CN107937424A - 双基因融合增强植物抗蚜性的方法 - Google Patents

双基因融合增强植物抗蚜性的方法 Download PDF

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CN107937424A
CN107937424A CN201711082638.4A CN201711082638A CN107937424A CN 107937424 A CN107937424 A CN 107937424A CN 201711082638 A CN201711082638 A CN 201711082638A CN 107937424 A CN107937424 A CN 107937424A
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aphid
coo2
plant
gene
atpase subunit
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唐克轩
马嘉伟
潘琪芳
付雪晴
张婷婷
钱虹妹
赵静雅
马亚男
王宇婷
孙小芬
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Shanghai Jiaotong University
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Priority to PCT/CN2018/114295 priority patent/WO2019091390A1/zh
Priority to CN201811316873.8A priority patent/CN109988761B/zh
Priority to US16/760,447 priority patent/US11208668B2/en
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Abstract

一种双基因融合增强植物抗蚜性的方法，通过从桃蚜(Myzus×persicae)的V‑ATPase subunit E基因和COO2基因片段中构建得到上述具有抗蚜虫功能的基因，通过构建表达载体并将该载体转入农杆菌，以该农杆菌侵染小麦幼胚，在小麦中表达产生V‑ATPase subunit E和COO2双基因的dsRNA，从而达到趋避蚜虫的目的。此外，实验证明通过转化双基因融合载体的转基因小麦抗虫效果高于分别转化单基因的转基因小麦。本发明利用基因工程手段获得的抗虫转基因植物具有只对目标害虫有效，而对非危害生物没有影响的优点，植物表达产生的抗虫物质存在于植物体内，不会对环境造成污染，且成本低，利于推广。

Description

双基因融合增强植物抗蚜性的方法
技术领域
本发明涉及的是一种生物工程领域的技术，具体是一种基于V-ATPase subunit E和COO2 双基因融合增强植物抗蚜性的方法。
背景技术
蚜虫是农业生产中的主要害虫之一，我国小麦主要种植地区小麦生育期间气温升高，出 现“暖冬”，致使蚜虫危害猖獗，每年在小麦生产上因蚜虫及其传播的病毒所造成的经济损失十 分严重。目前植物介导的RNAi技术作为农作物抗虫研究的热点之一，应用前景十分广阔。此 技术就是通过在寄主植物中表达相应昆虫特异基因的dsRNA，昆虫食用寄主植物后，其相应的 基因被沉默而表达量下降，造成昆虫的死亡，从而起到控制害虫危害的作用。因此利用植物介 导的RNAi技术培育抗蚜品种能够达到安全、持久、高效防治蚜虫的目的，具有重要的实际应 用价值。
发明内容
本发明提出一种双基因融合增强植物抗蚜性的方法，利用了V-ATPase通过水解ATP产 生能量从而为各种生命活动提供所需能量以及桃蚜唾液腺酶类基因COO2在蚜虫合成酶过程上 的重要特性，利用基因工程手段获得的抗虫转基因植物具有只对目标害虫有效，而对非危害生 物没有影响的优点，植物表达产生的抗虫物质存在于植物体内，不会对环境造成污染，且成本 低，利于推广。
本发明是通过以下技术方案实现的：
本发明涉及一种具有抗蚜虫功能的基因dsRNA，其具体融合自蚜虫的V-ATPasesubunit E和COO2基因片段，其核苷酸序列如Seq ID No.3所示。
所述的Seq ID No.3，通过将蚜虫的V-ATPase subunit E(Seq ID No.1所示)、COO2基因 (Seq ID No.2所示)以及V-ATPase subunit E和COO2基因部分片段融合片段基因合成。
