CN116064589B - 烟草NtPht1-7基因突变体及其应用 - Google Patents
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Abstract
本发明涉及烟草NtPht1‑7基因突变体及其应用，属于基因工程技术领域。本发明将NtPht1‑7基因经甲基磺酸乙酯(EMS)诱变引起烟草NtPht1‑7基因编码区第648位碱基发生G‑>A的突变，导致氨基酸发生从色氨酸到终止突变的变化，将该NtPht1‑7基因突变体命名为NtPht1‑7，其编码区核苷酸序列如SEQ ID NO.1所示，该基因发生突变后会导致烟草叶片中总砷离子含量降低。砷离子含量是烟草安全性的重要指标，因此该突变体材料在烟草上具有极大的育种价值。
Description
技术领域
本发明属于植物分子生物学领域，具体涉及一种烟草NtPht1-7基因突变体NtPht1-7及其分子鉴定方法和应用。
背景技术
伴随着我国经济的发展，自然环境遭到了极大的破坏。其中土壤和水污染是环境污染的主要形式，目前我国部分地区土壤中重金属含量超标，危害食品安全和人的健康。砷(As)是一种常见的环境毒物和人类致癌物，地壳岩石圈上砷的平均含量很低；但人类活动，如采矿、冶炼、污灌、施肥、杀虫剂和防腐剂的施用，都可能造成土壤砷污染。砷对动、植物具有强烈的毒害作用，砷污染在全世界，尤其在东南亚已成为一个严峻的环境问题。
砷并非植物生长过程的必需营养元素。在砷污染的土壤上种植的作物生长发育受到抑制，产量与品质降低，并造成作物体内砷的积累。若作物食用部分砷含量超标，人食用后砷随血液分布于全身各组织器官，引起相应的病变。因此，研究砷对植物的毒害作用及植物对砷的吸收转运机理，对食品安全和人类健康意义重大。
烟草中对人体有害的成分主要包括焦油、尼古丁和重金属等成份。砷是烟草中有害重(类)金属元素之一,在抽吸过程中可通过主流烟气进入人体和环境,对人体造成危害,对环境造成污染。1990年，在美国健康基金会的烟草致癌物清单(通常被称为“Hoffmann清单”)中将砷列入烟草44种有害成分之一。目前我国部分地区农业土壤中不同程度地存在砷的污染，因此,烟草在种植过程中，也可能会受到砷的污染，进而砷会在烟草中富集。香烟中的砷主要源于种植烟草的土壤、烟草杀虫剂，烟叶中的重金属在烟叶加工成卷烟后再以烟气的形式进入人体,对人的健康造成严重的影响。
为保护消费者身体健康，需持续推进烟草产品的降焦减害工作，必须降低烟叶中重要危害物重(类)金属的含量。培育烟叶中砷含量降低的烟草品种，是在烟草种植过程中减少烟叶中砷积累的一种非常有效的办法。
发明内容
随着分子生物学技术的发展，采用分子手段突变烟草中特定的转运砷离子的通道蛋白编码基因是解决这一问题的一种手段。因此通过EMS诱变的方法在栽培烟草中直接突变砷离子转运相关基因是创制低砷离子含量烟草品种的有效方法。
本发明采用如下技术方案实现。
一种烟草NtPht1-7基因突变体，本发明所述烟草NtPht1-7基因突变体的核苷酸序列如SEQ ID NO.1所示。
本发明所述烟草NtPht1-7基因突变体为烟草NtPht1-7基因编码区第648位碱基发生G->A的突变，导致氨基酸发生从色氨酸到终止突变的变化，从而造成该基因的提前终止。
本发明所述烟草NtPht1-7基因突变体是由以下引物对扩增得到，所述引物对的核苷酸序列为：
上游引物TTTTAACAGGCGGAATAGTTGCTC，SEQ ID NO.2所示；
下游引物GTTCCTAGCAAGTGAAGTCCATGA，SEQ ID NO.3所示。
本发明PCR反应条件为：PCR扩增体系：50μL体系，Template DNA1μL；primer-F(10μmol/L)1μL、primer-R(10μmol/L)1μL、5×buffer 10μL、dNTP mixture(10mmol/L)1μL；PCR扩增条件：①98℃，5min；②98℃30s；③58℃30s；④72℃30s；②-④35cycles；⑤72℃5min；4℃Forever。
本发明上述的烟草NtPht1-7基因突变体在制备低砷离子含量材料中的应用。本发明的目的是提供一种烟草NtPht1-7基因突变体NtPht1-7及其应用。
本发明的第一个目的是这样实现的，与野生型烟草NtPht1-7基因序列表中SEQ IDNO.4所示的核苷酸序列相比，突变体含有的NtPht1-7基因编码序列的第648位碱基发生G->A的突变，导致氨基酸发生从色氨酸到终止突变的变化，从而造成该基因的提前终止。
EMS诱变烟草种子：
先将烟草种子用次氯酸钠清洗消毒，然后用蒸馏水洗干。
再将烟草种子用磷酸盐缓冲液浸泡，以增加种子的萌发率。
将浸泡处理后获得的烟草种子放置于0.5％的甲基磺酸乙酯(EMS)溶液中浸泡10-15小时，然后进行离心，再吸取EMS溶液仅保留离心管底部的烟草种子。
将烟草种子用蒸馏水漂洗50次，充分洗去EMS溶液，将EMS废液用氢氧化钠处理，以免造成污染。
筛选获得突变体植株：
提取突变体烟草DNA；
以突变体材料的DNA为模板设计特异引物进行PCR扩增；
引物为NtPht1 F:TTTTAACAGGCGGAATAGTTGCTC
NtPht1 R：GTTCCTAGCAAGTGAAGTCCATGA
将扩增获得的PCR产物送交公司测序，分析测序结果获得突变体材料，挑选目标基因编码区发生碱基突变的烟株为候选突变体材料；
将候选突变体材料自交收种获得M2种子；
种植M2种子获得M2突变体植株，用特异引物鉴定突变体，最终获得纯合突变植株。
检测纯合突变体烟株中总砷离子含量。
本发明的有益效果在于，本发明将NtPht1-7基因经甲基磺酸乙酯(EMS)诱变引起烟草NtPht1-7基因编码区第648位碱基发生G->A的突变，导致氨基酸发生从色氨酸到终止突变的变化，将该NtPht1-7基因突变体命名为NtPht1-7，其编码区核苷酸序列如SEQ IDNO.1所示，该基因发生突变后会导致烟草叶片中总砷离子含量降低。砷离子含量是烟草安全性的重要指标，因此该突变体材料在烟草上具有极大的育种价值。
附图说明
图1突变体目标基因片段PCR扩增条带电泳图；
图2纯合突变体Sanger测序结果图；
图3突变体与野生型烟草植株总砷离子含量对照图；
具体实施方式
下面结合实施例和附图对本发明做进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或者替换，均属本发明的保护范围。
实施例1：烟草突变材料的获得
先将烟草种子用次氯酸钠清洗消毒，然后用蒸馏水洗干。
再将烟草种子用磷酸盐缓冲液浸泡，以增加种子的萌发率。
将浸泡处理后获得的烟草种子放置于0.5％的甲基磺酸乙酯(EMS)溶液中浸泡10-15小时，然后进行离心，再吸取EMS溶液仅保留离心管底部的烟草种子。
将烟草种子用蒸馏水漂洗50次，充分洗去EMS溶液，将EMS废液用氢氧化钠处理，以免造成污染。
实施例2：筛选获得NtPht1-7突变体
以烟草野生型材料的DNA为模板设计特异引物进行PCR扩增；
引物为NtPht1-7 F:TTTTAACAGGCGGAATAGTTGCTC
NtPht1-7 R：GTTCCTAGCAAGTGAAGTCCATGA
PCR反应条件为：
将扩增获得的PCR产物在0.8％的琼脂糖凝胶电泳，电泳结束后，采用Qiagen公司PCR产物纯化试剂盒，按照产品说明回收纯化所述的PCR产物，并送Invitrogen公司测序，验证序列结果。
将候选突变体材料自交收种获得M2种子；
种植M2种子获得M2突变体植株，用目标基因特异引物进行PCR扩增反应，对PCR产物进行Sanger测序，鉴定突变体，最终获得纯合突变植株。
实施例3：总砷离子含量测定
用浓硝酸和过氧化氢于微波消解仪中进行消解，用电感耦合离子体质谱仪(ICP-MS)测定总砷含量。
