CN109207486B - 马铃薯cisr2基因及其在抗低温糖化中的应用 - Google Patents
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Abstract
本发明提供了马铃薯CISR2基因及其在抗低温糖化中的应用；通过马铃薯基因组数据库中公布的序列，以低温糖化抗性基因型马铃薯“10908‑06”4℃贮藏15d后的块茎cDNA为模板，克隆了CISR2基因，并构建超量表达载体，遗传转化鄂马铃薯3号进行转基因功能验证，发现超量表达CISR2基因能够提高其抗低温糖化的能力。
Description
技术领域
本发明属于分子遗传育种领域，具体的说是马铃薯CISR2基因及其在抗低温糖化中的应用。
背景技术
马铃薯(Solanum tuberosum L.)是茄科茄属草本植物，是继小麦、玉米和水稻之后的第四大粮食作物，在保障粮食安全和促进经济发展方面起重要作用。目前，全球有150多个国家种植马铃薯，分布仅次于玉米，成为第二个分布最广泛的农作物(谢从华，2012)。全球食用马铃薯消费从鲜薯转向增值的马铃薯加工食品，我国是世界上马铃薯第一大生产国，但是马铃薯油炸加工用等专用型品种稀缺，不能满足加工业发展的需要(金黎平等，2003)。在马铃薯加工业中，为了延长加工周期，预防马铃薯块茎发芽、失水皱缩和病害传播，通常贮藏在10℃以下。但是低温会加速淀粉裂解转化为还原糖(葡萄糖和果糖)，即低温糖化。马铃薯油炸产品色泽变异约93％是由还原糖含量决定的(Roe et al.,1990；Pritchard and Adam,1994)。在高温油炸加工期间，还原糖和游离的氨基酸进行MailardReaction，使油炸产品色泽加深，且产生对人体有害的致癌物质丙烯酰胺(Friedman,2003；Tareke et al.,2002；Shepherd et al.,2010)，这是油炸加工业长期以来面临的一大难题。现已有研究发现通过减少块茎中还原糖含量来降低丙烯酰胺的形成是一种有效的途径(Amrein et al.,2003；Biedermann-Brem et al.,2003)。
现有技术存在的问题：根据RNA-seq数据及共表达实验筛选出CISR2转录因子，目前，关于CISR2在马铃薯块茎低温糖化代谢过程中的作用并无相关文献报道。
发明内容
鉴于现有技术的不足，本发明提供了马铃薯CISR2基因及其在抗低温糖化中的应用；通过马铃薯基因组数据库中公布的序列，以低温糖化抗性基因型马铃薯10908-06 4℃贮藏15d后的块茎cDNA为模板，克隆了CISR2基因，并构建超量表达载体，遗传转化鄂马铃薯3号进行转基因功能验证，发现超量表达CISR2基因能够提高其抗低温糖化的能力。
为了达到上述目的，本发明采用了如下的技术方案：
马铃薯RNA-seq分析与低温糖化基因筛选：
本发明选取了低温糖化抗性基因型“10908-06”和低温糖化敏感基因型“鄂马铃薯3号(E3)”，种植于温室。马铃薯块茎收获后，室温遮光放置10天，选单个薯块重量大于50g、无病虫害和无机械损伤的块茎，分为两份，一份20℃贮藏0d、5d、15d，另一份4℃贮藏5d、15d，分别取样。取样时将块茎去皮切成小块速冻于液氮，保存于-70℃。参照天根生化科技(北京)有限公司(DP441)多糖多酚植物总RNA提取试剂盒提取上述所取样品的RNA，送于华大基因进行转录组测序(参考CN201510010458.X、CN201010213759.X、CN201610639522.5)。分析低温糖化抗性和敏感基因型贮藏块茎低温应答的表达谱数据，发现CISR2在抗感材料中的表达模式不一致，表达谱数据结果如下表所示(基因的表达量单位为FPKM)。
马铃薯CISR2基因，其通过如下步骤克隆得到：
(1)引物设计
根据马铃薯基因组序列信息(PGSC0003DMG400002620)，用SnapGene2.3.2软件设计扩增该基因CDS序列的引物。
(2)RNA提取与反转录
参照天根(DP441)多糖多酚植物总RNA提取试剂盒提取低温糖化抗性基因型马铃薯10908-06 4℃贮藏15d后的块茎RNA，根据TOYOBO反转录试剂盒(Code No.FSQ-101)进行反转录获得cDNA。
(3)PCR反应和克隆载体构建
根据合成的引物以上述cDNA为模板PCR扩增CISR2基因，胶回收扩增片段，进行TOPO克隆，菌液检测并测序，测序正确后命名为TOPO-CISR2。
马铃薯CISR2基因在抗低温糖化中的应用，其功能验证方法包括如下步骤：
(1)植物表达载体构建
将TOPO-CISR2质粒与超量表达载体pJCV55进行LR反应，随后转化大肠杆菌，PCR检测，测序。测序正确的目标载体命名为pJCV55-CISR2。
