,而CHS基因的表达水平在30 min内较强,随后反而稍弱。LeMBF1可能通过激活PR1、HSP90、Apx2和Zat基因的表达提高植物的耐热性。
Transcriptional coactivators play a crucial role in eukaryotic gene expression by communicating between transcription factors and/or other regulatory components and the basal transcription machinery. They are divided into two classes: transcriptional coactivator that recruit or possess enzymatic activities that modify chromatin structure and transcriptional coactivator that recruit the general transcriptional machinery to a promoter where a transcription factor(s) is bound. Multiprotein bridging factor 1 (MBF1) belong to the Second class. MBF1 is a highly conserved transcriptional coactivator involved in the regulation of diverse processes such as endothelial cell differentiation, hormone-regulated lipid metabolism, central nervous system development, and His metabolism. MBF1 proteins from different organisms interact with transcription factors such as c-Jun, GCN4, and ATF1, or with different nuclear receptors, and link them with the TATA-binding protein. The plant MBF1s participate regulating involved in the development of organisms and the response to environment conditions. The expression of MBF1 in transgenic plants regulates a variety of signal transduction pathways and augments the accumulation of a number of defense transcripts. MBF1 protein could be used to enhance the tolerance of plants to different multiple stresses, such as high temperature, drought, pathogen invasion dyeing. In order to cultivate a high resistance to transgenic crops has provided a new path. However there is not relative report on MBF1 gene in tomatoes. We overexpressed this gene in tomato to check its defense ability and know clearly about the principal in molecular level. The main contents and results of the experiment are as follows:
1. Getting regenerated tomato
MBF1 gene was ligated to pDH51 cut with PstI. A fragment including 35S promoter, MBF1 gene and 35S terminator was excised from pDH51-MBF1 with EcoRI, the fragment was ligated to pBIN19 cut with EcoRI, named after pB1N19-MBF1. AC++ tomato cotyledon as explants, the MBF1 transformation of tomato was mediated by Agrobacterium LBA4404. Some regenerated tomatos have been obtained after screened by 50 mg/L Kan and 500 mg/L Sm.
2. Southern blotting analysis
The Southern blotting analysis indicated that all transformants had been transformed. Eight lines displayed multiple copies and three lines displayed single copy.
3. Northern analysis
The line 04, 05, 08 and 09 were chosen for Northern analysis with LeMBF1 as probe suggested that the LeMBF1 is over expressed in the 4 lines. 4. Down-stream gene analysis
We cloned tomato Actin, PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat gene from tomato. The expression level of PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6 obviously increased and the expression levels of CHS, PR2 and Pti4 showed no change in MBF1-overexpressing plants. Therefore, we suggested that expression of LeMBF1 upregulates the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6.
5. Biotic stress experiments
Brevibacliius brevis was infiltrated into AC++ and transgenic plant leaves. Bacteria was extracted from leaves, plated on agar plates, and scored for cfu cm-2. The result showed that MBF1-overexpressing plants were more resistant than wild type plants to bacterial growth. AC++ and transgenic plant leaves were inoculated with freshly harvested Fusarium graminearum conidial spore suspension. All samples were harvested. The above cloned PR genes were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that LeMBF1, PR1, PR2, PR4, PR6 and Pti4 mRNA level enhanced in AC++ plants. The expression levels of LeMBF1, PR2, PR6 and Pti4 obviously increased in transgenic plants, however, PR1 mRNA level weakly decreased. Compared with AC++ plants, expression of LeMBF1, PR2, PR4 and Pti4 mRNAs were significantly higher in transgenic plants, however, PR1 and PR6 mRNAs levels were lower. LeMBF1 expression enhances the tolerance of transgenic plants to pathogen by activating the expression of several PR genes and Pti4.
