CN110105439B - 海州常山NAC基因CtNAC2及其应用 - Google Patents
海州常山NAC基因CtNAC2及其应用 Download PDFInfo
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- CN110105439B CN110105439B CN201910530511.7A CN201910530511A CN110105439B CN 110105439 B CN110105439 B CN 110105439B CN 201910530511 A CN201910530511 A CN 201910530511A CN 110105439 B CN110105439 B CN 110105439B
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- nac
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Abstract
本发明公开了海州常山NAC基因CtNAC2及其应用。本发明选取两个种源的海州常山,从海州常山组织中克隆得到一个新的NAC基因,命名为CtNAC2,其核苷酸序列如SEQ ID NO.1所示,表达蛋白的氨基酸序列如SEQ ID NO.2所示,并通过qRT‑PCR技术对海州常山不同组织中CtNAC2基因表达量及海州常山盐胁迫不同处理时间下叶片中CtNAC2基因表达量进行测定分析,结果表明NAC基因CtNAC2在海州常山的茎和根中的表达较高。在盐胁迫处理下,反应72h时基因表达量最高,参与海州常山茎和根的生长发育和盐胁迫的响应,可用于在植物的抗逆基因工程改良,应用前景广阔。
Description
技术领域
本发明属于基因工程技术领域,具体涉及海州常山NAC基因CtNAC2及其应用。
背景技术
NAC基因家族广泛分布于植物界各个物种,是植物所特有的最大转录因子家族之一(周鸿慧等,2017),NAC基因家族有A、B、C、D、E五个亚区,起初在矮牵牛中被发现,之后在其他植物中也发现NAC基因家族(段奥其等,2018),现已证明NAC基因家族广泛参与植物发育、植物生物和非生物胁迫应答,且可增强植物对胁迫的防御和耐受能力(Tak H et al.,2018),如白桦中BpNAC012可以增强盐胁迫和渗透胁迫耐受性(Hu et al.,2018)。
海州常山(Clerodendrum trichotomum Thunb.),又名臭梧桐,为马鞭草科(Verbenaceae)赦桐属(Clerodendrum L.)灌木或小乔木,其花果美丽,是一种夏季观花、冬季观果的优良观赏花木(杨秀莲等,2019)。此外海州常山具有药用功能,其叶、枝干和根均可入药,具有抗高血压、关节炎、风湿和抗炎等作用(胡海军等,2014;et al.,2017)。沿海土壤盐渍化一直是一个世界性问题,选择合适的沿海盐碱植物一直是恢复沿海生态的关键措施(He et al.,2014;Li et al.,2010)。海州常山对于不良非生物胁迫具有良好抗性,拥有耐盐(岳远征等,2018)、耐水淹(曾德静等,2013)、耐高温(陈燕等,2015)和耐干旱(魏娟等,2009)等优良性状,尤其对盐胁迫有着优良的抗性,是一种可用于恢复沿海盐碱地生态的耐盐植物(李娅等,2017)。海州常山作为优良的耐盐植物,目前关于其在盐胁迫下分子响应机制的研究仍较为缺乏。探明海州常山在盐胁迫下的分子响应机制,对于海州常山在生态恢复上具有十分重要的意义。
发明内容
发明目的:针对技术的不足,本发明的目的是提供海州常山NAC基因CtNAC2,以实现NAC基因在海州常山盐胁迫应答响应机制的进一步研究;本发明的另一目的是提供海州常山NAC基因CtNAC2的表达蛋白;本发明的再一目的是提供上述基因在提高海州常山盐胁迫耐受性中的应用,所述基因在海州常山的茎和根中的表达较高。在盐胁迫处理下,反应72h时基因表达量最高,参与海州常山茎和根的生长发育和盐胁迫的响应,可用于在植物的抗逆基因工程改良,应用前景广阔。
技术方案:为了实现上述发明目的,本发明采用的技术方案为:
海州常山NAC基因CtNAC2,其核苷酸序列如SEQ ID NO.1所示。
海州常山NAC基因CtNAC2的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
所述海州常山NAC基因CtNAC2在提高海州常山盐胁迫耐受性中的应用。
所述海州常山NAC基因CtNAC2的表达载体。
所述海州常山NAC基因CtNAC2的宿主菌。
所述海州常山NAC基因CtNAC2克隆引物为:上游引物:5′-CATCGCCATCATCAGCAC-3′;下游引物:5′-CCGCCACATAATCACCG-3′。
