本发明涉及农业生物技术领域,具体地涉及烟粉虱med隐种染色质重塑因子btbrm2及其编码基因和应用。
背景技术:
烟粉虱(bemisiatabaci(gennadius))属节肢动物门,昆虫纲,半翅目,粉虱科,小粉虱属,是一种世界范围内重要的农业害虫。该虫寄主范围极广,可以通过取食植物汁液、分泌蜜露诱发煤污病、传播植物病毒等方式危害豆科、茄科、菊科、葫芦科、锦葵科以及十字花科等600余种经济作物,是我国作物生产必须应对的重大有害生物。烟粉虱是一个包含36个以上隐种的物种复合体。目前我国已有的烟粉虱隐种有15种,包括2个入侵种和13个本地种。入侵种med隐种在我国全国范围内广泛分布。而本地种仅分布在我国局部地区,如asiaii1隐种主要分布在中南部局部地区如海南、广东、广西、台湾、贵州、浙江等地,并且没有向其他地区扩散的趋势。
随着全球温度的变化,烟粉虱med隐种在不同地理环境下的成功适应,已经广泛引起了人们对其入侵机制的理论探讨,对于其温度适应性的分子机制更是近几年来的研究热点之一。对烟粉虱进行热激选择的实验中发现,入侵种烟粉虱2代内存活率显著提高,表明存活能力的快速提高是其在恶劣环境生存的重要策略。这种短期内生物体对环境变异产生的响应机制与表观遗传有关。表观遗传过程会增加有机体对非温度胁迫和其它环境挑战的进化潜能的响应,这与全球环境变暖的背景是高度相关的。
染色质重塑(chromatinremodeling)是目前表观遗传领域的研究热点之一,它能驱动核小体的置换或重新排列,改变染色质的空间构象。atp依赖的染色质重塑是染色质重塑复合体利用atp水解释放的能量来改变染色质结构,从而调控真核生物基因表达的一种重要的表观遗传调控机制。染色质重塑在生物防御相关基因的表达调控中发挥着关键作用,而染色质重塑因子brm能参与环境胁迫对生物逆境的调控,在调节非生物胁迫相关基因的表达中起重要作用。但其在烟粉虱温度胁迫中的作用仍未知。
rna干扰(rnainterference,rnai)现象是一种进化上保守的抵御转基因或外来病毒侵犯的防御机制。将与靶基因的转录产物mrna存在同源互补序列的双链rna(doublestrandrna,dsrna)导入细胞后能特异性地降解该mrna,从而产生相应的功能表型缺失。rnai广泛存在于生物界,通过rnai技术沉默昆虫体内某些基
因,使昆虫的某些能力增强或丧失,也能在特定时间抑制其功能基因表达使昆虫的发育停留在某个阶段,进而达到利用或防治其危害的目的。由于烟粉虱个体体积小,注射dsrna不易操作、费时等限制,而饲喂dsrna具有简便、易于操作等特点而在烟粉虱研究中得以应用。
技术实现要素:
本发明的目的是提供一种烟粉虱med隐种染色质重塑因子。
本发明的再一目的是提供上述烟粉虱med隐种染色质重塑因子的编码基因。
本发明的再一目的在于提供含有上述烟粉虱med隐种染色质重塑因子的编码基因的重组表达载体。
本发明的再一目的在于提供含有上述烟粉虱med隐种染色质重塑因子的编码基因的重组菌株。
本发明的再一目的在于提供上述烟粉虱med隐种染色质重塑因子的编码基因的应用。
根据本发明的具体实施方式,首次克隆出烟粉虱med隐种染色质重塑基因btbrm2,其cdna全长核苷酸序列如seqidno:1所示:
atggcagctcccactcaagacaatctgaatgctttgcagcacgctatagattcaatggaagagaaaggtatgcaagaggatcctagatattctcagctcctcgctatcagagctcgtcaaaataacatggagccccctcgacttcctggatcgcaaggattttgcgtatcctctgatagcccatcgaattccaagcaagttttgagtcccatccagttgcaacagttacgagtccaaatcatggcatatcgagttctcgctcggaatcagccgttgcctcatcaacttgcagtagcggttcaagggaaacgaatggactcgtctctcattcaaccgggtctgggacctccacctctcatgcctccaacctccggtggaaacactccaagcccacaagcacgacccactgattcaagcaatgaaaactcatctggaatgagtgcagctcctgtgtaccctgtcgcacccccaacttcacccaacgtaccccaaggacctccgccatctcagttgcccccagtaaccccacctcccccacagtcatcctcaacgatggctccacctcctgctgttccagcaccacctgctccatctgttcctgcccctctcccaccccctgctctcaattcaacaccccctgcgcccctcatgcctcccatttcagcaccgacaccacctgtacccttgcagccaccagtgacgtcacacgtccatatcgccccacccatgcctcggctccccatcacgagtcatcccgtcatggttgtccctcctcctcctaactcaatgctgaaaccgatgcccagtccagttgttctgcctcctccgccaccaccaccccccaatccgcagcaacttacgcagcaacttcagttacaaaaacagaacagagttacgactattcctcgtccaactgggatcgaccctatactaattttacaagaaagagaaaatagagtcgcagctcgcattgcccatcatatggagaagctctccaatcttccgtcaacagtgtcggatgaaattcgcctcaaagctcagatcgaacttcgtgctttgagaatcttgaattttcaacgtcagctccgaagcgagatcatatcttgtgctcgcagagattcaaccctggaaacagccgtcaatatgaaagcctacaagcgagtcaaaacccaaggattaaaagaatctcgcgcgacagaaaagttggaaaagcagcagaaactggaagcagagaggaaacggagacaaaaacatcaagaatatttggcagccatcattcagcacggtaaagactttaaagaataccatcggaataatcaagcgaggatcgtccggttgaataaagccatgcttagttaccatgtgaatgcggagcgggagcagaagaaagaacaggagaggatcgaaaaggaacgaatgagacgcctaatggcagaagatgaagagggttacaggaaactcatcgatcaaaagaaagacaagagactggcattcttactgtctcaaactgacgaatacatcacaaacttgacagaaatggtcaaacaacacaaagttgaacaaaaacgtaaacttgcagagaaagcgaaagaagacaagaggaaaaagaaagaaactgaggatgcggagcggagagcttgcgtaatggaaatggaaagtgggaaaaaattgaagggagaagaagctcccttgatggtggacctaggcacttggctggaagctcatcctggttgggaagcatgtgaggactcagaggcagagactgaagaagagtctgattctgattccgatcaaaatatcgaagaaaaaattgaagaagtcaaagaagagaagaaaccaaaagaagaacttgatccgaaagaagttgttgaaaaagcgaaaagtgaagacgatgaatacaaaaatacctccgaagaacttacctactacagtattgctcacacaatcaatgaaattgtgacagaacaagcctctatcatggttaatggtaaattaaaagaatatcaaatcaagggacttgagtggctagtctctctgtacaataataatctcaatggcatcctggccgatgaaatgggtcttggaaaaaccatccaaacaatcgcattgatcacatacttgatggagatgaaaaaagtaaacggtccatatttaatcatcgtacctctttccaccctttcaaattgggtgttggagtttgaaaaatgggccccatctgtaaatgttgttgcgtacaaaggctcaccagctgtcagacgtgcacttcaagcacaaatgagatcatctaaattcaatgtgctactcacaacgtatgaatacgttatcaaggataaagctgttctagccaagttgcactggaagtacatgatcatcgatgaagggcatcgaatgaagaatcaccattgcaaattgacacaagtgctaaattcacattatgttgttccgcaaaggctccttttgactggaactccgttgcaaaataagttgcccgaactgtgggctctcctcaattttcttcttccgtctattttcaaatcctgctcaacatttgaacagtggttcaatgctccttttgccacaactggtgaaaaagtggaattaaatgaggaagaaactattttgatcattcgccgtctacataaagttttacgtccatttttgctgcgacgtttgaagaaagaagtcgagtctcaacttccagacaaaatcgaatatatcgtaaaatgtgatatgtcagggctccagagagtcttgtataggcacatgcaaagtaaaggggtccttctaactgatggctctgaaaaaggtaacaagggcaaaggtggagccaaagcactgatgaatacaattgtccaacttcgcaagttgtgcaatcatccattcctgtttcaacatatcgaagagaagttctgcgatcatatcggctgttcgtcaaatggtgttgtaagtggacctgacctttatcgagtctctggtaagtttgaactattggatcgaattttgccaaagttgaaggccaccaatcaccgagtacttctattttgtcaaatgacccagctcatgagcataatggaggattatttcaactggcgaggattctcgtaccttcgtcttgatggaacaaccaaagcggaagatcgaggggatttattgaaaaaatttaacagcgccaccagtgaatatttcatattcttactgagcacacgagctggtggacttggcttaaatcttcaagctgcagacactgtagttattttcgactctgattggaatccccatcaggatttacaagcgcaagatcgagcacatcgaattggtcaaaagaacgaggtacgagtattgaggttaatgacggtcaattccgttgaagaacgaatcttagcagcagctcgatacaaattgaacatggatgaaaaagtcatccaggcaggcatgttcgatcagaagtctactggaactgagaggcagcagtttttgcatagtattttgcatcaagatgatgcagaagatgaggaagagaatgaggttccggatgatgaaactgtaaatcagatgattgctcgcagtgaagctgaatttgacacttttcaaaaaatggatgctgaaagacgcaaagaagagtctaagggcaaaaaatcccggcttattgaggagagtgaactaccagactggttggttaaagatgacgcagaggttgaagcttggacgtacgaacaggaagaagtgcagatgggacgaggctcaaggacaagaaaggaaattgactactcagacagcatgacagaaaaggaatggttaaaggccattgatgatggtattgatgactttgatgagattgaagatgaagtgaaagtgaagaaaactcgcaaacggcgcaaaaaagaagaggaggaagaggaaccggccaaaaaacgaagaaatggtacagagaagaatccgcaagagaaaacaccacctgctaattcaggagcggatgctcgaatgaagaagcaaatgcataaattgatgacgattgttgtagagtacactgaacctcaagattcgcgtgttctcagtgaaccattcatgaaactaccatctcgtcgtgaacttcctgattattatgaagtcatcaagaaacccctcgacatcaagaaaattctcacaaaaattgatgaaggaaagtacgaggaactggatgatctggagcgcgatttcatgcaattatgcaagaatgcgcagttgtataatgaagaagcatcactcatctatgaagactcaattgtactgcagtctgtttttacgaacgctcgtcagcgtttggaaagtgaagaggaagaagctccggaggaagaagaaaaagcggcacctgctgaagaggaagcctcatctggagcagaatcgtcctctgtgaaaatgaaattaaaattgaaggcatcaaccaaagcgccgaaaaattccgatgccaaaactgatacaaagtctaccccacgatcaaggaaaagaacctcgaaaaaatacataagtgacgatgagaatgaagatgatgccgaatcaagtaatggctaa