(2)马铃薯遗传转化
将pJCV55-CISR2植物表达载体转入农杆菌菌株LBA4404中，参照Sihuaijun等(2003)的方法进行遗传转化。
(3)抗低温糖化功能验证
将获得的转基因株系及对照E3试管苗种植于温室，薯块收获后，将鲜重大于50g的块茎分别于4℃贮藏30d，然后取样测定CISR2的相对表达量、还原糖含量(RS content)、蔗糖含量(Suc content)及酸性转化酶活性(VI activity)，上述测定采用张迟2007的方法。
附图说明
图1为4℃贮藏30d转基因块茎中CISR2基因的相对表达量；
图2为4℃贮藏30d转基因块茎中还原糖及蔗糖含量；
图3为4℃贮藏30d转基因块茎中转化酶活性。
具体实施方式
为使发明的目的、技术方案和优点更加清楚，下面结合附图对本发明的具体实施方式进行详细说明。这些优选实施方式的示例在附图中进行了例示。附图中所示和根据附图描述的本发明的实施方式仅仅是示例性的，并且本发明并不限于这些实施方式。
在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明的技术方案，在附图中仅仅示出了与根据本发明的方案密切相关的结构和/或处理步骤，而省略了关系不大的其他细节。
实施例1
该实施例提供马铃薯CISR2基因及其克隆方法，具体包括如下步骤：
(1)引物设计
本发明中的CISR2基因根据马铃薯基因组数据库公布的序列(PGSC0003DMG400002620)设计引物扩增该基因的CDS全长序列，引物序列如下：
CISR1F(5’-CCACATGGCGGAAGTTGAAGTACA-3’)
CISR1R(5’-TCAACAAGCCCATTTCAGTG-3’)
另外设计了CISR2的定量引物，引物序列如下：
qCISR1F(5’-CCGCTAGGGCACATGATGTT-3’)
q CISR1R(5’-TTTGACAATGGACGAGGCAA-3’)
(2)RNA提取与反转录
参照天根(DP441)多糖多酚植物总RNA提取试剂盒提取低温糖化抗性基因型马铃薯10908-06块茎4℃贮藏15d后的块茎RNA，根据TOYOBO反转录试剂盒(Code No.FSQ-101)进行反转录获得cDNA。
(3)PCR反应和克隆载体构建
通过PCR的方法，以低温糖化抗性基因型马铃薯10908-06为模板，扩增CISR2的CDS序列(741bp)，反应程序为：95℃，30s；95℃，15s；55℃，15s；72℃，30s；33个循环(2-4步)；72℃，10min；4℃ forever。胶回收相应目标片段后连接至TOPO载体上，然后转化至大肠杆菌DH5α，挑斑，菌液检测，送往英潍捷基(上海)贸易有限公司测序验证，阳性质粒提取。
实施例2
该实施例是将实施例1中得到的CISR2基因进行低温糖化抗性验证，具体验证步骤如下：
(1)植物表达载体构建
将TOPO-CISR2质粒与超量表达载体pJCV55进行LR反应，随后转化大肠杆菌，PCR检测，测序。测序正确的目标载体命名为pJCV55-CISR2，然后该质粒热激转化至农杆菌LBA4404中，菌检获得阳性克隆。
(2)马铃薯遗传转化
马铃薯遗传转化的方法参照Si等(Si et al.,2003)方法进行，采用的农杆菌菌株为LBA4404，受体材料为马铃薯E3，具体方法：1)马铃薯试管苗E3扩繁于8％MS固体培养基(含有活性炭)，结薯培养室(22℃，8h光/16h暗光周期)进行培养，生长6-8周的试管薯用于遗传转化；2)将含有目标质粒的农杆菌划线于LB固体培养基上(含有相应抗生素)进行活化，挑取单克隆于20mLYEB液体培养基中(含有相应抗生素)，28℃ 200rpm培养24h，取2mL菌液于40mL YEB液体培养基中(含有相应抗生素)，28℃ 200rpm进行亚培养，直至OD600约为0.5(约6h)，5000rpm，离心6min，去上清，10mL 3％MS液体培养基重悬；3)加入少量3％MS液体培养基于培养皿内，将试管薯置于皿中横切，厚度约1-2mm；切好的薯片放入重悬液中侵染10min，中途轻轻地摇一下，弃菌液，将薯片置于有滤纸的培养皿中，菌液吸干后转入P1共培养培养基(3％MS固体培养基+0.2mg/L GA3+0.2mg/L IAA+0.5mg/L 6-BA+2mg/L ZT，pH5.8)，23℃培养箱黑暗培养48h；4)将暗培养后的薯块转至P2分化培养基(3％MS固体培养基+0.2mg/L IAA+0.2mg/L GA3+2mg/L ZT+0.5mg/L 6-BA+75mg/L Kan+400mg/L Cef)，23℃培养箱培养(16h光/8h暗光周期)；5)大约1周后减去薯块的侧芽。将薯块中间的抗性芽接种至P3生根培养基(3％MS+50mg/L Kan+400mg/L Cef)，抗性芽长大后进行二次生根，随后进行阳性检测。