有益效果:与现有技术相比,本发明选取两个种源的海州常山,从海州常山组织中克隆得到一个新的NAC基因,命名为CtNAC2,并通过qRT-PCR技术对海州常山不同组织中CtNAC2基因表达量及海州常山盐胁迫不同处理时间下叶片中CtNAC2基因表达量进行测定分析,结果表明NAC基因CtNAC2在海州常山的茎和根中的表达较高。在盐胁迫处理下,反应72h时基因表达量最高,参与海州常山茎和根的生长发育和盐胁迫的响应,可用于在植物的抗逆基因工程改良,应用前景广阔。
附图说明
图1是海州常山不同组织中CtNAC2基因表达量图;
图2是海州常山盐胁迫不同处理时间下叶片中CtNAC2基因表达量图。
具体实施方式
下面结合具体实施例进一步说明本发明,但这些实例并不用来限制本发明。
以下实施例所使用的主要供试材料为:采用种植在南京林业大学白马教学研究基地内的泰安、盐城两个海州常山种源。
实施例1
1海州常山组织器官的选取
采样时间为2018年9月13日。在天气情况理想条件下,采集海州常山组织样品。选择生长良好、高度和长势较为一致的3棵植株(作为3个生物学重复)进行采样,每株分别采集根、茎、叶、花、果等组织,采集后放入无菌无酶的离心管中,并迅速用液氮速冻带回实验室,置-80℃超低温冰箱保存备用。
2胁迫处理材料的准备
采集海州常山组织样品时,同时采集两个种源的插穗,将插穗扦插于混合基质(珍珠岩∶蛭石∶泥炭∶沙=1∶1∶1∶1∶2)中,置于培养室培养(25℃,60%RH),定期浇灌养护,保证正常生长,待插穗生长出4对以上叶片后使用。所有胁迫处理均在人工气候培养箱中完成(宁波东南仪器厂RDN-1000-3),待处理前一周将海州常山扦插苗放入每瓶培养基均为150mL的1/4MS培养基中,移入人工气候培养箱中适应一周,设置光照时间为14h,昼夜温度为25/21℃,光照强度为180mmol/m2/s,相对湿度为60%。将插穗置于含1.0%NaCl的培养基中(每瓶均为150mL培养基,参考海州常山耐盐阈值0.8%NaCl浓度确定本实施例所使用的NaCl浓度),于胁迫0h、2h、6h、12h、24h、48h和72h采集插穗叶片。
3海州常山CtNAC2克隆
海州常山总RNA提取均采用北京艾德莱生物有限公司的EASY spin Plus试剂盒,提取的RNA先经过用1.5%的琼脂糖凝胶电泳验证RNA的完整性,所有OD260/OD280在1.8~2.0之间,OD260/OD230>2.0,所有样品中RNA至少2条清晰条带,18S/28S条带大致为2∶1才会被采用。用Mettler公司生产的核酸检测仪UV5NANO检测RNA质量及浓度。使用北京全式金公司的EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix反转录试剂盒试剂盒合成cDNA第一条链。
根据已有转录组测序数据库获得一条海州常山NAC基因全长,使用primerpremier 5.0设计引物,NAC转录因子基因全长克隆正向引物5′至3′端为CATCGCCATCATCAGCAC,反向引物5′至3′端为CCGCCACATAATCACCG,引物设计原则、PCR反应体系及反应程序参照王晰等人的方法(王晰等,2018,http://kns.cnki.net/kcms/detail/detail.aspx?filename=NNXB201804021&dbcode=CJFD&dbname=CJFD2018&v=)等,通过切胶回收,将产物连接转化,挑选3个阳性克隆送至南京金斯瑞生物有限公司测序。
测序后获得长度为1438bp,如SEQ ID NO.1所示,将此序列命名为CtNAC2,编码蛋白为328个氨基酸组成的序列,如SEQ ID NO.2所示。
4海州常山CtNAC2表达分析
使用TaKaRa公司的Ex TaqTM染料进行qRT-PCR,荧光定量仪型号为赛默飞公司的plied Biosystems StepOne PCR System。使用primer premier 5.0设计引物,正向引物5′至3′端为TTCGGGGAGGGAAGTTCAG,反向引物5′至3′端为AGACATCGCCATCATCAGCA,内参选用课题组之前筛选的海州常山组织和盐胁迫下适用的内参基因UBCE2(引物合成序列:F:GCAAAGGCTGATTGATGAGATTC R:CCTCAACATTGTCTTGGGTGG)和RPL(引物合成序列F:AGTCAATGGTGGCGATGTAGC R:CCCTTGGTCACTCCGATAATGT)),荧光实时定量引物设计原则、PCR反应体系及程序参照母洪娜等人的方法(Mu et al.,2017,https://link.springer.com/article/10.1007%2Fs12041-017-0769-8)。
选取这两个种源海州常山叶、花、果、根和茎作为组织样品并选取两个种源海州常山扦插苗盐胁迫下的叶片作为胁迫处理样品,使用UBCE2和RPL作为内参基因,进行实时荧光定量PCR。结果如图1所示,在组织中,CtNAC2基因在茎中表达最多,根中表达量其次,之后是果、叶和花,在花中的基因表达量最少,泰安种源CtNAC2在茎中的基因表达量是花中的20.83倍,盐城种源CtNAC2在茎中的基因表达量是花中的25.38倍。总体来说泰安种源和盐城种源CtNAC2基因表达量在相同的组织部位下,泰安种源的基因表达量高于盐城种源的基因表达量。