本发明涉及一种双基因融合增强植物抗蚜性的方法，通过从桃蚜(Myzus×persicae)的 V-ATPase subunit E基因、COO2基因以及V-ATPase subunit E基因和COO2基因部分序列融合 片段分别构建得到具有抗蚜虫功能的基因，通过构建表达载体并将其分别转入农杆菌，以该农 杆菌侵染小麦幼胚，分别在小麦中表达产生V-ATPase subunit E、COO2基因以及V-ATPase subunit E和COO2双基因的dsRNA，从而达到趋避蚜虫的目的。
所述的表达载体，通过构建玉米ubiquitin-1启动子驱动V-ATPase subunit E，COO2基 因以及V-ATPase subunit E基因和COO2融合基因沉默载体，即RNAi载体。
所述的ubiquitin-1启动子具体为：组成型启动子。
本发明涉及一种重组表达载体，即RNAi载体，其基于dsRNA构建得到。
所述的dsRNA包括：
1)由Seq ID No.1所示的在植物中表达的核苷酸和其反向互补序列；
2)由Seq ID No.1所示的在植物中表达的核苷酸和其反向互补序列；
3)由Seq ID No.3所示的在植物中表达的核苷酸和其反向互补序列，由Seq IDNo.1所示 的核苷酸和Seq ID No.2所示的核苷酸组成。
所述的抗蚜性包括：
1)在防治蚜虫中的应用或在制备防治蚜虫产品中的应用；
2)在促进蚜虫死亡中的应用或在制备促进蚜虫死亡产品中的应用；
3)在抑制蚜虫生长中的应用或在制备抑制蚜虫生长产品中的应用。
技术效果
本发明的实验证明所得在小麦中表达桃蚜相关基因V-ATPase subunit E、COO2以及 V-ATPase subunit E和COO2融合片段表达cDNA的序列的dsRNA，导致麦蚜产生致死效应， 并且V-ATPase subunit E和COO2融合片段表达cDNA的序列的dsRNA抑制蚜虫效果最佳。
与现有技术相比，本发明对于适用于本发明的植物没有特别的限制，只要其适合进行基 因的转化操作，如各种农作物、花卉植物或林业植物等。
附图说明
图1为本发明pDE1005:proUBI:V-ATPase subunit E+COO2载体构建示意图；
图2为实施例中转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦PCR 鉴定示意图；
图中：M：Marker III；+：pDE1005:proUBI:V-ATPase subunit E+COO2质粒；CK：野生型Fileder春小麦；1-1，1-3，1-6，2-3，2-3，2-6，3-2，3-4，3-6，4-1，4-3，4-4，5-3， 5-5，5-8：转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦植株，简称VC。
图3为实施例中转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦 V-ATPase subunit E+COO2基因半定量结果示意图；
图中：M：Marker III；+：pDE1005:proUBI:V-ATPase subunit E+COO2质粒；CK：野生型Fileder春小麦；1-1，1-3，1-6，2-3，2-3，2-6，3-2，3-4，3-6，4-1，4-3，4-4，5-3， 5-5，5-8：转ubiquitin-1启动子驱动V-ATPase subunit E+COO2基因T1代小麦植株，简称VC。
具体实施方式
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子 克隆：实验室手册(New York:Coldspringharbor Laboratory Press,1989)中所述的条件，或按照 制造厂商所建议的条件。所采用的试剂盒均为市售或公开渠道可以获得的试剂盒。
实施例1
含V-ATPase subunit E和COO2双基因的RNAi载体的合成：分别合成桃蚜(Myzus×persicae)的V-ATPase subunit E基因片段，COO2基因片段以及V-ATPase subunit E和COO2双 基因片段，分别构建发夹结构的正向基因，内含子序列和反向的基因，构建到pDE1005载体 的多克隆位点上，分别获得pDE1005:proUBI:V-ATPase subunit E，pDE1005:proUBI:COO2和 pDE1005:proUBI:V-ATPase subunit E+COO2基因沉默载体，基因由上海生工生物工程公司合 成。
实施例2
根癌农杆菌介导V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2双基因
RNAi载体转化Fielder春小麦
1.1.幼胚的预培养
开花授粉后13～14d的未成熟种子(幼胚大小1.0～1.2mm)，用70％酒精表面消毒1～ 2min，15％的次氯酸钠灭菌15min，无菌水冲洗4～5次。