(3)抗低温糖化功能验证
将3个OE-CISR2(3、5、8)超量转基因株系和对照E3同时种植于温室，转基因株系和对照在田间农艺性状上无显著差异。将收获的转基因块茎和对照E3先20℃避光保存10d，选单个薯块重量大于50g的4℃贮藏30d，分别取样。
首先提取了4℃贮藏30d后的块茎的RNA，反转录得到其cDNA，实时荧光定量RT-qPCR检测CISR2的相对表达水平(见图1)，和对照相比，CISR2的表达水平显著升高。
还原糖的含量是判断马铃薯抗低温糖化能力的重要指标，因此对处理后的转基因株系和对照测定了还原糖及蔗糖含量，还原糖含量采用二硝基水杨酸法测定，蔗糖含量采用国家卫生部测定标准(GB5009，8.85)测定。OE-CISR2超量转基因株系块茎4℃贮藏30d后，还原糖的含量与对照相比显著降低(见图2)，蔗糖含量和对照相比无明显差异(见图2)。同时又对处理后的转基因株系和对照测定了酸性转化酶活性，OE-CISR2超量转基因株系块茎4℃贮藏30d时，酸性转化酶活性与对照相比显著降低(见图3)。现有实验结果表明，超量表达CISR2基因极大的提高了马铃薯抗低温糖化的能力。
与现有技术相比，本发明的有益效果为：
(1)转基因株系及对照E3的块茎4℃贮藏30d后，OE-3、OE-5及OE-8株系中还原糖含量显著低于对照E3，表明超量表达CISR2能够有效的减少低温贮藏下还原糖的累积。
(2)转基因株系及对照E3的块茎4℃贮藏30d后，OE-3、OE-5及OE-8株系中转化酶活性显著低于对照E3，表明超表达CISR2基因能在低温贮藏过程中有效的抑制酸性转化酶的活性。
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。
序列表
<110> 西南大学、华中农业大学
<120> 马铃薯CISR2基因及其在抗低温糖化中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 741
<212> DNA
<213> 马铃薯(Solanum tuberosum)
<400> 1
atggcggaag ttgaagtaca aatttcagaa acagagagct cttctaatac taatacctct 60
gtttcttctt cttcgtcttc gttgtccgct gattccatgc acaaatcggt ttttgattcg 120
tcaaacaaat taccgaaagg gaagaaacgg ccgacgaaga aacagaaggc tattactaat 180
agtgagagta gacatcaaat ttacagagga gttaggatga gaagttgggg gaaatgggta 240
tctgaaattc gtgaaccgag gaagaaatca cgtatttggc tcggtactta tcctacagca 300
gaaatggccg ctagggcaca tgatgttgca gcggtgagta taaaaggaaa atccgccatt 360
ctcaatttcc ctcatctaat cgactcgttg cctcgtccat tgtcaaattc acctagagat 420
gttcaagctg ctgctgctaa agcagcatca atgagggacc caccttcttc agcctcatcg 480
tcctcgtcct cgtcctcatc ctcaacaaca tcaacgggat ctgaagagct ttgtgagatt 540
attgaactgc ctaatttagc agaaagtgac gattcaaaaa ctgagttgcg gccgagtgac 600
tcagttgaag gactgctgta ctcgccgtgg tgggcagatc atagtacaga tttttgtggt 660
tattttctgg agcagtctgc agctggcgct ggggaaagtt taatttcttg cagctttgag 720
acactgaaat gggcttgttg a 741
Claims (2)
1.马铃薯CISR2基因在降低马铃薯低温贮藏下的还原糖积累中的应用，其特征在于所述CISR2基因CDS序列如序列表SEQ ID NO：1所示。
2.马铃薯CISR2基因在抑制马铃薯低温贮藏下酸性转化酶活性中的应用，其特征在于所述CISR2基因CDS序列如序列表SEQ ID NO：1所示。
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