CN108660141B - NtCNGC1基因在烟草抗青枯病中的应用 - Google Patents
NtCNGC1基因在烟草抗青枯病中的应用 Download PDFInfo
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Abstract
本发明公开了一种NtCNGC1基因在烟草抗青枯病中的应用，所述NtCNGC1基因的核苷酸系列如SEQ ID NO:1所示，本发明的NtCNGC1基因在烟草中超表达能够显著提高烟草对青枯病菌的抗性，该基因在烟草抗青枯病基因工程中具有十分重要的应用价值。
Description
技术领域
本发明涉及一种NtCNGC1基因在烟草抗青枯病中的应用，属于植物基因工程领域。
背景技术
青枯劳尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是世界范围的毁灭性土传病害，给烟草生产带来巨大损失。目前国内外对烟草抗青枯病的分子机理研究甚少，对控制青枯病的抗性基因数目、作用机制、烟草与青枯菌互作机理还不清楚。从转录组水平上分析和挖掘烟草品系中可能存在的抗青枯病基因，不仅能从分子水平上加深对烟草与青枯菌互作的认识，还将有助于青枯病抗源合理利用和抗病育种工作。
发明内容
本发明要解决的技术问题是：提供一种NtCNGC1基因在烟草抗青枯病中的应用，以提高烟草对青枯病的抗性，降低青枯病给烟草生产带来的经济损失。
本发明的技术方案是：NtCNGC1基因在烟草抗青枯病中的应用，所述NtCNGC1基因的核苷酸系列如SEQ ID NO:1所示。
进一步的，将NtCNGC1基因与pBI121质粒融合，构件植物表达载体，再将植物表达载体转入农杆菌菌株中，通过农杆菌介导的叶盘法转化，经卡那霉素筛选，获得抗性植株，进一步检测获得阳性植株，即得到对青枯病有高抗性的烟株。
本发明的有益效果是：通过在烟草中超表达NtCNGC1基因能够显著提高烟草对青枯病菌的抗性，该基因在烟草抗青枯病基因工程中具有十分重要的应用价值。
附图说明
图1为烟草NtCNGC1基因的PCR扩增结果，其中，M：DM 10000 marker；1：cDNA扩增产物；2：基因组DNA扩增产物。
图2为NtCNGC1的基因结构示意图，其中，方框为外显子，水平线为内含子，开放阅读框和非翻译区分别用黑色方框和白色方框表示，ATG为起始密码子，TGA为终止密码子，数字为内含子或外显子的长度(bp)。
图3为植物CNGCs的系统进化树，其中，At：拟南芥；Nt：烟草；Hv：大麦；Mt：蒺藜苜蓿；Os：水稻。拟南芥CNGCs的名称根据帕斯卡命名法命名；水稻CNGCs的名称按照Nawaz命名方式命名。各基因的GenBank登录号如下：AtCNGC1(NP_200125)，AtCNGC2(NP_197045)，AtCNGC3(NP_566075)，AtCNGC4(NP_851188)，AtCNGC5(NP_851209)，At CNGC6(NP_565560)，AtCNGC7(NP_173051)，AtCNGC8(NP_173408)，AtCNGC9(NP_194785)，AtCNGC10(NP_001184885)，AtCNGC11(NP_182167)，AtCNGC12(NP_850454)，AtCNGC13(NP_192010)，AtCNGC14(NP_850056)，AtCNGC15(NP_180393)，AtCNGC16(NP_190384)，AtCNGC17(NP_194765)，AtCNGC18(NP_196991)，AtCNGC19(NP_188396)，AtCNGC20(NP_566585)，OsCNGC2(NP_001057767)，OsCNGC4(ABF97880)，OsCNGC5(ABA98416)，OsCNGC7(NP_001047485)，OsCNGC8(ABA95858)，OsCNGC9(NP_001063911)，OsCNGC12(NP_001048268)，OsCNGC14(NP_001051331)，OsCNGC15(NP_001172593)，OsCNGC16(NP_001055973)，HvCBT1(CAA05637)，NEC1(AAY58312)，NtCBP4(AF079872)，NtCBP7(AF33669)，MtCNGC19(XP_003620101)，MtCNGC20(XP_003610842)。通过Clustal W序列比对，采用Mega 4.0软件(Neighbor-Joining法)构建植物CNGCs的系统进化树。
图4为烟草接种青枯病菌病后NtCNGC1基因的表达分析。
图5为NtCNGC1基因在不同组织中的表达分析。
图6为pBI35S:NtCNGC1重组质粒结构图。
图7为超量表达NtCNGC1提高烟草青枯病抗性，其中，A：非转基因植株叶片接种后10d的发病症状；B：转基因植株叶片接种后10d的发病症状；C：转基因植株(右侧3株)和非转基因植株(左侧3株)接种后14d的发病症状。
