CN106834534B - 检测柑橘铁螯合还原酶家族基因的方法 - Google Patents
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Abstract
本发明公开了一种检测柑橘铁螯合还原酶家族基因的方法，包括步骤：1)选取1年生的资阳香橙、卡里佐枳橙和枳的实生苗用作组织表达分析的材料；2)提取各实生苗根、茎、叶组织的RNA；3)以步骤2)提取的RNA为模板，反转录合成cDNA第一条链；4)荧光定量PCR；5)采用2△Ct法分析试验所得数据。本发明检测柑橘铁螯合还原酶家族基因的方法，通过特定的引物和反应体系，分别鉴定出了5种铁螯合还原酶基因在各种柑橘的不同部位的表达情况，有利于研究柑橘的耐缺铁特性。
Description
技术领域
本发明涉及一种铁螯合还原酶基因检测技术领域，特别涉及一种检测柑橘铁螯合还原酶家族基因的方法。
背景技术
在植物中，铁作为卟啉的主要构成成分和许多重要辅酶的辅基成分，如血红蛋白、过氧化物酶、黄素蛋白、铁氧还蛋白等，直接参与光合作用、呼吸作用、氮素同化、激素合成、活性氧的产生和清除、抵御病原物入侵等过程。由于铁是合成叶绿素所必需的元素，植物缺铁会造成叶绿体形态结构异常，叶绿素含量减少，光合速率降低，植株叶片失绿变黄、生长迟缓甚或死亡，造成农产品产量降低和品质变劣，给农业生产带来严重损失。
虽然地壳中铁的含量丰富(约为5％)，但土壤中的有效铁含量却很低。植物吸收铁的主要形式是还原铁(Fe2+)，而在中性或偏碱的土壤中，铁是以氧化态铁(Fe3+)存在，能供植物利用的还原铁(Fe2+)非常有限。Bienfaint、Romheld及Marscher等总结了高等植物在缺铁胁迫过程中形成的两种适应性机理——机理Ⅰ和机理Ⅱ。机理Ⅰ植物包括双子叶植物和非禾本科的单子叶植物；机理Ⅱ植物仅包括单子叶禾本科植物。现有的研究证明，机理Ⅰ植物吸收铁的机理是：先通过H+-ATPase向细胞外分泌H+，酸化土壤，促进根际周围Fe3+的溶解，再利用铁螯合还原酶(Ferric chelate reductase,FRO)将三价铁(Fe3+)还原为亚铁(Fe2 +)，在铁运载蛋白(iron-regulated transporter,IRT)的作用下将亚铁(Fe2+)运输到根细胞中而获得铁营养。
铁螯合还原酶是机理Ⅰ植物吸收铁的关键酶。在模式植物拟南芥中存在8个FROs的编码基因，AtFRO2首先在拟南芥中被克隆出来，编码蛋白具有8个跨膜螺旋结构，在缺铁诱导的根表皮中大量表达。AtFRO3在根和叶中都可表达，在缺铁胁迫时，在拟南芥的侧根、根尖、维管束部位大量表达。AtFRO6定位在细胞质膜上，AtFRO7定位在叶绿体膜上，AtFRO8定位在线粒体膜上，AtFRO1和AtFRO4在拟南芥中的表达量很低。将拟南芥的铁螯合还原酶基因AtFRO2和AtFRO6分别转化大豆和烟草，提高了转基因植物根和叶中铁螯合还原酶活性，转基因植物的叶绿素含量上升，对缺铁胁迫的耐性增强。酵母的铁螯合还原酶基因refre1在烟草和水稻中的异源表达也提高了转基因植物的耐缺铁能力。
柑橘属于铁吸收利用机理I植物，西南大学裴炎课题组研究结果表明，在缺铁胁迫条件下，与植物铁吸收相关的铁螯合还原酶活性在香橙中增加约20倍，而在枳中仅增加3倍，并克隆出资阳香橙铁螯合还原酶基因FRO2；且香橙根系在铁胁迫前后分泌质子的能力显著强于枳。我们分析发现柑橘中还存在4个新的铁螯合还原酶基因，分别为FRO1、FRO3、FRO4和FRO5。鉴定这五种柑橘铁螯合还原酶基因将利于柑橘耐缺铁的研究。
发明内容
有鉴于此，本发明的目的是提供一种检测柑橘铁螯合还原酶家族基因的方法，以鉴定柑橘中的5种铁螯合还原酶基因。
本发明检测柑橘铁螯合还原酶家族基因的方法，包括以下步骤：
1)选取资阳香橙、卡里佐枳橙和枳的根、茎和叶用作表达分析的材料；
2)分别提取资阳香橙、卡里佐枳橙和枳的根、茎、叶组织的RNA；
3)以步骤2)提取的RNA为模板，反转录合成cDNA第一条链；
4)荧光定量PCR；
