CN109295076A - 一种人工合成的Bt抗虫基因JBT-FC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‑FC及其应用，属于基因工程领域的一种Bt蛋白。本发明人工合成的Bt抗虫基因JBT‑FC，其核苷酸序列用SEQ ID No 1所示。它较其密码子优化前更容易获得稳定表达的转基因植株。本发明JBT‑FC抗虫基因为玉米害虫防治提供了一种新的途径，转基因玉米表现出较高的玉米螟抗性，为今后利用该基因培育抗虫植物提供研究基础。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种人工合成的Bt抗虫基因JBT-FC及其应用。
背景技术
玉米是世界上三大粮食作物之一，是我国主要的粮食作物与经济作物。虫害严重威胁玉米的种植生长，影响玉米产量的提升，由于其具有发生代数不稳定，抗药性较强等特点，化学方法对虫害的防治效果并不理想。传统育种方法限制了现有抗虫玉米种质的培育进程。基因工程技术为解决这一难题提供了重要途径。由于该方法具有安全、有效、可降低投资和减少环境污染等诸多优点,基因工程技术为解决玉米螟危害提供了重要途径。但是随着转基因抗虫玉米种植规模不断扩大，转基因抗虫玉米仍然存在很多问题。抗虫范围相对较小，而危害玉米的害虫极多，玉米害虫逐渐对抗虫玉米产生抗性。
苏云金芽孢杆菌的毒蛋白(简称Bt-toxin)基因是目前世界上应用最为广泛的抗虫基因。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。当被昆虫吞食后,在昆虫中肠碱性环境下,经蛋白酶的作用，Bt-toxin被降解产生毒性小肽,并和中肠上皮细胞纹缘膜上的受体特异结合,然后插入细胞膜造成细胞膜穿孔,破坏细胞内外的渗透平衡,导致细胞膨胀而裂解,使得昆虫停止取食并最终死亡。
自第一次分离克隆到Bt基因以后,人们一直在试图将该基因转移进植物体并实现表达,以获得能够抗虫的转基因植物。但是Bt基因在植物中的表达并不稳定，而且抗虫效果不理想。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，在转化前需根据受体的密码子特征进行优化，使抗虫基因适合在植物受体细胞中高表达是关键。因此，对天然Bt基因进行改造，使其在植物中稳定高效表达，对获得杀虫率高的转基因抗虫植物是十分必要的。
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-FC。
本发明的第二个目的是提供人工合成的Bt抗虫基因JBT-FC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-FC，所述Bt抗虫基因JBT-FC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-FC在制备抗虫植物中的应用。
本发明的优点：人工合成的Bt抗虫基因JBT-FC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-FC可在转基因玉米中稳定遗传和表达，且表达量高，所得转基因玉米表现出较高的抗虫性。本发明JBT-FC抗虫基因为玉米害虫防治提供了一种新的途径，为今后利用该基因培育抗虫植物提供研究基础。
附图说明
图1为JBT-FC基因的植物表达载体。
图2为转JBT-FC基因的玉米RT-PCR检测结果。
具体实施方式
以下结合具体实施例进一步阐述本发明。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-FC基因的设计改造
根据苏云金芽孢杆菌Cry1Ac蛋白不同区域的活性分析及玉米基因组特点，我们对Cry1Ac蛋白氨基酸序列对应的密码子及编码框进行了优化改造，设计出一条新的基因，命名为JBT-FC，新基因JBT-FC中G的含量为19.52％；C的含量为24.05％；T的含量为26.71％，A的含量为29.71％，Bt抗虫基因JBT-FC(简称JBT-FC基因)的核苷酸序列如SEQ ID No：1所示。
实施例2 JBT-FC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-FC基因，同时在该基因的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-FC基因进行酶切，并回收JBT-FC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-FC新基因的植物表达载体pCAMBIA3301-JBT-FC(见图1)。
实施例3 JBT-FC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-FC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4μLpCAMBIA3301-JBT-FC质粒与农杆菌100μL混匀，冰浴15min，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400μL YEP液体培养基，于28℃振荡培养3h，8000r/min，28℃，离心收集菌体，弃300μL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左右，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-FC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-FC特异性引物进行扩增，扩增出的片段大小为1767bp(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:atgttatctgcttatactat(SEQNO.2)；下游:gaccgggatgaactcaattt(SEQ NO.3)。产物经0.9％琼脂糖凝胶电泳检测。如图2所示，泳道1为Marker，泳道2-9为转基因植株，泳道10为非转基因对照，泳道11为质粒pCAMBIA3301-JBT-FC阳性对照。RT-PCR检测结果表明，JBT-FC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-FC基因玉米植株。