具体实施方式
一、实施例
实验材料和试剂：
强致病力烟草青枯病菌由烟草行业分子遗传重点实验室分离鉴定并继代保存于TTC培养基。烟草青枯病抗病品种DB101和易感品种红花大金元由贵州省烟草科学研究院良种繁育中心提供，采用漂浮育苗，待烟苗长至2～3片真叶时，移栽至花盆中(泥炭土：蛭石：珍珠岩＝1：1：1)培养。
1、NtCNGC1基因的克隆
根据GenBank数据库中的烟草CNGC1基因序列(登录号：XM_016655150)(CNGC1基因核苷酸序列如SEQ ID NO:1所示)设计克隆引物CNGC1-F和CNGC1-R(表1)。提取DB101烟草叶片总RNA，并反转录为cDNA，作为模版对烟草CNGC1基因进行PCR扩增、克隆和测序。同时，从DB101烟草叶片中提取基因组DNA，作为模版进行PCR扩增、克隆和测序。
2、NtCNGC1基因的生物信息学分析
采用SMART在线工具http://smart.embl-heidelberg.de/检索保守模体；采用PlantCARE在线工具http://bioinformatics.psb.ugent.be/webtools/plantcare/html/预测启动子的顺时作用元件；采用ClustalX 2.0软件对齐所有CNGC序列；采用MEGA4.0软件构建进化树，方法为邻接法，自举检测次数为1000。
3、NtCNGC1基因的荧光定量PCR表达分析
当烟苗长至5～6片真叶时，选取DB101和红花大金元生长状况一致的烟苗，接种青枯病菌，以蒸馏水处理烟苗作为对照。分别在接种后3、6、12、24和48h取接种叶片作为检测样品；并在DB101苗期和旺长期，取中部叶、茎和根作为检测样品，所有样品液氮速冻保存。采用Trizol试剂盒(Invitrogen公司)提取各样品的总RNA，DNaseⅠ除去基因组DNA后，采用反转录试剂盒(TaKaRa公司)将其反转录成cDNA，荧光定量PCR进行烟草CNGC1基因的表达分析。基因扩增上游引物为CNGC1-rF、下游引物为CNGC1-rR，以烟草β-actin基因作为内参，内参基因扩增中所用上下游引物为Actin-rF和Actin-rR(表1)。各处理样品进行3次重复。采用2-ΔΔCT法进行基因相对定量分析[。
表1 NtCNGC1基因的克隆和表达分析引物
4、载体构建
根据NtCNGC1基因的序列设计合成特异性引物对CNGC1-121F和CNGC1-121R，引物末端分别引入XbaⅠ和SacⅠ酶切识别位点。以克隆入pGEM-T Easy载体的NtCNGC1基因为模板扩增目标基因。用PrimeSTAR高保真酶进行PCR扩增然后将XbaⅠ和SacⅠ双酶切片段插入农杆菌植物表达载体pBI121相应酶切位点，获得35S驱动的植物表达载体pBI35S:NtCNGC1，最后将重组质粒转入农杆菌菌株LBA4404中。
5、基因转化
采用农杆菌介导的叶盘法转化红花大金元。经卡那霉素(Kan)多重筛选，获得Kan抗性植株，进一步用NPTⅡ特异引物进行PCR鉴定。PCR鉴定阳性植株抽提RNA用qRT-PCR方法检测表达量。
6、转基因植株青枯病抗性鉴定
将转基因植株进行大量无性繁殖，生根后移栽到温室塑料钵中种植，当烟苗长至5～6片真叶时，选取红花大金元转基因植株和非转基因对照植株进行接种。
二、结果与分析：
1、NtCNGC1基因的序列特征
分别以烟草基因组DNA和cDNA为模板，用引物对CNGC1-F/CNGC1-R扩增出目的条带(图1)。测序结果表明，NtCNGC1基因组DNA扩增产物长度为6519bp，cDNA扩增产物长度为2445bp，其中编码区全长为2154bp。该基因具7个内含子，第7内含子最长，为952bp，第6内含子次之，为659bp，第2内含子最短，仅86bp；8个外显子中，第2外显子最长，为565bp，其次为第7外显子，长度为476bp，第5外显子最短，仅112bp(图2)。
2、NtCNGC1编码蛋白的特征
NtCNGC1编码717个氨基酸，等电点为9.35，分子量为83kD。SMART在线工具检索发现，NtCNGC1编码蛋白有6个保守结构域，分别位于97～118，133～152，185～207，257～279，380～402和488～619氨基酸残基位置，N端为5个连续的跨膜结构域(Transmembraneregion)，C端为环化核苷酸结合域(Cyclic nucleotide-monophosphate binding domain，cNMP)。
3、NtCNGC1基因的上游启动子分析
启动子预测结果表明，位于NtCNGC1基因起始密码子ATG的5′区上游1500bp的启动子核苷酸序列具有多种顺式作用元件(表2)，包括与光响应、真菌激发子响应、热响应、干旱响应、伤口响应、水杨酸响应、厌氧诱导、防御与应激反应、病原菌应答、木质部诱导性表达和韧皮部阻遏性表达、代谢调节及器官发育等相关元件。
表2 NtCNGC1基因上游调控区顺式作用元件
4、NtCNGC1的系统进化分析