主要仪器设备
a)7500a型电感耦合等离子体质谱仪(美国AGILENT公司)；
b)Mars5型微波消解仪(美国CEM公司)。
实验器皿：
——Millipore Simplicity型超纯水器(美国MILLIPORE公司)；
——Mettler AE 163型电子天平(感量0.0001g瑞士梅特勒-托利多公司)；——50mL塑料容量瓶；
——消解罐。
试剂(备注：以下所有水均为实验室制备的蒸馏水或超纯水)
a)水，实验室制备的蒸馏水或超纯水
b)浓硝酸，65％(质量分数)
b-1)硝酸溶液，5％体积分数
移取12.5mL浓硝酸于237.5mL水中混匀。
b-2)硝酸溶液，20％体积分数
移取50mL浓硝酸于200mL水中混匀。
c)过氧化氢，30％(质量分数)
d)标准溶液
d-1)标准空白溶液
5％硝酸溶液b-1)
d-2)标准储备液
砷，介质5％硝酸溶液b-1)。
d-3)标准工作溶液
准确移取不同体积的标准储备液d-2)至不同的塑料容量瓶中，用5％硝酸溶液b-1)稀释定容。得到不同浓度砷的标准工作溶液。
f)高纯度氩气，纯度应不低于99.999％。
分析步骤
a)称取0.2-0.3g烟样于消解罐中(精确至0.1mg)；
b)依次加入5mL浓硝酸和2mL过氧化氢，旋紧密封，置于微波消解仪中进行消解,待温度降至室温后取出消解罐。同时做空白试验；
c)将消解罐中试样转至50mL塑料容量瓶，用水冲洗消解罐3-4次，清洗液一并转移至容量瓶中。用水定容，摇匀后得试样液；
d)用电感耦合离子体质谱仪(ICP-MS)测定总砷含量。
结果的计算与表述
a)砷含量的计算
试样中的砷含量，按式(1)进行计算：
式中：
X——试样中砷的含量，单位为微克每克(μg/g)
c——试样溶液浓度，单位为微克每升(μg/L)；
c0——试样空白溶液浓度，单位为微克每升(μg/L)；
V——试样溶液体积，单位为毫升(mL)；
m——试样质量，单位为克(g)；
w——试样中水分含量，％。
EMS烟草突变体的砷胁迫下杀青烟叶中总砷离子含量
在未受砷胁迫处理(沙与植烟基质1:1等体积混合)的土壤中,没有突变的野生烟草云烟87杀青烟叶中总砷离子平均含量为0.50mg/Kg，NtPht1-7基因编码区第648位点发生G到A突变的烟草EMS突变体NtPht1-7的砷离子平均含量为0.46mg/Kg；在砷胁迫处理(沙与砷含量高的矿区土1:1等体积混合)的土壤中，没有突变的野生烟草云烟87杀青烟叶中总砷离子平均含量为0.76mg/Kg，NtPht1-7基因编码区648位点发生G到A突变的烟草EMS突变体NtPht1-7的总砷离子平均含量为0.56mg/Kg(图3)。砷胁迫土壤中突变体材料NtPht1-7的杀青烟叶中总砷离子含量降低了26.12％，总砷离子有显著的降低，因此该EMS突变体材料对于烟草育种具有重要的价值。
SEQ ID NO.1：
ATGGCTAAAGATCAATTACAAGTACTAAATGCACTAGATGTAGCAAAAACACAATTATACCATTTCACTGCAATTGTAAT
TGCTGGAATGGGATTTTTTACTGATGCATATGACCTATTTTGCATTTCATTAGTCACAAAATTACTTGGCCGAATTTACT
ATCATCATCACGAAAATGCACCAAAACCAGGTATTCTCCCTCCTCCTATCGCGGCTGCTGTTAATGGCGTGGCGTTTGTT
GGTACTCTTTCGGGGCAACTGTTTTTCGGGTGGCTAGGTGATAAATTAGGGAGAAAAAAAGTTTATGGAATGACACTTAT
GCTTATGGTTATTTGTTCAATAGCTTCTGGACTTTCATTTGGTAAAACACCTAATGGTGTTATAACCACTTTATGTTTTT