6. High temperature experiments
AC++ and transgenic plants were incubated at 39°C. Survival rate of AC++ and transgenic plants in response to heat stress, the result showed enhanced basal thermotolerance of MBF1-expressing plants. Leaves were harvested. LeMBF1, PR1, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of LeMBF1, PR1, HSP90, Apx2, CHS and Zat obviously increased in AC++ and transgenic plants. Compared with AC++ plants, expression of LeMBF1, PR1, HSP90, Apx2 and Zat mRNAs were significantly higher in transgenic plants. CHS mRNAs levels was higher within 30 min, thereafter, became lower. LeMBF1 expression enhances the thermotolerance of transgenic plants by activating the expression of PR1, HSP90, Apx2 and Zat.
1、再生番茄植株的获得
将LeMBF1基因与经Pst I酶切的pDH51载体连接形成中间载体,再经EcoR I酶切获得了包含35S启动子、MBF1基因、35S终止子的片段,此片段与经EcoR I酶切的pBIN19载体连接,构成双元表达载体质粒pBIN19-MBF1。以AC++番茄子叶为外植体,通过农杆菌LBA4404介导,将CaMV 35S启动子调控下的MBF1基因转入番茄,经过50 mg/L Kan及500 mg/L的Sm筛选,获得一部分生根的再生植株。
2、Southern分析
选择11个转化株系进行Southern分析,结果表明在这些转化株系中有8个是多拷贝,有3个是单拷贝。
3、Northern分析
选择04、05、08和09进行Northern分析,检测结果显示这4个株系中LeMBF1均获得表达。
4、下游基因检验
克隆番茄Actin、PR1、PR2、PR4、PR6、Pti4、HSP90、Apx2、CHS和Zat基因的片段,以Actin为内参,在AC++植株和转基因植株中进行半定量RT-PCR分析。结果显示:1) HSP90、Apx2、Zat、PR1、PR4和PR6基因的表达水平在LeMBF1超表达植株中均明显增强,LeMBF1正调控这些基因;2) CHS,PR2和Pti4基因的表达水平在AC++植株和转基因植株中没有明显变化,表达量基本一致。
5、生物胁迫实验
用芽孢杆菌侵染AC++和转基因植株的叶片,利用平板菌落计数法检测叶片上的菌落总数。结果显示:转基因植株对细菌的抵抗力显然比AC++植株强。用赤霉菌侵染AC++和转基因植株的叶片,以Actin为内参进行半定量RT-PCR分析下游防卫反应基因的表达。结果显示:1)在AC++植株中,LeMBF1、PR1、PR2、PR4、PR6和Pti4基因的表达水平明显增强。2)在转基因植株中,LeMBF1、PR2、PR4、PR6和Pti4基因的表达水平明显增强,但PR1基因的表达水平轻微减弱。3)与AC++植株相比,转基因植株中LeMBF1、PR2、PR4和Pti4基因的表达水平明显较高,而是PR1和PR6基因的表达水平反而低。LeMBF1可能通过激活部分PRs和Pti4基因的表达提高植物的抗病性。
6、高温实验
将AC++植株和转基因植株放置于39°C高温,统计植株在高温下的存活率。结果显示:转基因植株的耐热性比AC++植株强。利用半定量RT-PCR分析高温条件下防卫反应相关基因在AC++和转基因植株中的表达。结果显示:1)在AC++和转基因植株中,LeMBF1、PR1、HSP90、Apx2、CHS和Zat基因的表达水平均明显升高。2)与AC++植株相比,转基因植株中LeMBF1、PR1、HSP90、Apx2和Zat基因的表达水平明显较强,而CHS基因的表达水平在30 min内较强,随后反而稍弱。LeMBF1可能通过激活PR1、HSP90、Apx2和Zat基因的表达提高植物的耐热性。
Transcriptional coactivators play a crucial role in eukaryotic gene expression by communicating between transcription factors and/or other regulatory components and the basal transcription machinery. They are divided into two classes: transcriptional coactivator that recruit or possess enzymatic activities that modify chromatin structure and transcriptional coactivator that recruit the general transcriptional machinery to a promoter where a transcription factor(s) is bound. Multiprotein bridging factor 1 (MBF1) belong to the Second class. MBF1 is a highly conserved transcriptional coactivator involved in the regulation of diverse processes such as endothelial cell differentiation, hormone-regulated lipid metabolism, central nervous system development, and His metabolism. MBF1 proteins from different organisms interact with transcription factors such as c-Jun, GCN4, and ATF1, or with different nuclear receptors, and link them with the TATA-binding protein. The plant MBF1s participate regulating involved in the development of organisms and the response to environment conditions. The expression of MBF1 in transgenic plants regulates a variety of signal transduction pathways and augments the accumulation of a number of defense transcripts. MBF1 protein could be used to enhance the tolerance of plants to different multiple stresses, such as high temperature, drought, pathogen invasion dyeing. In order to cultivate a high resistance to transgenic crops has provided a new path. However there is not relative report on MBF1 gene in tomatoes. We overexpressed this gene in tomato to check its defense ability and know clearly about the principal in molecular level. The main contents and results of the experiment are as follows:
1. Getting regenerated tomato
MBF1 gene was ligated to pDH51 cut with PstI. A fragment including 35S promoter, MBF1 gene and 35S terminator was excised from pDH51-MBF1 with EcoRI, the fragment was ligated to pBIN19 cut with EcoRI, named after pB1N19-MBF1. AC++ tomato cotyledon as explants, the MBF1 transformation of tomato was mediated by Agrobacterium LBA4404. Some regenerated tomatos have been obtained after screened by 50 mg/L Kan and 500 mg/L Sm.
2. Southern blotting analysis
The Southern blotting analysis indicated that all transformants had been transformed. Eight lines displayed multiple copies and three lines displayed single copy.
3. Northern analysis
The line 04, 05, 08 and 09 were chosen for Northern analysis with LeMBF1 as probe suggested that the LeMBF1 is over expressed in the 4 lines. 4. Down-stream gene analysis
We cloned tomato Actin, PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat gene from tomato. The expression level of PR1, PR2, PR4, PR6, Pti4, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6 obviously increased and the expression levels of CHS, PR2 and Pti4 showed no change in MBF1-overexpressing plants. Therefore, we suggested that expression of LeMBF1 upregulates the expression levels of HSP90, Apx2, Zat, PR1, PR4 and PR6.
5. Biotic stress experiments
Brevibacliius brevis was infiltrated into AC++ and transgenic plant leaves. Bacteria was extracted from leaves, plated on agar plates, and scored for cfu cm-2. The result showed that MBF1-overexpressing plants were more resistant than wild type plants to bacterial growth. AC++ and transgenic plant leaves were inoculated with freshly harvested Fusarium graminearum conidial spore suspension. All samples were harvested. The above cloned PR genes were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that LeMBF1, PR1, PR2, PR4, PR6 and Pti4 mRNA level enhanced in AC++ plants. The expression levels of LeMBF1, PR2, PR6 and Pti4 obviously increased in transgenic plants, however, PR1 mRNA level weakly decreased. Compared with AC++ plants, expression of LeMBF1, PR2, PR4 and Pti4 mRNAs were significantly higher in transgenic plants, however, PR1 and PR6 mRNAs levels were lower. LeMBF1 expression enhances the tolerance of transgenic plants to pathogen by activating the expression of several PR genes and Pti4.
6. High temperature experiments
AC++ and transgenic plants were incubated at 39°C. Survival rate of AC++ and transgenic plants in response to heat stress, the result showed enhanced basal thermotolerance of MBF1-expressing plants. Leaves were harvested. LeMBF1, PR1, HSP90, Apx2, CHS and Zat were detected by Semiquantitative RT-PCR analysis with Actin gene as a reference in AC++ and transgenic plants. The results showed that the expression levels of LeMBF1, PR1, HSP90, Apx2, CHS and Zat obviously increased in AC++ and transgenic plants. Compared with AC++ plants, expression of LeMBF1, PR1, HSP90, Apx2 and Zat mRNAs were significantly higher in transgenic plants. CHS mRNAs levels was higher within 30 min, thereafter, became lower. LeMBF1 expression enhances the thermotolerance of transgenic plants by activating the expression of PR1, HSP90, Apx2 and Zat.
引文
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