CN109266659A - 一种人工合成的Bt抗虫基因JBT-AC及其应用 - Google Patents
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Abstract
本发明公开了一种人工合成的Bt抗虫基因JBT‑AC，所述Bt抗虫基因JBT‑AC的核苷酸序列用SEQ ID No 1所示。Bt抗虫基因JBT‑AC较其密码子优化前更容易获得高表达的转基因植株。抗虫基因JBT‑AC可在转基因玉米中稳定遗传和表达，且表达量高，所得转基因玉米抗虫性好。
Description
技术领域
本发明属于抗虫基因工程领域，具体涉及一种人工合成的Bt抗虫基因JBT-AC及其应用。
背景技术
玉米是主要粮食作物之一，全世界有超过10亿的人口以玉米为主食。虫害是造成玉米减产的主要原因之一。在现代农业中，使用化学杀虫剂为减少虫害损失起到了巨大作用。但是,化学杀虫剂的使用会不可避免的造成环境污染以及威胁人类健康。利用转基因技术培育具有抗虫性状的品种是一种控制害虫、减少环境污染的有效方法。
Bt-toxin是苏云金芽孢杆菌在孢子形成时期所产生的一种杀虫毒素，苏云金芽孢杆菌的毒蛋白基因(简称Bt-toxin)是目前世界上应用最为广泛的抗虫基因。
虽然早期的转抗虫基因植物或多或少都对昆虫有些抗性，但杀虫蛋白在植株上的表达量很低，抗虫效果不理想。其主要原因是目前使用的杀虫晶体蛋白大多来源于细菌，与高等植物结构基因正常的序列相比，基因含有较多的AT碱基和ATTTA重复序列。AT富含区在高等植物中被认为是不能表达的内含子，ATTTA重复序列的mRNA的稳定性差，二者都不能在高等植物中编码。此外，Bt基因中的偏爱密码子与植物中的不同，以及共同存在的一些不稳定元件如poly(A)信号序列、切割序列、终止序列等都直接影响着Bt基因在植物中的mRNA特性。
抗虫基因虽然对人畜无毒，不破坏环境，但目前普遍存在获得的转基因植物抗虫效率低的问题。抗虫基因大多来源于细菌等微生物，其密码子与植物有较大差异，因此，对天然Bt基因进行改造，合成新的抗虫基因，使抗虫基因适合在植物受体细胞中表达，从而提高转基因植株的杀虫性是目前急需解决的问题。
发明内容
本发明的目的是克服现有技术的不足，提供一种人工合成的Bt抗虫基因JBT-AC。
本发明的第二个目的是提供人工合成的Bt抗虫基因JBT-AC在制备抗虫植物中的应用。
本发明的技术方案概述如下：
一种人工合成的Bt抗虫基因JBT-AC，所述Bt抗虫基因JBT-AC的核苷酸序列用SEQID No 1所示。
上述的人工合成的Bt抗虫基因JBT-AC在制备抗虫植物中的应用。
本发明的优点：Bt抗虫基因JBT-AC较其密码子优化前更容易获得高表达的转基因植株。抗虫基因JBT-AC可在转基因玉米中稳定遗传和表达，且表达量高，所得转基因玉米抗虫性好。
附图说明
图1为JBT-AC基因设计过程。
图2为JBT-AC基因的植物表达载体。
图3为转JBT-AC基因的玉米RT-PCR检测结果。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。
下述实施例中所用方法如果没有特别的说明，均为常规方法，所用术语和缩写均是本领域技术人员通用的术语和缩写。
天塔五号玉米种子购自天津科润津丰种业有限责任公司。本申请以玉米种为例，实验证明，其它植物也可以用于本发明。
C58农杆菌感受态购自武汉淼灵生物科技有限公司。
含有基因Bar的质粒pCAMBIA3301购自武汉淼灵生物科技有限公司。
实施例1 JBT-AC基因的设计改造
本发明是在苏云金芽孢杆菌中所发现的对鳞翅目害虫有特异杀虫活性的杀虫晶体蛋白基础上，根据苏云金芽孢杆菌Cry1Ac蛋白和Cry4Aa蛋白不同区域的活性分析及玉米基因组特点，我们对两种蛋白对应的密码子及编码框进行了优化改造，分别命名为Cry1Ac-J和Cry4Aa-J。接着将Cry1Ac-J的DomainⅠ与Cry4Aa-J的DomainⅡ和DomainⅢ进行融合(见图1)，获得一条新的基因，Bt抗虫基因JBT-AC(简称JBT-AC基因)的核苷酸序列如SEQ IDNo：1所示。
实施例2 JBT-AC基因的植物表达载体构建
按照实施例1的方案我们设计合成了JBT-AC基因，同时在基因的5’端添加了BamHI的酶切位点，3’端添加了SalI的酶切位点。用BamHI、SalI对JBT-AC基因进行酶切，并回收JBT-AC基因片段，然后与BamHI、SalI酶切过得的已有植物表达载体pCAMBIA3301进行连接，得到含有JBT-AC基因的植物表达载体pCAMBIA3301-JBT-AC(见图2)。
实施例3 JBT-AC基因转化农杆菌。