柑橘中各铁螯合还原酶基因的荧光定量引物分布于基因的不同外显子区，引物的序列如下：
FRO1基因的上游引物为5'-AGGCACCCTTTACTACCTCCTAC-3'，下游引物为5'-GACAAGACTTCCCTTCGTTGATA-3'；
FRO2基因的上游引物为5'-CATGCTGGGCATTTCTCTTCTT-3'，下游引物为5'-GTTTCGCTCCATTCTAGCACCT-3'；
FRO3基因的上游引物为5′-CCAGCTTTGAGATTCTGTTGGT-3′，下游引物为5′-GGTACTTGAGTTGCCATGTGTT-3′；
FRO4基因的上游引物为5′-GACCTGAGCTAAAGAGGATGCT-3′，下游引物为5′-GCCAGACCAGATGAACAGATTG-3′；
FRO5基因的上游引物为5′-CCATTTTACAGGGCACGACAT-3′，下游引物为5′-ATTATTGGCCAAAGAGAGCAGC-3′；
PCR反应体系为：SYBR GreenⅠ20μL，iQ SYBR MIX 10μL，浓度为10μmol·L-1的上游引物和下游引物各0.3μL，cDNA 5μL，ddH2O 4.4μL；
选取看家基因Actin为内参基因，采用温度循环为94℃-60℃-72℃三步法进行qRT-PCR，PCR反应程序为：95℃预变性1min；94℃10s，60℃15s，72℃15s，40个循环；
5)采用2△Ct法分析试验所得数据。
本发明的有益效果：
本发明检测柑橘铁螯合还原酶家族基因的方法，通过特定的引物和反应体系，分别鉴定出了5种铁螯合还原酶基因在各种柑橘的不同部位的表达情况，有利于研究柑橘的耐缺铁特性。
附图说明
图1为FROs基因在资阳香橙中的表达情况；
图2为FROs基因在卡里佐枳橙中的表达情况；
图3为FROs基因在枳中的表达情况。
具体实施方式
下面结合附图和实施例对本发明作进一步描述。
本实施例检测柑橘铁螯合还原酶家族基因的方法，包括以下步骤：
1)选取1年生的资阳香橙、卡里佐枳橙和枳的实生苗用作组织表达分析的材料；
2)使用由北京艾德莱科技有限公司生产的RN09-EASY spin植物RNA快速提取试剂盒，提取各实生苗根、茎、叶组织的RNA；当然在不同实施例中也可选用其它种类的植物RNA提取试剂盒；
3)以步骤2)提取的RNA为模板，反转录合成cDNA第一条链；本实施例中cDNA第一条链的合成采用的反转录试剂盒为宝生物(大连)公司生产的
RT ReagentKit(宝生物工程(大连)有限公司，TaKaRa Code:DRR037A)；当然在不同实施例中cDNA的合成也可采用其它的反转录试剂盒；
4)荧光定量PCR；
柑橘中各铁螯合还原酶基因的荧光定量引物分布于基因的不同外显子区，这样避免了PCR过程中基因组DNA的影响，引物的序列如下：
FRO1基因的上游引物为5'-AGGCACCCTTTACTACCTCCTAC-3'，下游引物为5'-GACAAGACTTCCCTTCGTTGATA-3'；
FRO2基因的上游引物为5'-CATGCTGGGCATTTCTCTTCTT-3'，下游引物为5'-GTTTCGCTCCATTCTAGCACCT-3'；
FRO3基因的上游引物为5′-CCAGCTTTGAGATTCTGTTGGT-3′，下游引物为5′-GGTACTTGAGTTGCCATGTGTT-3′；
FRO4基因的上游引物为5′-GACCTGAGCTAAAGAGGATGCT-3′，下游引物为5′-GCCAGACCAGATGAACAGATTG-3′；
FRO5基因的上游引物为5′-CCATTTTACAGGGCACGACAT-3′，下游引物为5′-ATTATTGGCCAAAGAGAGCAGC-3′；
PCR反应体系为：SYBR GreenⅠ20μL，iQ SYBR MIX 10μL，浓度为10μmol·L-1的上游引物和下游引物各0.3μL，cDNA 5μL，ddH2O 4.4μL；
选取看家基因Actin为内参基因，采用温度循环为94℃-60℃-72℃三步法进行qRT-PCR，PCR反应程序为：95℃预变性1min；94℃10s，60℃15s，72℃15s，40个循环；
本实施例中所有PCR反应均设置3次生物学重复；
5)采用2△Ct法分析试验所得数据，2△Ct法即每百万看家基因中FROs数目＝106×2Ct (Actin)－Ct(FRO)。
本实施例检测柑橘铁螯合还原酶家族基因的方法，通过特定的引物和反应体系，分别鉴定出了5种铁螯合还原酶基因在各种柑橘的不同部位的表达情况(具体见说明书附图1-3)，有利于研究柑橘的耐缺铁特性。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
Claims (1)
1.一种检测柑橘铁螯合还原酶家族基因的方法，其特征在于：包括以下步骤：
1)选取资阳香橙、卡里佐枳橙和枳的根、茎和叶用作表达分析的材料；
2)分别提取资阳香橙、卡里佐枳橙和枳的根、茎、叶组织的RNA；
3)以步骤2)提取的RNA为模板，反转录合成cDNA第一条链；
4)荧光定量PCR；
柑橘中各铁螯合还原酶基因的荧光定量引物分布于基因的不同外显子区，引物的序列如下：
FRO1基因的上游引物为5'-AGGCACCCTTTACTACCTCCTAC-3'，下游引物为5'-GACAAGACTTCCCTTCGTTGATA-3'；
FRO2基因的上游引物为5'-CATGCTGGGCATTTCTCTTCTT-3'，下游引物为5'-GTTTCGCTCCATTCTAGCACCT-3'；
FRO3基因的上游引物为5′-CCAGCTTTGAGATTCTGTTGGT-3′，下游引物为5′-GGTACTTGAGTTGCCATGTGTT-3′；
FRO4基因的上游引物为5′-GACCTGAGCTAAAGAGGATGCT-3′，下游引物为5′-GCCAGACCAGATGAACAGATTG-3′；
FRO5基因的上游引物为5′-CCATTTTACAGGGCACGACAT-3′，下游引物为5′-ATTATTGGCCAAAGAGAGCAGC-3′；
PCR反应体系为：SYBR GreenⅠ 20μL，iQ SYBR MIX 10μL，浓度为10μmol·L-1的上游引物和下游引物各0.3μL，cDNA 5μL，ddH2O 4.4μL；
选取看家基因Actin为内参基因，采用温度循环为94℃-60℃-72℃三步法进行qRT-PCR，PCR反应程序为：95℃预变性1min；94℃10s，60℃15s，72℃15s，40个循环；
5)采用2△Ct法进行计算分析试验所得数据，2△Ct法即每百万看家基因中铁螯合还原酶基因数目＝106×2Ct(Actin)－Ct(FRO)。
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| WO2002079756A2 (en) * | 2001-03-30 | 2002-10-10 | Trustees Of Dartmouth College | Novel molecules of the multi-drug and toxin efflux (mate) protein family and uses thereof |
| CN1482245A (zh) * | 2003-06-20 | 2004-03-17 | 西南农业大学 | 香橙铁螯合还原酶基因 |
| CN104195223A (zh) * | 2014-05-28 | 2014-12-10 | 中国农业大学 | 一种用于鉴定耐缺铁苹果砧木的方法 |
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| CN1482245A (zh) * | 2003-06-20 | 2004-03-17 | 西南农业大学 | 香橙铁螯合还原酶基因 |
| CN104195223A (zh) * | 2014-05-28 | 2014-12-10 | 中国农业大学 | 一种用于鉴定耐缺铁苹果砧木的方法 |
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