荞麦FtMATE和FtFRDL2的耐铝毒功能研究
荞麦FtMATE和FtFRDL2的耐铝毒功能研究[20200614171457]
摘要：在酸性土壤中（pH<5.0），铝（Al3+）是限制作物产量的主要影响因素，对植物有较大毒害。不同植物受铝毒害程度不同，部分植物甚至能够高抗铝和高积累铝，例如荞麦。有研究显示，荞麦吸收的铝在根部及叶片中主要以铝-草酸复合物的形式存在，而铝在木质部中则以铝-柠檬酸复合物的形式存在[2]。这暗示着在铝从根部向木质部转运的过程中存在一个配体交换的过程。而介导这个过程的基因及其调控机制尚不明确。大量研究表明，MATE家族的一些基因与有机酸的合成分泌密切相关 [3]，因而我们推测在荞麦中MATE基因参与铝从根部向地上部的转运过程。我们通过RNA-seq的方法获得部分荞麦MATE家族基因的片段序列，利用RACE技术获得其完整的序列信息。再通过荧光定量PCR的方法研究其受铝诱导的程度与表达部位，发现两个MATE基因在根部强烈地受铝诱导，而在地上部基本不受铝诱导。时间梯度处理实验表明2小时处理表达有轻微上升，而在处理6小时后表达量达到最大。浓度梯度处理在10μM与100μM诱导的表达基本相同，铝对基因的表达没有剂量效应。同时，基因表达的表达具有铝特异性，不能被其他金属（Cu，Cd，La）诱导表达。RNAi和酵母异源表达的载体构建已经完成，可进一步构建转基因植株，直接研究基因在抗铝毒中发挥的作用。
*好棒文|www.hbsrm.com +Q: *351916072*
关键字:荞麦；有机酸分泌；MATE基因；抗铝毒
目录
Abstract 6
引言
引言
1 材料与方法 7
1．1材料 7
1．1．1植物材料 7
1．1．2载体 7
1．1．3菌株 7
1．1．4试剂盒 7
1．1．5 培养基 7
1．2 MATE全长扩增 7
1．2．1 3′RACE 7
1．2．2 5′RAC 7
1．2．3转化 7
1．2．4荞麦DNA提取 7
1．2．5 荞麦RNA提取 7
1．2．6 反转录 7
1．2．7 全长扩增 8
1．3 基因结构分析 8
1．4 启动子扩增 8
1．5 荞麦水培 8
1．5．1 浸种 8
1．5．2 催芽 8
1．5．3 培养 8
1．5．4 处理 8
1．6 铝处理与相对表达测定 8
1．6．1 地上部表达测定 8
1．6．2 RNA-seq结果的相对表达 8
1．6．3 根部浓度梯度处理与表达 8
1．6．4 铝特异性试验 9
1．6．5 时间梯度处理 9
1．7 RNAi载体构建 9
1．7．1 基因片段扩增 9
1．7．2 BP重组反应 9
1．7．3 LR重组反应 9
1．8 酵母异源表达载体构建 9
1．8．1 基因片段扩增 9
1．8．2 酶切 9
1．8．3 重组 10
1．8．4 转化 10 *好棒文|www.hbsrm.com +Q: *351916072*
r /> 2 结果与分析 10
2．1 基因扩增 10
2．2 基因结构 10
2．3 启动子扩增 10
2． 4 RNA-seq的根部相对表达 11
2． 5 根部浓度梯度处理 11
2． 6 基因表达的特异性 12
2．7 时间梯度处理 12
2．8 地上部表达 13
2． 9 RNAi载体构建 13
2．10 转酵母载体构建 13
3 讨论 14
参考文献： 15
荞麦FtMATE和FtFRDL2基因的耐铝毒功能研究
生命基地 朱仪方
Gene Function of FtMATE and FtFRDL2 in Aluminium resistant in Buckwheat (Fagopyrum tataricum)
Student majoring in the college of life science and technology
Yifang Zhu
Supervisor Huang Chao-Feng
Abstract： In acid soil(pH<5.0), Aluminium is the main influence factor that inhibit crop yield and have great poison to plant. Al have different toxic to plant, some plant can both tolerant and accumulate Al, such as buckwheat. It is reported that, buckwheat absorption of aluminum in root and leaf mainly exists in the form of aluminium - oxalic acid complex, and aluminum in the xylem is in the form of aluminium - citric acid compound. It is suggested that there is a process which used for ligand exchange when Al is transport from root to xylem. While the gene which mediate this process and its trgulation mechanism of this process is still unknown. A large number of studies have shown that some members of MATE family is closely related to the synthesis of organic acid secretion, so we suspect in buckwheat MATE involved in Al transportation from root to leaf. We obtained p *好棒文|www.hbsrm.com +Q: *351916072*
art of buckwheat MATE family gene fragment sequence by the method of RNA - seq, using RACE technology to obtain the complete sequence information. By real-time fluorescence quantitative PCR to study the degree of induce and expression tissue, founding that two MATE genes were induced greatly by Al in root but can not induced in leaf. Time course experiment showed that when treated by Al for 2 hours , expression of two genes have slight rise, and when treated for 6 hours, expression of two genes reached the maximum. Concentration gradient treatment showed that Al treatment have no dosage effect for the same express level when treated with 10μM and 100μM. The expression are specific to Al for other mental cation(Cu, Cd, La)can not induce genes exspress. Constrcut of RNAi and yeast heterologous expression vector have been finished and can be used for construct transgene plant for study the function of two genes in buckwheat.
摘要：在酸性土壤中（pH<5.0），铝（Al3+）是限制作物产量的主要影响因素，对植物有较大毒害。不同植物受铝毒害程度不同，部分植物甚至能够高抗铝和高积累铝，例如荞麦。有研究显示，荞麦吸收的铝在根部及叶片中主要以铝-草酸复合物的形式存在，而铝在木质部中则以铝-柠檬酸复合物的形式存在[2]。这暗示着在铝从根部向木质部转运的过程中存在一个配体交换的过程。而介导这个过程的基因及其调控机制尚不明确。大量研究表明，MATE家族的一些基因与有机酸的合成分泌密切相关 [3]，因而我们推测在荞麦中MATE基因参与铝从根部向地上部的转运过程。我们通过RNA-seq的方法获得部分荞麦MATE家族基因的片段序列，利用RACE技术获得其完整的序列信息。再通过荧光定量PCR的方法研究其受铝诱导的程度与表达部位，发现两个MATE基因在根部强烈地受铝诱导，而在地上部基本不受铝诱导。时间梯度处理实验表明2小时处理表达有轻微上升，而在处理6小时后表达量达到最大。浓度梯度处理在10μM与100μM诱导的表达基本相同，铝对基因的表达没有剂量效应。同时，基因表达的表达具有铝特异性，不能被其他金属（Cu，Cd，La）诱导表达。RNAi和酵母异源表达的载体构建已经完成，可进一步构建转基因植株，直接研究基因在抗铝毒中发挥的作用。
*好棒文|www.hbsrm.com +Q: *351916072*
关键字:荞麦；有机酸分泌；MATE基因；抗铝毒
目录
Abstract 6
引言
引言
1 材料与方法 7
1．1材料 7
1．1．1植物材料 7
1．1．2载体 7
1．1．3菌株 7
1．1．4试剂盒 7
1．1．5 培养基 7
1．2 MATE全长扩增 7
1．2．1 3′RACE 7
1．2．2 5′RAC 7
1．2．3转化 7
1．2．4荞麦DNA提取 7
1．2．5 荞麦RNA提取 7
1．2．6 反转录 7
1．2．7 全长扩增 8
1．3 基因结构分析 8
1．4 启动子扩增 8
1．5 荞麦水培 8
1．5．1 浸种 8
1．5．2 催芽 8
1．5．3 培养 8
1．5．4 处理 8
1．6 铝处理与相对表达测定 8
1．6．1 地上部表达测定 8
1．6．2 RNA-seq结果的相对表达 8
1．6．3 根部浓度梯度处理与表达 8
1．6．4 铝特异性试验 9
1．6．5 时间梯度处理 9
1．7 RNAi载体构建 9
1．7．1 基因片段扩增 9
1．7．2 BP重组反应 9
1．7．3 LR重组反应 9
1．8 酵母异源表达载体构建 9
1．8．1 基因片段扩增 9
1．8．2 酶切 9
1．8．3 重组 10
1．8．4 转化 10
r /> 2 结果与分析 10
2．1 基因扩增 10
2．2 基因结构 10
2．3 启动子扩增 10
2． 4 RNA-seq的根部相对表达 11
2． 5 根部浓度梯度处理 11
2． 6 基因表达的特异性 12
2．7 时间梯度处理 12
2．8 地上部表达 13
2． 9 RNAi载体构建 13
2．10 转酵母载体构建 13
3 讨论 14
参考文献： 15
荞麦FtMATE和FtFRDL2基因的耐铝毒功能研究
生命基地 朱仪方
Gene Function of FtMATE and FtFRDL2 in Aluminium resistant in Buckwheat (Fagopyrum tataricum)
Student majoring in the college of life science and technology
Yifang Zhu
Supervisor Huang Chao-Feng
Abstract： In acid soil(pH<5.0), Aluminium is the main influence factor that inhibit crop yield and have great poison to plant. Al have different toxic to plant, some plant can both tolerant and accumulate Al, such as buckwheat. It is reported that, buckwheat absorption of aluminum in root and leaf mainly exists in the form of aluminium - oxalic acid complex, and aluminum in the xylem is in the form of aluminium - citric acid compound. It is suggested that there is a process which used for ligand exchange when Al is transport from root to xylem. While the gene which mediate this process and its trgulation mechanism of this process is still unknown. A large number of studies have shown that some members of MATE family is closely related to the synthesis of organic acid secretion, so we suspect in buckwheat MATE involved in Al transportation from root to leaf. We obtained p *好棒文|www.hbsrm.com +Q: *351916072*
art of buckwheat MATE family gene fragment sequence by the method of RNA - seq, using RACE technology to obtain the complete sequence information. By real-time fluorescence quantitative PCR to study the degree of induce and expression tissue, founding that two MATE genes were induced greatly by Al in root but can not induced in leaf. Time course experiment showed that when treated by Al for 2 hours , expression of two genes have slight rise, and when treated for 6 hours, expression of two genes reached the maximum. Concentration gradient treatment showed that Al treatment have no dosage effect for the same express level when treated with 10μM and 100μM. The expression are specific to Al for other mental cation(Cu, Cd, La)can not induce genes exspress. Constrcut of RNAi and yeast heterologous expression vector have been finished and can be used for construct transgene plant for study the function of two genes in buckwheat.
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