烟粉虱med隐种染色质重塑基因btbrm2编码氨基酸序列如seqidno:2所示:
maaptqdnlnalqhaidsmeekgmqedprysqllairarqnnmepprlpgsqgfcvssdspsnskqvlspiqlqqlrvqimayrvlarnqplphqlavavqgkrmdssliqpglgppplmpptsggntpspqarptdssnenssgmsaapvypvapptspnvpqgpppsqlppvtppppqssstmapppavpappapsvpaplpppalnstppaplmppisaptppvplqppvtshvhiappmprlpitshpvmvvppppnsmlkpmpspvvlppppppppnpqqltqqlqlqkqnrvttiprptgidpililqerenrvaariahhmeklsnlpstvsdeirlkaqielralrilnfqrqlrseiiscarrdstletavnmkaykrvktqglkesrateklekqqkleaerkrrqkhqeylaaiiqhgkdfkeyhrnnqarivrlnkamlsyhvnaereqkkeqeriekermrrlmaedeegyrklidqkkdkrlafllsqtdeyitnltemvkqhkveqkrklaekakedkrkkketedaerracvmemesgkklkgeeaplmvdlgtwleahpgweacedseaeteeesdsdsdqnieekieevkeekkpkeeldpkevvekakseddeykntseeltyysiahtineivteqasimvngklkeyqikglewlvslynnnlngilademglgktiqtialitylmemkkvngpyliivplstlsnwvlefekwapsvnvvaykgspavrralqaqmrsskfnvllttyeyvikdkavlaklhwkymiideghrmknhhckltqvlnshyvvpqrllltgtplqnklpelwallnfllpsifkscstfeqwfnapfattgekvelneeetiliirrlhkvlrpfllrrlkkevesqlpdkieyivkcdmsglqrvlyrhmqskgvlltdgsekgnkgkggakalmntivqlrklcnhpflfqhieekfcdhigcssngvvsgpdlyrvsgkfelldrilpklkatnhrvllfcqmtqlmsimedyfnwrgfsylrldgttkaedrgdllkkfnsatseyfifllstragglglnlqaadtvvifdsdwnphqdlqaqdrahrigqknevrvlrlmtvnsveerilaaaryklnmdekviqagmfdqkstgterqqflhsilhqddaedeeenevpddetvnqmiarseaefdtfqkmdaerrkeeskgkksrlieeselpdwlvkddaeveawtyeqeevqmgrgsrtrkeidysdsmtekewlkaiddgiddfdeiedevkvkktrkrrkkeeeeeepakkrrngteknpqektppansgadarmkkqmhklmtivveytepqdsrvlsepfmklpsrrelpdyyevikkpldikkiltkidegkyeelddlerdfmqlcknaqlyneeasliyedsivlqsvftnarqrleseeeeapeeeekaapaeeeassgaesssvkmklklkastkapknsdaktdtkstprsrkrtskkyisddeneddaessng
上述氨基酸序列具有brm蛋白典型的结构特征:hsa结构域(406-478aa)、brk结构域(541-585aa)、atp酶域(dexdc:661-853aa和helicc:1022-1106aa)、snac结构域(1201-1266aa)以及bromo结构域(1325-1436aa)。