实验中所用的农杆菌为C58菌株，重组质粒载体pCAMBIA3301-JBT-AC上构建有筛选基因Bar。将保存的C58农杆菌感受态从-80℃冰箱拿出100μL，放在冰上，取出2μLpCAMBIA3301-JBT-AC质粒与农杆菌100μL混匀，冰浴13min，放入冰上预冷的电击杯中，1500V，电击转化。将菌液吸入1.5mL离心管中，加入500μL YEP液体培养基，于28℃振荡培养3h，8000r/min，28℃，离心收集菌体，弃400μL上清，重悬菌体，涂板于28℃暗培养2天，筛选出转化成功的转化用农杆菌单菌落。
实施例4农杆菌侵染液的制备
从平板上挑取转化用农杆菌单菌落接种于YEP液体培养基中(含100mg/L卡那霉素)，28℃、180r/min振荡培养过夜，当农杆菌生长至对数生长期时(OD600为0.6，20℃，5000r/min离心7min收集菌体，用等体积侵染培养基重悬菌体，得到农杆菌侵染液。
YEP培养基：10g/L蛋白胨+10g/L酵母提取物+5g/L NaCl，pH为7.0。
侵染培养基：1/2MS+65g/L蔗糖+36.5g/L葡萄糖+150μmol/L乙酰丁香酮，pH为5.3。
实施例5转抗虫基因JBT-AC玉米的获得。
1玉米芽尖的转化。选取整齐饱满的天塔五号玉米种子，70％酒精浸泡1min，0.1％升汞消毒12min，无菌水清洗3次后接入发芽培养基，26℃，黑暗条件下培养。待苗长至4-6cm时，在无菌条件下切除幼叶和胚芽鞘以露出芽尖生长点，并用灭过菌的手术刀轻轻划伤用于侵染。将玉米芽尖浸泡在农杆菌侵染液中(含有150μmol/L乙酰丁香酮)，并置于真空干燥器中，在50kPa压力下侵染20分钟，侵染完后弃去菌液，用无菌滤纸吸去玉米芽尖表面多余的菌液，继续放入发芽培养基中培养3天，得到玉米小苗。
发芽培养基：1/2MS+30g/L蔗糖+7.5g/L琼脂，pH为5.7。
2转化苗的移栽和筛选。洗去玉米小苗根部的培养基，移入装有珍珠岩、蛭石、营养土的体积比为1:2:2的花盆中，放于温室培养。待小苗长到5-7cm时将250mg/L的草胺膦水溶液，10mL/株，喷洒在叶片上进行抗性苗的筛选。
3抗性植株的RT-PCR分子检测。用非转基因对照植株叶片和转基因植株叶片(本实施例步骤2获得的叶片)提取总RNA，反转录合成的cDNA作为模板，用JBT-AC特异性引物进行扩增，扩增出的片段大小为1752bp(SEQ ID No：1)。扩增反应程序为:94℃3min；(94℃30s，58℃30s，72℃30s，32个循环)；72℃延伸7min。引物为上游:atgattgatatctctttatc(SEQNO.2)；下游:gaccgggatgaactcaattt(SEQ NO.3)。产物经0.9％琼脂糖凝胶电泳检测。如图3所示，泳道1为Marker，泳道2为质粒pCAMBIA3301-JBT-AC阳性对照，泳道3为非转基因对照，泳道4-14为转基因植株。RT-PCR检测结果表明，JBT-AC基因已经成功整合入玉米植物基因组中，表明成功获得了转JBT-AC基因玉米植株。
实施例6转JBT-AC基因玉米植株抗虫性鉴定
将获得的转基因苗种植于温室，并且全生育期不施农药，花后16天观察植株抗虫性，以非转基因的植株作为阴性对照。田间观察时，只要植株上出现叶片有横排小虫孔、苞叶、花丝被蛀食、茎秆、穗柄、穗轴被蛀食等，均认为不抗虫。植株无明显虫害，植株健壮，认为抗虫。统计结果见下表。
注：“+”代表检测为阳性；“-”代表检测为阴性；“有”代表植株上能看到虫的危害症状，如叶片有横排小虫孔、苞叶、花丝被蛀食、茎秆、穗柄、穗轴被蛀食；“无”代表植株没有玉米螟的危害症状。
上述结果表明：18株转JBT-AC基因玉米植株中抗虫植株为15株，抗虫率为83.33％，与非转基因玉米植株相比，转JBT-AC基因玉米植株表现为抗虫，且抗虫效果好。
序列表
<110> 天津大学
<120> 一种人工合成的Bt抗虫基因JBT-AC 及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1752
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgattgata tctctttatc cttgactcaa tttcttctct cagagttcgt tcctggtgct 60
ggctttgtcc tcggactggt agacataatt tgggggatct tcggtccctc gcagtgggat 120
gcctttttag tgcaaataga gcagttgatt aatcaacgta tcgaagagtt cgcacgcaac 180
caggcgataa gtcggcttga aggcctcagc aatctatatc aaatttacgc tgagtctttt 240
cgggaatggg aggccgaccc aaccaacccg gcactgagag aagagatgag catccagttc 300