1.2.农杆菌与幼胚愈伤组织的共培养
室温下，3500rpm离心10min收集农杆菌菌体，尽去上清，用1/10WCC重悬液(MS 基本培养基)以1：2比例重悬。将小麦幼胚转移至农杆菌菌液中侵染30min，将愈伤组织转移至灭菌培养皿中的无菌滤纸上，25℃黑暗条件下共培养2d(幼胚)。
共培养2d的幼胚愈伤组织转移到IESX1培养基(MS基本培养基(含MS维生素)+30gL-1蔗糖+2.0mg L-1dicamba+250mg L-1Cb+5mg L-1PPT，pH 5.8)，25℃黑暗培养2周，然后 转移到IESX2培养基(MS基本培养基(含MS维生素)+30g L-1蔗糖+2.0mg L-1dicamba+250 mg L-1Cb+10mg L-1PPT，pH 5.8)，25℃黑暗培养2-3周。
1.3.抗性再生植株的筛选
筛选后的幼胚愈伤组织转移到IEFH培养基(MS基本培养基(含MS维生素)+20g L-1蔗 糖+0.2mg L-1 2,4-D+250mg L-1Cb+5mg L-1PPT，pH＝5.8)，25℃、光照培养3～4周。
筛选后的成熟胚愈伤组织转移到XCFH分化培养基(MS基本培养基(不含MS维生素)+ 20g L-1蔗糖+10.0mg L-1B1维生素+1.0mg L-1B3维生素+1.0mg L-1B6维生素+2.0 mgL-1甘氨酸+5.0mg L-1谷氨酰胺+0.2mg L-1IAA+250mg L-1Cb+5mg L-1PPT，pH 5.8)上，25℃光照培养3～4周，分化植株。
将长至2～3cm的幼苗移至生根壮苗培养基(1/2MS培养基(含MS维生素)+20g L-1蔗 糖+250mg L-1Cb+5mg L-1PPT，pH 5.8)上，25℃、光照条件下培养3～4周。移栽生长健壮的抗性植株到花盆中。
转基因小麦的PCR检测
利用阳性鉴定的PCR引物对目的基因进行检测。结果表明，利用所设计的PCR特异引 物，能扩增出特异DNA片段。而以非转化小麦基因组DNA为模板时，没有扩增出任何片段。
本实施例将所述的植物表达载体转化根癌农杆菌，分别获得用于转化小麦的含V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2基因植物过量表达载体的根癌农杆菌菌株， 利用所构建的根癌农杆菌菌株转化小麦幼胚，获得经PCR检测的转基因小麦植株。
实施例3
V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2双基因RNAi载体T1代小 麦进行抗蚜虫性鉴定。
将同一虫龄的麦蚜分别接于待测转基因植株和3株野生型小麦展开的嫩叶上，每株接 10头，培养10天后，统计叶片上蚜虫数量。结果表1所示，与野生型对照相比，V-ATPasesubunit E，COO2以及V-ATPase subunit E+COO2双基因RNAi转基因小麦植株获得了显著提高的抗蚜性。
V-ATPase subunit E，COO2以及V-ATPase subunit E+COO2双基因RNAi转基因小麦植 株抗虫效果比较，V-ATPase subunit E+COO2双基因RNAi转基因小麦植株抗虫效果最佳。
表1 V-ATPase subunit E转基因小麦对蚜虫的抗性鉴定
表2 COO2转基因小麦对蚜虫的抗性鉴定
表3 V-ATPase subunit E+COO2双基因转基因小麦对蚜虫的抗性鉴定
结果显示，五个转V-ATPase subunit E和COO2双基因RNAi载体T1代小麦植株上蚜虫 数量显著低于野生型对照，并且与V-ATPase subunit E和COO2RNAi载体T1代小麦植株抗虫 效果相比，效果更佳。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式 对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围 内的各个实现方案均受本发明之约束。
序列表
<110> 上海交通大学
<120> 双基因融合增强植物抗蚜性的方法
<130> f-b000e
<141> 2017-11-07
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 622
<212> DNA
<213> 桃蚜 (Myzus × persicae V-ATPase subunit E)
<400> 1
gtattgagtt gttggcacat aagaataaaa taaaaatttg taacacactg gaatcgcgat 60
cataactcaa caaccgtgta ttcttatttt atttttaaac attgtgtgac cttagcgcta 120