如图2所示,盐胁迫下CtNAC2基因表现为0~2h表达量有小幅上升,泰安种源2h时基因表达量为对照的6倍,盐城种源2h时的基因表达量是对照的5倍。2~12h有下降趋势,12~72h基因表达量增加,72h时基因表达量达到最高,泰安种源72h时CtNAC2基因表达量是0h的12.27倍,盐城种源72h时CtNAC2基因表达量是0h的8.34倍,相同处理条件及相同处理时间下,CtNAC2基因的表达量泰安种源多于盐城种源。
序列表
<110> 南京林业大学
<120> 海州常山NAC基因CtNAC2及其应用
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1438
<212> DNA
<213> 海州常山(Clerodendrum trichotomum Thunb.)
<400> 1
catctctgat attcattctt ctatacacaa gataatttca acacactctc cgccccctgc 60
caccacaacg ccgccacata atcaccgtga aaaatgagca gtttgagcac ggtggaggcg 120
aagctgccac cggggtttcg gttccaccca agagacgaag aactcatatg tgattacctg 180
atgaaatgga tctcgggttg cgcccacccg cttcctctct tgatcgaagt tgaccttaac 240
aagtgcgagc cttgggacat tcctgacgtt gcatgtgttg ggagcagaga atggtatttt 300
tacagccaaa gagacaggaa atatgcaacc gggctccgaa caaatcgggc cacaatatct 360
ggctactgga aagccactgg caaggatcgg cccgtaattc gtaagggaat cctcgttgga 420
atgcggaaga ctctggtatt ctaccagggc cgggctccca aggggagaaa aagcgattgg 480
gttatgcatg agtttcgcca tgaaggaatt catgggcccg gcccgaaacc tccttctccc 540
gtcaaggaag attgggttct atgccgagtg ttcttcaaaa gccgatctga aatttcatcc 600
agaacacaaa acatgaacaa cgaagtacca ccttcttcac catctctgcc gccattaatg 660
gactccttca ctggattcgg cccaacaacc actcagcccg gcccaagtga caaccagtac 720
caagagcagg tgccctgctt ctccattttc aacaacacaa accaagcaaa ccctaatttc 780
tctctcctcg cccacatgga ctcactggag atgcatccca ccaccaagaa catgccaaac 840
tatggaggaa caatccctga tcatcttgga atttataatt ctccaccgtt gatgaacttt 900
aatccttctt cttgtgataa gaagatgatc agatcagttt tgaatcatct gacaaaaatg 960
gaaagtgggc atgatcagat cagtattaag ggtggttctc caagcttcgg ggagggaagt 1020
tcagacagtt acttgtctga agtgggattg tcttctatgt ggaatcacta ctcatgatgc 1080
ggtttggatc ctccgttaca tattgtgcta cgtcatatgt gacattactt ttaatatatg 1140
ctacagttaa ttagcaatgt catccattat atatgacgtt tgataattgt aatagaacag 1200
aggatgtaat accaaaaatt atggagaata tcatgtgctg atgatggcga tgtctagttt 1260
gcaatttcaa gaatggagtt ctgcccaaca acgtcaaatt tagtatgttg tgtgacattg 1320
tttatagtta gctatagcgg tgtgtatgta tattgcatgt atacatacat cttaaaagtc 1380
taatgttaga aaatatgtaa attgaatata tttctatctt gccttggtaa tttgtcag 1438
<210> 2
<211> 328
<212> PRT
<213> 海州常山(Clerodendrum trichotomum Thunb.)