实施例6转JBT-FC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：22株转JBT-FC基因玉米植株中抗虫植株为18株，抗虫率为81.82％，与非转基因玉米植株相比，转JBT-FC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-FC 及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1767
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgttatctg cttatactat tgttgtcggt accgtattga caggctttgg attcacgact 60
cctcttgggc tcgccctaat cggttttggc accctgatac ccgtgttatt cccagcacaa 120
gatcagtcca atacatggtc agactttatt acgcaaacta aaaacatcat aaagaaagaa 180
attgcctcga cctacatcag taatgctaac aagatattga atcgtagctt caacgttatt 240
tctacatatc ataatcacct taaaacgtgg gagaacaatc cgaaccctca gaatactcaa 300
gatgtccgca cccagatcca actcgtacat taccactttc agaacgtgat acccgaacta 360
gttaattcct gtccaccgaa cccttcagac tgcgattatt acaatattct ggtcttatcg 420
agttatcaag ccgcaaactt gcatcttagc gtactccgag acgtgtctgt tttcggacag 480
cggtgggggt ttgatgcggc tacaatcaat tccagataca acgacctaac gaggctgata 540
ggtaattata ctgatcactg tgtcaagtgg tacaacgtag gcttagacaa attgcgtgga 600
tcatcgctta atctcattaa gaccacaccc gatagtaacc tagacgggaa tatcaactgg 660
aatacgtata acactgaatt caaaactgaa tttacccgtg agatttatac agctttagtt 720
gaatctcctt cctcaaagtc gatcgccgca ttggaggcgg ctgaagatag tcttacgata 780
cgcaaacccc atctcttcga ctacctacaa ggtattgagt ttcacactcg actccagcca 840
ggctatttcg gaaaggatag ctttaattac tggtctggga actatgtctc cacccggccg 900
tcaatcggtt cgaatgacat aattacaagt cctttctacg gcaacaaaag tttaggattg 960
cttacgcatc aaatccagct caattctaac gtatataaga cttccataac cgatacatca 1020
tcgcccagta atagagtgac gaaaatgcta ggggttacta aggtcgaatt tagccaatac 1080
aacgaccaga ccgatgaggc ctctacacaa acgtatgact ccaaaaataa gaacattttc 1140
ggtctgccaa tcttaaaaag gcgtgaagag cagggcaatc cgactttgtt tcctaccgta 1200
aacgattaca cacacatact ttcatatgtg acgaattacg cagaatgttt cctcatgcaa 1260
ggatcgcgcg ggactattcc cgttctaacc tggacacata gtagcgtgga caagcttaaa 1320
aatactattt ataccgatgc tatcacacaa gttcctgccg tcaagtctaa ctttttaaat 1380
gcaacggcga aagtaataaa gggtcccggc ttcactggag gggacttggt gcgtcttaac 1440
tcctcaggta ccctctcggg ccgcatggaa attcagtgta aaacaagtat ctttaatgat 1500
ccaacgcgaa gctacttcat acggattaga tatgcttcta acgggtccgc caatactagg 1560
gcagttatct caaacaatga ctttctagtc atatacgtac cggcgaccgc tacatcgctg 1620
gataacttaa gtattcctgg ggtggccgag ttgggtatgg cacttaatcc cacgttcagc 1680
ggcaacaata tcataccaac tattggagcg gaatcttttg tttccaacga gaagatctat 1740
atagacaaaa ttgagttcat cccggtc 1767
<210> 2
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
atgttatctg cttatactat 20
<210> 3
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
gaccgggatg aactcaattt 20
Claims (2)
1.一种人工合成的Bt抗虫基因JBT-FC，其特征是所述Bt抗虫基因JBT-FC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-FC在制备抗虫植物中的应用。
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