对温度胁迫下btbrm2基因的表达模式进行分析,实时荧光定量pcr结果表明,烟粉虱med隐种经过低温胁迫处理5h后,btbrm2基因的表达量显著升高;高温胁迫处理1h后,btbrm2基因的表达量即显著升高。而短时高、低温胁迫处理后,烟粉虱本地种asiaii1隐种该基因的表达量显著降低。比较不同隐种在同一温度胁迫下btbrm2基因的表达量发现,高、低温胁迫处理后med隐种btbrm2基因的表达量显著高于asiaii1隐种。
本发明提供了上述烟粉虱med隐种染色质重塑基因btbrm2的应用。对烟粉med隐种进行rnai,结果显示,饲喂dsbtbrm2的med隐种成虫的高温击倒时间显著降低,低温冷致昏恢复时间显著增加,说明btbrm2基因在烟粉虱med隐种的温度耐受性中起关键作用。上述烟粉虱med隐种染色质重塑基因btbrm2可用于破坏烟粉虱温度耐受性,进而可以用于烟粉虱的防治。
本发明首次从烟粉虱med隐种中克隆得到染色质重塑基因btbrm2,明确了温度胁迫下btbrm2基因在烟粉虱med隐种与其他隐种中表达模式的差异。此外,降低该基因的表达直接降低了med隐种对高、低温的耐受能力。所获得的结果为进一步研究烟粉虱med隐种的温度耐受性机理与表观遗传之染色质重塑的关系奠定基础,可用于破坏烟粉虱温度耐受性,进而用于防治烟粉虱的危害及扩散。
附图说明
图1烟粉虱med隐种btbrm2基因中的保守结构域;
图2显示不同温度处理1h(a)、3h(b)和5h(c)btbrm2基因在烟粉虱med隐种与asiaii1隐种中表达模式的差异;
图3显示饲喂btbrm2基因dsrna、饲喂dsegfp、饲喂10%蔗糖溶液和ck条件下btbrm2的表达量变化情况;
图4显示btbrm2基因的dsrna处理对烟粉虱med隐种成虫耐热性(a)和耐寒性(b)的影响:比较饲喂btbrm2基因dsrna、饲喂dsegfp、饲喂10%蔗糖溶液和ck条件下烟粉虱med隐种成虫高温击倒时间和低温冷致昏恢复时间。
具体实施方式
实施例1:烟粉虱med隐种btbrm2基因全长cdna序列克隆
分别取不同温度胁迫条件下烟粉虱成虫200头放入1.5ml的离心管,液氮冷冻后利用研磨棒将其研成粉末,然后提取rna,-80℃保存以备用。根据全式金反转录试剂盒(one-stepgdnaremovalandcdnasynthesissupermix)说明,对提取的rna进行反转录合成cdna。以cdna为模板,设计引物,进行pcr扩增。设计引物如表1、表2所示:
表1
表2
实验发现,利用表1中的引物,pcr扩增获得烟粉虱med隐种btbrm2基因的全长cdna。而利用表2中的引物,无法得到目的基因序列。
利用表1序列,通过pcr扩增,获得btbrm2基因的cdna序列全长为4548bp,所得的基因具有如seqidno:1所示的核苷酸序列,该基因编码1515个如seqidno:2所示的氨基酸序列。克隆获得的基因编码的氨基酸序列的保守结构域分析表明,其具有brm蛋白典型的结构特征:包括406-478位点的hsa结构域、541-585位点的brk结构域、661-853位点的dexdc结构域、1022-1106位点的helicc结构域、1201-1266位点的snac结构域以及1325-1436位点的bromo结构域。该基因的保守结构域如图1所示。
实施例2:分析btbrm2基因的表达特性
(1)提取不同温度胁迫下烟粉虱成虫的rna及合成cdna
选取初羽化的烟粉虱med隐种和asiaii1隐种成虫,分别在0、12、26、35、40℃下对烟粉虱成虫进行胁迫处理。各3个生物学重复,共3000头成虫,胁迫结束后立即置于液氮中冷冻3分钟后-80℃保存。按照实例1的方法提取rna并反转录成cdna。
(2)荧光定量pcr检测不同温度下btbrm2表达量:
设计荧光定量pcr的btbrm2基因和两个内参基因(ef1-α、β-tublin)的引物:
brm2-qf:aaaaccatccaaacaatcgc
brm2-qr:tttcaaactccaacacccaa
ef1-α-f:tagccttgtgccaatttccg
ef1-α-r:ccttcagcattaccgtcc
β-tub-f:tgtcaggagtaacgacgtgtttg
β-tub-r:ttcgggaacggtaagtgctc
反应体系为20μl:2×transstarttmgreenqpcr10.0μl、roa0.4μl、cdna模板1.0μl、primer-f0.4μl、primer-r0.4μl、ddh2o7.8μl。