根据CNGC序列中的2个功能结构域，比较NtCNGC1与全部拟南芥CNGC基因家族成员和部分烟草、大麦、蒺藜苜蓿、水稻中假定的CNGC之间的亲缘关系，结果表明(图3)，CNGC主要分成5个亚组(Ⅰ、Ⅱ、Ⅲ、Ⅳa、Ⅳb)，其中，Ⅰ、Ⅱ、Ⅲ亲缘关系很近，它们与另外两个亚组Ⅳa和Ⅳb的关系较远。NtCNGC1与拟南芥抗病正调控因子AtCNGC11、AtCNGC12和AtCNGC11/12嵌合体同属于CNGC家族的第Ⅰ亚组，而拟南芥抗病负调控因子AtCNGC2和AtCNGC4属于CNGC家族的第Ⅳb亚组。
5、NtCNGC1基因的表达分析
为了验证NtCNGC1基因的转录组表达结果，利用荧光定量PCR检测NtCNGC1响应青枯病菌侵染的表达情况。在烟草青枯病菌侵染后，抗病品种DB101中NtCNGC1基因的表达量从3h就有上调表达趋势，而感病品种红花大金元在12h才表现出上调表达，且表达量前者明显高出后者(图4)。荧光定量PCR进一步检测NtCNGC1基因在抗病烟草DB101中的组织表达情况，发现在苗期和旺长期NtCNGC1在根中表达量最高，茎中其次，叶中最低(图5)。
6、植物表达载体构建及转基因植株的获得
NtCNGC1基因编码区扩增片段用XbaⅠ和SacⅠ双酶切后插入到pBI121质粒的相应位点，获得35S驱动的植物表达载体，最后将重组质粒转入农杆菌菌株LBA4404中，图6为pBI35S:NtCNGC1重组质粒结构图。通过农杆菌介导的叶盘法转化，经卡那霉素(Kan)多重筛选，获得Kan抗性植株，进一步经过PCR和qRT-PCR的检测，获得阳性植株。
7、超量表达NtCNGC1转基因植株晚疫病抗病性鉴定
为了解烟草超量表达NtCNGC1基因是否会影响青枯病抗性，将3个超量表达转基因株系和对照株系种植于人工气候室塑料钵中。当烟苗长至5～6片真叶时，采用温室苗期叶片人工注射接种法对转基因株系和对照株系进行青枯病抗性鉴定。接种青枯病原菌后，每天观察接种叶片和整株的发病情况。接种10d后转基因株系和对照株系叶片接种点均形成坏死病斑，同时对照株系的整个叶片泛黄萎蔫(图7-A)；接种14d后对照株系表现出明显枯萎(图7-B)，而转基因株系变化不大，表明NtCNGC1基因超量表达提高了烟草对青枯病的抗性。
结论：在烟草中超表达NtCNGC1能够显著提高烟草对青枯病菌的抗性。核苷酸系列表：
SEQUENCE LISTING
序列表
<110> 贵州省烟草科学研究院
<120> NtCNGC1基因在烟草抗青枯病中的应用
<160> 1
<210> 1
<211> 2154
<212> DNA
<213> NtCNGC1基因
<400> 1
ATGATGAATC TCAAACAAGA TAAATATGTA AGGTTTGAGG ACTGGAAGTC AGAAGAGTCA 60
TCTTTCAACT CTCCAAATAA CAGGCCATTT CATATCAGAA AACCATCATT TAGTTTGTTG 120
ATGAGTAACA TTAGAAGAAG GCTTGAGAGT GGTTCTGAAA GAATTAGTAG TTGGAGAAAA 180
TCAATACGCG TCCATCCTCT AACCGCTAAA CCAACAAAAG ATCAATCTAT CTCGTCAAAG 240
AAACAAATTC TTGATCCTCA GGGGCGATTT CTTCAGCAAT GGAACAAAAT ATTTGTATTG 300
ATTTGTACAA TTGCAGTGTC ATTGGATCCA CTATTCTTCT ACATACCTGT CATTGATAAC 360
GAAAACAAGT GCCTTGATTT GGACACGACG TTAAAGATCA CTGCTTGTGT TCTACGTTCG 420
ATCACTGATC TTTTCTATAT CTTTCACATT ATCTTGCAAT TTCGTACTGG TTTTATCCCT 480
CCTTCTTCTC GAGTATTTGG AAGGGGTGAG TTGATTGAAG ATTCCTCTGC TATAGCCAAG 540
AGATATTTGA AATCTTATTT CATTGTTGAT ATTTTAGCAG TTCTTCCACT TCCACAGATT 600
GTGATATTGA TTATCAGTCC AAGTGTGAAT AGCCCGATTT CTCTAGCGAC GAAAGAAATC 660
TTGAAGATTG TCATTTTTGT CCAATATGTT CCAAGAATAT TTAGAATTTA TCCATTGTAT 720
AAAGAAGTAA CAAGAACTGC AGGCTTATTT ACAGAAACGG CATGGGGTGG AGCTGCTTTC 780
AACCTTTTCC TTTACATGTT AGCCAGTAAT GTAGTCGGAG CCTTCTGGTA CTTGATCTCA 840
GTAGAACGCC AAGATACATG TTGGCGCGAT GCATGTGATA AGATTGACTC ATGTTCTTTA 900