tggaactgat tgctcagcag ttggtaccag ctgtacgaac tgcattgttc ggtcgtaatc 180
accttgacta acgagtcgtc aaccatggtc gacatgcttg acgtaacaag ccagcattag 240
caaacagaaa attcgctgaa taatcgcagt aaatccctac agcaataata tgcgtattaa 300
gtttgtcttt taagcgactt attagcgtca tttagggatg tcgttattat acgcataatt 360
aaacaaaaat aaaacattct atttaaaata ttttttctca ttccatttgg taactatgta 420
tttgttttta ttttgtaaga taaattttat aaaaaagagt aaggtaaacc attgatacat 480
attgcataaa ttataaccat cccatctata catagtttag gttcaataca caattattta 540
taacgtattt aatattggta gggtagatat gtatcaaatc caagttatgt gttaataaat 600
attttgagtc ttaaaactca ga 622
<210> 2
<211> 718
<212> DNA
<213> 桃蚜 (Myzus × persicae COO2)
<400> 2
atgggaagtt acaaattata cgtagccgtc atggcaatag ccatagctgt agtacaggaa 60
tacccttcaa tgtttaatat gcatcggcag taccgttatc ggtatcgaca tcatgtcctt 120
gttagatgcg attggtctgc cgctgaaccg tacgatgagc aggaagaagc gtctgtcgaa 180
caatctacgc taaccagacg gcgacttggc atgctactcg tccttcttcg cagacagctt 240
ttaccgatgg agcaccgtca gtgcgatgaa tacaaatcga agatctggga caaagcattt 300
aatggctacc tcgtggcagt cacgctactt atgtttagct tctagaccct gtttcgtaaa 360
agcaaccagg aggctatgca gctgatggaa ctaacgttta atacaggtaa ggaattaggc 420
tcgttggtcc tccgatacgt cgactacctt gattgcaaat tatgtccatt ccttaatccg 480
tccaacgaag tgtgctcgga cacgacgcgg gccattttta acttcatcga tgtgatggcc 540
aggttgcttc acacgagcct gtgctgcgcc cggtaaaaat tgaagtagct acactaccgg 600
accaaccaga acgcccatta ctcgctgggt atgatgaaca agatgttggc gttcatcatt 660
ggttggtctt gcgggtaatg agcgacccat actacttgtt ctacaaccgc aagtagta 718
<210> 3
<211> 1340
<212> DNA
<213> 桃蚜 (Myzus × persicae V-ATPase subunit E和COO2双基因)
<400> 3
gtattgagtt gttggcacat aagaataaaa taaaaatttg taacacactg gaatcgcgat 60
cataactcaa caaccgtgta ttcttatttt atttttaaac attgtgtgac cttagcgcta 120
tggaactgat tgctcagcag ttggtaccag ctgtacgaac tgcattgttc ggtcgtaatc 180
accttgacta acgagtcgtc aaccatggtc gacatgcttg acgtaacaag ccagcattag 240
caaacagaaa attcgctgaa taatcgcagt aaatccctac agcaataata tgcgtattaa 300
gtttgtcttt taagcgactt attagcgtca tttagggatg tcgttattat acgcataatt 360
aaacaaaaat aaaacattct atttaaaata ttttttctca ttccatttgg taactatgta 420