aatgatatga actccgcgtt aacaacggct atacctttgt ttgccgttca aaattatcag 360
gtcccccttc tctcagtata cgtgcaagca gcgaacctac atctgtcggt tttacgtgac 420
gtcagtgtat tcggacagcg ctgggggttt gatgctgcca ctattaatag ccgatataac 480
gacttgaccc ggcttatcgg taattacaca gattatgcag tgagatggta caacacgggc 540
ctcgaaaggg tttggggacc agactctcgt gattgggtcc gctataatca attccgacgg 600
gagctaactc tgaccgtatt agacatagtg gcgttgtttc cgaactacga ttccagaagg 660
gaattcaaaa ctgaatttac ccgtgagatt tatacagctt tagttgaatc tccttcctca 720
aagtcgatcg ccgcattgga ggcggctgaa gatagtctta cgatacgcaa accccatctc 780
ttcgactacc tacaaggtat tgagtttcac actcgactcc agccaggcta tttcggaaag 840
gatagcttta attactggtc tgggaactat gtctccaccc ggccgtcaat cggttcgaat 900
gacataatta caagtccttt ctacggcaac aaaagtttag gattgcttac gcatcaaatc 960
cagctcaatt ctaacgtata taagacttcc ataaccgata catcatcgcc cagtaataga 1020
gtgacgaaaa tgctaggggt tactaaggtc gaatttagcc aatacaacga ccagaccgat 1080
gaggcctcta cacaaacgta tgactccaaa aataagaaca ttttcggtct gccaatctta 1140
aaaaggcgtg aagagcaggg caatccgact ttgtttccta ccgtaaacga ttacacacac 1200
atactttcat atgtgacgaa ttacgcagaa tgtttcctca tgcaaggatc gcgcgggact 1260
attcccgttc taacctggac acatagtagc gtggacaagc ttaaaaatac tatttatacc 1320
gatgctatca cacaagttcc tgccgtcaag tctaactttt taaatgcaac ggcgaaagta 1380
ataaagggtc ccggcttcac tggaggggac ttggtgcgtc ttaactcctc aggtaccctc 1440
tcgggccgca tggaaattca gtgtaaaaca agtatcttta atgatccaac gcgaagctac 1500
ttcatacgga ttagatatgc ttctaacggg tccgccaata ctagggcagt tatctcaaac 1560
aatgactttc tagtcatata cgtaccggcg accgctacat cgctggataa cttaagtatt 1620
cctggggtgg ccgagttggg tatggcactt aatcccacgt tcagcggcaa caatatcata 1680
ccaactattg gagcggaatc ttttgtttcc aacgagaaga tctatataga caaaattgag 1740
ttcatcccgg tc 1752
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgattgata tctctttatc 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaccgggatg aactcaattt 20
Claims (2)
1.一种人工合成的Bt抗虫基因JBT-AC，其特征是所述Bt抗虫基因JBT-AC的核苷酸序列用SEQ ID No 1所示。
2.权利要求1的人工合成的Bt抗虫基因JBT-AC在制备抗虫植物中的应用。
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