TTCGATTTTGGCTTGGTTTTGGTATTGGTGGCGATTACCCTTTATCCGCGACGATTATGTCTGAATATGCAAATAAAAAG
ACTCGTGGGGCTTTTATTGCTGCTGTTTTTGCAATGCAAGGTTTTGGTATTTTAACAGGCGGAATAGTTGCTCTTATTGT
TGCTGGTGCATTTAAAAATGCTTACCCTTCACCAATTTATTCAGTAAACCCTAAAGATTCAACACCACCTGAAGCTGATT
ATGTTTGAAGAATTATTGTTATGTTTGGTGCAATTCCAGCTTTACTTACTTATTATTGGCGTATGAAGATGCCTGAAACT
GCCCGTTACACGGCCTTAGTCGCGAAAAATGCTGAAAAAGCTGCTGCTGATATGTCCAAAGTATTGAACGTTGAAATTGA
AGTAGAGACAAATAAAGTTGAAGAAAAACGACATGATTTTGGTTTGTTTACTAAGGAATTTCTTCGTCGTCATGGACTTC
ACTTGCTAGGAACAACGAGTACATGGTTTTTATTAGACATTGCTTTTTACAGTCAAAATCTTTTTCAGAAAGATATATTT
AGTAAAATTGGATGGATTCCTCATCCAGAAACGATGAACGCGTTAGATGAAGTTTTCAAGATTGCAAAGGCACAGACTCT
TATTGCACTTTGTAGTACTGTTCCAGGTTACTGGTTTACAGTAGCATTTATTGATAAAATGGGTCGATTTGCTATACAAT
TAATGGGATTCTTTTTCATGACAGTGTTCATGTTTGCTTTAGCCATTCCATATAATCACTGGACACAAAAGGAAAACAGA
ATAGGGTTTGTTATTATGTATTCACTTACCTTTTTCTTCGCTAATTTTGGTCCAAATGCAACAACATTTGTTGTACCCGC
TGAGATTTTTCCAGCTAGGTTAAGATCAACGTGCCACGGAATATCAGCAGCAGCTGGAAAAGCAGGAGCAATTGTTGGGG
CATTTGGATTTTTATATGCAGCTCAATCTACAGATCCATTAAAGGTTGATGCTGGTTATCCAACTGGTATAGGTGTGAAA
AATGCACTTATTGTTCTTGGTTGTGTTAATTTACTTGGAATGTTGTTTACATTCTTGGTGCCAGAATCCAAAGGAAAATC
ATTGGAAGAAATGTCTAAGGAAAATGAAGGAGAAGAAGAAAATTATGAAAAAGACACTAAGGCAGAGAATGCACAAACAA
TTCCACTTTAA
SEQ ID NO.2：
TTTTAACAGGCGGAATAGTTGCTC
SEQ ID NO.3：
GTTCCTAGCAAGTGAAGTCCATGA
SEQ ID NO.4:
ATGGCTAAAGATCAATTACAAGTACTAAATGCACTAGATGTAGCAAAAACACAATTATAC
CATTTCACTGCAATTGTAATTGCTGGAATGGGATTTTTTACTGATGCATATGACCTATTT
TGCATTTCATTAGTCACAAAATTACTTGGCCGAATTTACTATCATCATCACGAAAATGCA
CCAAAACCAGGTATTCTCCCTCCTCCTATCGCGGCTGCTGTTAATGGCGTGGCGTTTGTT
GGTACTCTTTCGGGGCAACTGTTTTTCGGGTGGCTAGGTGATAAATTAGGGAGAAAAAAA
GTTTATGGAATGACACTTATGCTTATGGTTATTTGTTCAATAGCTTCTGGACTTTCATTT