GACAACTTAT ACTGTGGAGG AAACAGAAAC GGAAACGCTT TGCTTCTAAA TTCTTCTTGC 960
CCTCTCCTGA AATCAGAAGA TATAAAAGAT CCAAATGACT TTGATTTTGG AATATTTCTT 1020
GATGCTCTTC AGTTTCGGAT AGTAGAAAAG CAAAAATTCT GGTCCAAACT CTTCTATTGC 1080
TTTTGGTGGG GACTGAGAAA CTTAAGTTCT CTTGGCCAAA ACCTTAAGAC AAGCACCTTT 1140
GTTGGGGAGA TTCTCTTTGC TGTCTTCATT TCAATTATTG GGCTAATCTT GTTTTCCTTG 1200
CTTATTGGCA ATATGCAGAA ATATTTGCAG TCTATCACAG TAAGAGTAGA AGAGATGAGA 1260
GTGAGAAGGC GAGACGCAGA GCAATGGATG TCTCATCGCA TGCTTCCTGA TAATCTGAGA 1320
GCACGAATTA GAAGACATGA ACAGTACAAA TGGCAAGAAA CCAGGGGTGT AGAGGAAGAT 1380
TTACTTATTC ATAATCTTCC TAGAGACTTG AGAAGAGATT TAAAGCGCCA TCTCTGTTGG 1440
TCTTTGGTTA AAAGAGTTCC AATGTTTGAG AAAATGGATG AACAATTACT AGATGCAATG 1500
TGTGGTCGAC TTAAACCAGC ACTCTACACA GAGAAAAGTT TCATAATCCG AGAAGGCGAT 1560
CCAGTAGATG AGATGCTCTT TCTAATGAGA GGTACTCTAA TAACTATGAC AACAAATGGT 1620
GGAAGAACTG GTTTTTTCAA CTCTGTATCA CTCAAAGCTG GTGATTTCTG TGGAGAGGAG 1680
CTTCTTACTT GGGCTTTAGA CCCTCACACT TCCTCTAGTC TTCCTACTTC AACAAGAACA 1740
GTTCAAGCTG AAACTGATAT AGAAGCTTTT GCCCTCACTG CTGATGATCT CAAGTTTGTT 1800
GCCTCACAGT TTCGACGTCT TAATAGCAAA CAGCTTCAAC ATAGTTTCAG GCTCTACTCA 1860
CAGCAATGGA GGACATGGGG TGCATGCTTT ATACAAGTAG CATGGCGTCG ACATTGTAGG 1920
AACAAGCTTG AGAAATCTTT AAGAGAGGAA GAAGATAGAT TGCAGGTTGC ATTAGCAAAA 1980
GAGAGTACAA ATGCACCAAG TCTTGGAGCT ACCATTTATG CATCAAGATT TGCTGCTAAT 2040
GCGCTGCGCG CCTTGCGACG CAACCATACA ACTGGTGCCA AATTATCTCC CACACTACCT 2100
CTACTGCTTC AGAAACCAGC TGAACCAAAT TTCAGTGAGG AAAATCATTC ATGA 2154
Claims (2)
1.NtCNGC1基因在烟草抗青枯病中的应用，所述NtCNGC1基因的核苷酸序列如SEQ IDNO:1所示，所述NtCNGC1基因在烟草中超量表达。
2.根据权利要求1所述NtCNGC1基因在烟草抗青枯病中的应用，其特征在于：将NtCNGC1基因与pBI121质粒融合，构建植物表达载体，再将植物表达载体转入农杆菌菌株中，通过农杆菌介导的叶盘法转化，经卡那霉素筛选，获得抗性植株，进一步检测获得阳性植株，即得到对青枯病有抗性的烟草植株。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6033891A (en) * | 1999-03-09 | 2000-03-07 | Novo Nordisk Biotech, Inc. | Nucleic acids encoding polypeptides having cellobiose dehydrogenase activity |
| WO2001007596A1 (en) * | 1999-07-23 | 2001-02-01 | Wisconsin Alumni Research Foundation | Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death |