tttgttttta ttttgtaaga taaattttat aaaaaagagt aaggtaaacc attgatacat 480
attgcataaa ttataaccat cccatctata catagtttag gttcaataca caattattta 540
taacgtattt aatattggta gggtagatat gtatcaaatc caagttatgt gttaataaat 600
attttgagtc tatgggaagt tacaaattat acgtagccgt catggcaata gccatagctg 660
taaaactcag atacccttca atgtttaata tgcatcggca gtaccgttat cggtatcgac 720
tagtacagga agttagatgc gattggtctg ccgctgaacc gtacgatgag caggaagaag 780
atcatgtcct tcaatctacg ctaaccagac ggcgacttgg catgctactc gtccttcttc 840
cgtctgtcga attaccgatg gagcaccgtc agtgcgatga atacaaatcg aagatctggg 900
gcagacagct taatggctac ctcgtggcag tcacgctact tatgtttagc ttctagaccc 960
acaaagcatt tagcaaccag gaggctatgc agctgatgga actaacgttt aatacaggta 1020
tgtttcgtaa atcgttggtc ctccgatacg tcgactacct tgattgcaaa ttatgtccat 1080
aggaattagg ctccaacgaa gtgtgctcgg acacgacgcg ggccattttt aacttcatcg 1140
tccttaatcc gaggttgctt cacacgagcc tgtgctgcgc ccggtaaaaa ttgaagtagc 1200
atgtgatggc caccaaccag aacgcccatt actcgctggg tatgatgaac aagatgttgg 1260
tacactaccg gtggttggtc ttgcgggtaa tgagcgaccc atactacttg ttctacaacc 1320
cgttcatcat gcaagtagta 1340

Claims (7)

1.一种具有抗蚜虫功能的基因，其特征在于，融合自蚜虫的V-ATPase subunit E和COO2基因，其核苷酸序列如Seq ID No.3所示。
2.一种双基因融合增强植物抗蚜性的方法，其特征在于，通过从桃蚜(Myzus×persicae)的V-ATPase subunit E基因、COO2基因以及V-ATPase subunit E基因和COO2基因部分序列融合片段分别构建得到具有抗蚜虫功能的基因，通过构建表达载体并将其分别转入农杆菌，以该农杆菌侵染小麦幼胚，分别在小麦中表达产生V-ATPase subunit E、COO2基因以及V-ATPase subunit E和COO2双基因的dsRNA，从而达到趋避蚜虫的目的。
3.根据权利要求2所述的方法，其特征是，所述的表达载体，通过构建玉米ubiquitin-1启动子驱动V-ATPase subunit E，COO2基因以及V-ATPase subunit E基因和COO2融合基因沉默载体，即RNAi载体。
4.根据权利要求2所述的方法，其特征是，所述的ubiquitin-1启动子具体为：组成型启动子。
5.根据权利要求2所述的方法，其特征是，所述的侵染，即将小麦幼胚转移至农杆菌菌液中侵染后置于黑暗条件下的培养基上培养得到幼胚愈伤组织，进而经抗性再生植株筛选后得到小麦抗性苗。
6.根据权利要求2所述的方法，其特征是，所述的抗蚜性包括：
1)在防治蚜虫中的应用或在制备防治蚜虫产品中的应用；
2)在促进蚜虫死亡中的应用或在制备促进蚜虫死亡产品中的应用；
3)在抑制蚜虫生长中的应用或在制备抑制蚜虫生长产品中的应用。
7.一种重组表达载体，即RNAi载体，其特征在于，基于dsRNA构建得到；
所述的dsRNA包括：
1)由Seq ID No.1所示的在植物中表达的核苷酸和其反向互补序列；
2)由Seq ID No.1所示的在植物中表达的核苷酸和其反向互补序列；
3)由Seq ID No.3所示的在植物中表达的核苷酸和其反向互补序列，由Seq ID No.1所示的核苷酸和Seq ID No.2所示的核苷酸组成。
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