GGTAAAACACCTAATGGTGTTATAACCACTTTATGTTTTTTTCGATTTTGGCTTGGTTTT
GGTATTGGTGGCGATTACCCTTTATCCGCGACGATTATGTCTGAATATGCAAATAAAAAG
ACTCGTGGGGCTTTTATTGCTGCTGTTTTTGCAATGCAAGGTTTTGGTATTTTAACAGGC
GGAATAGTTGCTCTTATTGTTGCTGGTGCATTTAAAAATGCTTACCCTTCACCAATTTAT
TCAGTAAACCCTAAAGATTCAACACCACCTGAAGCTGATTATGTTTGGAGAATTATTGTT
ATGTTTGGTGCAATTCCAGCTTTACTTACTTATTATTGGCGTATGAAGATGCCTGAAACT
GCCCGTTACACGGCCTTAGTCGCGAAAAATGCTGAAAAAGCTGCTGCTGATATGTCCAAA
GTATTGAACGTTGAAATTGAAGTAGAGACAAATAAAGTTGAAGAAAAACGACATGATTTT
GGTTTGTTTACTAAGGAATTTCTTCGTCGTCATGGACTTCACTTGCTAGGAACAACGAGT
ACATGGTTTTTATTAGACATTGCTTTTTACAGTCAAAATCTTTTTCAGAAAGATATATTT
AGTAAAATTGGATGGATTCCTCATCCAGAAACGATGAACGCGTTAGATGAAGTTTTCAAG
ATTGCAAAGGCACAGACTCTTATTGCACTTTGTAGTACTGTTCCAGGTTACTGGTTTACA
GTAGCATTTATTGATAAAATGGGTCGATTTGCTATACAATTAATGGGATTCTTTTTCATG
ACAGTGTTCATGTTTGCTTTAGCCATTCCATATAATCACTGGACACAAAAGGAAAACAGA
ATAGGGTTTGTTATTATGTATTCACTTACCTTTTTCTTCGCTAATTTTGGTCCAAATGCA
ACAACATTTGTTGTACCCGCTGAGATTTTTCCAGCTAGGTTAAGATCAACGTGCCACGGA
ATATCAGCAGCAGCTGGAAAAGCAGGAGCAATTGTTGGGGCATTTGGATTTTTATATGCA
GCTCAATCTACAGATCCATTAAAGGTTGATGCTGGTTATCCAACTGGTATAGGTGTGAAA
AATGCACTTATTGTTCTTGGTTGTGTTAATTTACTTGGAATGTTGTTTACATTCTTGGTG
CCAGAATCCAAAGGAAAATCATTGGAAGAAATGTCTAAGGAAAATGAAGGAGAAGAAGAA
AATTATGAAAAAGACACTAAGGCAGAGAATGCACAAACAATTCCACTTTAA
以上所述的仅是本发明的部分具体实施例，方案中公知的具体内容或常识在此未作过多描述(包括但不仅限于简写、缩写、本领域惯用的单位)。应当指出，上述实施例不以任何方式限制本发明，对于本领域的技术人员来说，凡是采用等同替换或等效变换的方式获得的技术方案均落在本发明的保护范围内。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (2)
1.一种烟草NtPht1-7基因突变体，其特征在于，所述烟草NtPht1-7基因突变体的核苷酸序列如SEQ ID NO. 1所示。
2.如权利要求1所述的烟草NtPht1-7基因突变体的应用，为制备低砷离子含量的烟草。
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