| CN106811472A (zh) * | 2017-01-16 | 2017-06-09 | 福建农林大学 | NtRRS2基因及其在烟草抗青枯病中的应用 |
| CN107988251A (zh) * | 2017-11-29 | 2018-05-04 | 新疆农垦科学院 | 植物不饱和脂肪酸含量相关蛋白GhSAD在抗寒性鉴定过程中的应用 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468794B (zh) * | 2013-07-16 | 2015-03-18 | 贵州省烟草科学研究院 | 一种烟草青枯病分子检测引物、检测方法及应用 |
| CN104082124B (zh) * | 2014-06-30 | 2016-08-24 | 福建农林大学 | 一种高抗性烟草品种的选育方法 |
| CN105309082B (zh) * | 2015-10-21 | 2018-03-23 | 贵州省烟草科学研究院 | 一种烟草青枯病综合防控方法 |
| CN106754963B (zh) * | 2017-01-16 | 2019-09-20 | 福建农林大学 | NtRRS3基因及其在烟草抗青枯病中的应用 |
| CN107619830A (zh) * | 2017-09-26 | 2018-01-23 | 西南大学 | 一种植物抗病基因NtWRKY50及其在烟草抗青枯病中的应用 |
| CN111304213B (zh) * | 2019-12-31 | 2022-03-25 | 中国农业科学院生物技术研究所 | 甜橙CsCNGC2和CsCNGC4基因用于提高植物柑橘黄龙病菌抗性的应用 |
-
2018
- 2018-05-28 CN CN201810524616.7A patent/CN108660141B/zh not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6033891A (en) * | 1999-03-09 | 2000-03-07 | Novo Nordisk Biotech, Inc. | Nucleic acids encoding polypeptides having cellobiose dehydrogenase activity |
| WO2001007596A1 (en) * | 1999-07-23 | 2001-02-01 | Wisconsin Alumni Research Foundation | Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death |
| CN106811472A (zh) * | 2017-01-16 | 2017-06-09 | 福建农林大学 | NtRRS2基因及其在烟草抗青枯病中的应用 |
| CN107988251A (zh) * | 2017-11-29 | 2018-05-04 | 新疆农垦科学院 | 植物不饱和脂肪酸含量相关蛋白GhSAD在抗寒性鉴定过程中的应用 |
Non-Patent Citations (6)
| Title |
|---|
| An apple cyclic nucleotide-gated ion channel gene highly responsive to Botryosphaeria dothidea infection enhances the susceptibility of Nicotiana benthamiana to bacterial and fungal pathogens;Zhang W等;《Plant Sci》;20180203;第269卷;摘要，第2.5、3.3节 * |
| Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance;Sunkar R等;《Plant J》;20001130;第24卷(第4期);533-542 * |
| PREDICTED: Nicotiana tabacum cyclic nucleotide-gated ion channel 1-like (LOC107827918), mRNA;GenBank Database;《GenBank Database》;20160503;Accession No. XM_016655150 * |
| 一个受青枯病菌诱导的烟草功能基因NtCNGC1的克隆与表达分析;林世锋等;《中国烟草学报》;20180706;第24卷(第6期);86-92 * |
| 植物环核苷酸门控通道(CNGC)基因家族的结构与功能;王月等;《植物生理学通讯》;20071231;第43卷(第1期);1-8 * |
| 番茄CNGC基因家族的基因组水平鉴定及其抗病功能分析;蔡新忠;《中国学位论文全文数据库》;20150907;全文 * |
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