<400> 2
Lys Met Ser Ser Leu Ser Thr Val Glu Ala Lys Leu Pro Pro Gly Phe
1 5 10 15
Arg Phe His Pro Arg Asp Glu Glu Leu Ile Cys Asp Tyr Leu Met Lys
20 25 30
Trp Ile Ser Gly Cys Ala His Pro Leu Pro Leu Leu Ile Glu Val Asp
35 40 45
Leu Asn Lys Cys Glu Pro Trp Asp Ile Pro Asp Val Ala Cys Val Gly
50 55 60
Ser Arg Glu Trp Tyr Phe Tyr Ser Gln Arg Asp Arg Lys Tyr Ala Thr
65 70 75 80
Gly Leu Arg Thr Asn Arg Ala Thr Ile Ser Gly Tyr Trp Lys Ala Thr
85 90 95
Gly Lys Asp Arg Pro Val Ile Arg Lys Gly Ile Leu Val Gly Met Arg
100 105 110
Lys Thr Leu Val Phe Tyr Gln Gly Arg Ala Pro Lys Gly Arg Lys Ser
115 120 125
Asp Trp Val Met His Glu Phe Arg His Glu Gly Ile His Gly Pro Gly
130 135 140
Pro Lys Pro Pro Ser Pro Val Lys Glu Asp Trp Val Leu Cys Arg Val
145 150 155 160
Phe Phe Lys Ser Arg Ser Glu Ile Ser Ser Arg Thr Gln Asn Met Asn
165 170 175
Asn Glu Val Pro Pro Ser Ser Pro Ser Leu Pro Pro Leu Met Asp Ser
180 185 190
Phe Thr Gly Phe Gly Pro Thr Thr Thr Gln Pro Gly Pro Ser Asp Asn
195 200 205
Gln Tyr Gln Glu Gln Val Pro Cys Phe Ser Ile Phe Asn Asn Thr Asn
210 215 220
Gln Ala Asn Pro Asn Phe Ser Leu Leu Ala His Met Asp Ser Leu Glu
225 230 235 240
Met His Pro Thr Thr Lys Asn Met Pro Asn Tyr Gly Gly Thr Ile Pro
245 250 255
Asp His Leu Gly Ile Tyr Asn Ser Pro Pro Leu Met Asn Phe Asn Pro
260 265 270
Ser Ser Cys Asp Lys Lys Met Ile Arg Ser Val Leu Asn His Leu Thr
275 280 285
Lys Met Glu Ser Gly His Asp Gln Ile Ser Ile Lys Gly Gly Ser Pro
290 295 300
Ser Phe Gly Glu Gly Ser Ser Asp Ser Tyr Leu Ser Glu Val Gly Leu
305 310 315 320
Ser Ser Met Trp Asn His Tyr Ser
325
Claims (5)
1.海州常山NAC基因CtNAC2,其核苷酸序列如SEQ ID NO .1所示。
2.权利要求1所述的海州常山NAC基因CtNAC2的表达蛋白 ,其氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述的海州常山NAC基因CtNAC2在检测海州常山盐胁迫中的应用。
4.含有权利要求1所述的海州常山NAC基因CtNAC2的表达载体。
5.含有权利要求1所述的海州常山NAC基因CtNAC2的宿主菌。
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