反应条件:95℃30s;95℃5s,60℃30s,40个循环;绘制熔解曲线。
(3)数据分析
通过2-δδct方法计算基因的相对表达量。利用sas9.4软件对数据进行统计分析,不同温度处理之间的差异使用单因素方差分析(anova),并进行duncan检验;同一温度不同隐种间的差异使用独立样本t-test进行分析。数据以均值±标准误差表示,显著性检验水平**代表p<0.01,***代表p<0.001。
如图2显示,实时荧光定量pcr结果表明,烟粉虱med隐种经过低温胁迫处理5h(c)后,btbrm2基因的表达量显著升高;高温胁迫处理1h(a)后,btbrm2基因的表达量即显著升高。而短时高、低温胁迫处理后,烟粉虱本地种asiaii1隐种该基因的表达量显著降低。比较不同隐种在同一温度胁迫下btbrm2基因的表达量发现,高、低温胁迫处理后med隐种btbrm2基因的表达量显著高于asiaii1隐种。
实施例3:分析btbrm2基因对烟粉虱med隐种温度耐受性的影响
3.1合成dsrna
(1)设计合成加上t7启动子(下划线所示序列)的引物序列:
t7+btbrm2-f:5’-taatacgactcactatagggtgtgatatgtcagggct-3’
t7+btbrm2-r:5’-taatacgactcactatagggtactcggtgattggtgg-3’。由上海生工生物工程技术服务有限公司合成。
(2)总rna提取及cdna合成:同实施例1。
(3)t7引物pcr扩增、产物纯化,纯化所得的pcr产物即为合成dsrna的模板。使用试剂盒合成和纯化dsrna,按照试剂盒说明书进行操作。
3.2dsrna饲喂
parafilm膜预先用depc水处理以去除rna酶。在浓度为10%的蔗糖液中加入dsrna,浓度为0.3~0.5μg/μl。根据烟粉虱的取食特点,对parafilm膜夹营养液法做了相应的改进:取初羽化烟粉虱成虫约200头,放入两端通透的玻璃管中,用双层parafilm覆盖玻璃管上端,在两膜之间加入浓度为10%的蔗糖溶液200μl,并加入dsrna,并使其终浓度为0.3-0.5μg/μl,玻璃管下端用纱布覆上,保持通风透气。玻璃管周围及下端用黑色塑料包裹,使烟粉虱向顶端paraflim膜聚集更好的取食dsrna,将装置放入人工气候箱(温度26±0.5℃,24h光照,相对湿度为60-70%),饲喂3h。把完成饲喂的烟粉虱收集于指型管中,一组放入预热的高温水浴锅进行热击倒,并开始记录至烟粉虱不能自主站立的时间,处理温度为45℃。另一组放入低温水浴锅中进行冷击倒十分钟后取出记录冷致昏恢复时间,处理温度为-5℃。以未做任何处理(ck)、饲喂10%蔗糖溶液和饲喂含有dsegfp的10%蔗糖混合溶液(dsegfp终浓度为0.3-0.5μg/μl)的烟粉虱为对照,各处理均设置4个生物学重复。
通过2-δδct方法计算基因的相对表达量,结果如图3所示,饲喂dsbtbrm2可以显著降低btbrm2基因的表达。利用sas9.4统计软件分析饲喂不同溶液后烟粉虱med隐种成虫的高温击倒时间和低温冷致昏恢复时间,结果如图4所示,饲喂btbrm2基因dsrna的烟粉虱med隐种的高温击倒时间(a)显著低于ck组、饲喂dsegfp组和蔗糖组的击倒时间,而低温冷致昏恢复时间(b)显著高于三个对照组(p<0.05);所饲喂的靶序列片度为btbrm2基因特有的序列,由此确保了干扰效果为烟粉虱med隐种的目的btbrm2基因所产生,因此,说明btbrm2基因在烟粉虱med隐种温度耐受性中起着关键作用。
本发明从烟粉虱med隐种中克隆得到btbrm2基因的全长cdna,荧光定量pcr显示btbrm2基因在不同温度下在不同烟粉虱隐种中表达量存在差异;最后通过饲喂btbrm2基因dsrna,进而导致烟粉虱med隐种成虫的高温击倒时间显著缩短、低温冷致昏恢复时间显著延长。根据本发明的具体实施方式,试验结果明确了btbrm2基因在烟粉虱med隐种温度耐受性中起关键作用。本发明为进一步研究烟粉虱med隐种的温度耐受性机理与表观遗传之染色质重塑的关系奠定基础,可用于破坏烟粉虱温度耐受性,进而用于防治烟粉虱的危害及扩散。
序列表
<110>中国农业科学院植物保护研究所
<120>烟粉虱med隐种染色质重塑因子btbrm2及其编码基因和应用
<160>2
<170>siposequencelisting1.0
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