抗虫Bt基因cry1Ac甜菜叶绿体遗传转化与表达研究
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摘要
甜菜（Beta vulgaris L.）是我国重要的糖料作物，也是虫害非常严重的作物之一，尤其是甘蓝夜蛾的危害更为严重，化学防治达不到预期效果，还严重污染环境。随着人类生态及环保意识的加强，大力发展绿色农业已经成为新时代的要求，因而，培育高抗虫甜菜新品种成为一项重要研究课题，基因工程方法培育抗虫品种是害虫防治的有效途径。本论文首次尝试对未知叶绿体基因组序列的甜菜进行抗虫基因的叶绿体转化与表达研究。以克隆的甜菜叶绿体基因atpB/rbcL为同源片段，以作用鳞翅目的Bt基因cry1Ac为外源目的基因，以aadA基因为筛选标记基因构建了甜菜叶绿体定点整合转化载体，基因枪法转化甜菜，通过抗性筛选、分子检测及抗虫试验等，最终获得了甜菜抗虫转基因再生植株及后代，并对转基因植株后代抗虫基因遗传稳定性进行了研究，以及对叶绿体转化技术的机理进行了探索，最终建立了甜菜叶绿体转化体系。
通过对甜菜不同基因型、外植体及培养基激素组合等再生影响因素的筛选，优化了各类培养基配方，建立了以甜菜叶柄为外植体的高效不定芽直接再生体系，为转基因再生植株的获得打下了基础。通过抗性筛选试验确定了甜菜遗传转化中适宜的筛选剂（壮观霉素和卡那霉素）及其使用浓度（30mg/L和50mg/L），确定了甜菜叶绿体转化的筛选标记基因为aadA基因。
通过比较试验，借鉴已有植物叶绿体DNA提取方法优点，并结合甜菜特性（糖分含量较高），研究建立了甜菜叶绿体DNA分离纯化方法。试验证明：利用该方法获得的甜菜叶绿体DNA不仅纯度高，得率提高3倍以上，可直接用于分子生物学研究及基因组测序。
由于甜菜叶绿体基因组全序列未知，本研究首先利用高等植物叶绿体基因组在进化过程中的高度保守的特性，借助生物信息学软件分析了烟草、水稻、菠菜、玉米等高等植物的叶绿体基因组全序列资料，筛选到紧密连锁的atpB基因与rbcL基因、rps7基因与ndhB基因两个位点，并参照烟草、水稻和菠菜叶绿体基因组全序列资料中相应区段核苷酸序列，分别设计引物，PCR方法成功克隆了甜菜叶绿体中与光合作用有关的重要功能基因atpB和rbcL及rps7和ndhB的完整基因，测序、序列分析及同源性比较证实：得到的结果与预期相符。其中，atpB和rbcL基因序列已提交到GenBank，登录号分别为DQ067451、DQ067450，atpB和rbcL基因的克隆同时也为研究甜菜光合作用机理提供依据。
采用分子生物学手段成功构建了抗虫Bt基因cry1Ac甜菜叶绿体转化载体，该载体包含有同源片段atpB/rbcL、Bt基因cry1Ac表达盒（Prrn-cry1Ac-psbA3’）、筛选标记基因aadA表达盒（Prrn-aadA-TpsbA），采用基因枪法转化甜菜，通过多次继代及2轮分化与多次继代结合的抗性筛选，获得一批转基因抗性植株。建立了甜菜叶绿体遗传转化体系，为今后继续开展甜菜叶绿体遗传转化研究提供了依据。
转基因再生植株外源基因的PCR检测、Southern blot、Northern blot、Western blot，结果表明：抗虫Bt基因cry1Ac已定点整合到甜菜叶绿体基因组中，并得到了表达。以二龄末甘蓝夜蛾为试虫，通过人工饲虫法，分别检测克隆工程菌及转基因再生植株的杀虫性，结果表明：以克隆菌菌体蛋白涂抹甜菜叶片饲喂的二龄末幼虫，幼虫平均死亡率达到90%；以不同转基因再生植株叶片饲喂二龄末幼虫，幼虫死亡率达33.3%~66.7%。之所以出现差异，可能与转化体的同质化程度不同有关。众所周知：每个叶肉细胞含有数十个甚至数百个叶绿体，被整合外源基因的叶绿体数量越多，即转化体同质化程度越高，其外源基因表达量越高，杀虫性也越好。采用在抗性培养基上2轮筛选及多次继代方法对转基因植株进行同质化筛选研究，结果表明：转基因植株的同质化程度明显提高，加快了获得同质化纯系的进程。转基因甜菜后代抗虫基因的遗传稳定性和抗虫性分析表明，外源基因在转基因后代中得到稳定遗传并表达稳定的杀虫活性,幼虫死亡率仍达33.3%~55.6%。
为了使叶绿体转化技术能广泛应用，本论文对其机理进行了研究。以除草剂抗性基因bar为筛选标记基因，比较研究bar基因和aadA基因的筛选效率，结果表明：筛选标记基因是影响目的基因转化效率的因素；以本研究克隆的位于叶绿体基因组中反向重复序列上的rps7-rps12/ndhB基因为同源片段，比较研究同源片段位置与目的基因转化效率的关系，结果表明：同源片段的位置对目的基因转化效率影响差异不大。此研究结果为其他植物开展叶绿体转化研究提供理论依据。
Sugar beet is a important sugar crops in China, but it’s always infested bymany insect pests, especially Barathra brassicae L. The chemical insecticidescann’t exterminate the insects effectively and also have bad effect on theentironment. With the emphasis of ecological and environmental protection,ecological agriculture has become a significant demand in the future and insect-resistant genetic engineering has been an effective method for breeding insect-resistant sugar beet. In this research, insect-resistant gene was transformed andexpressed into the chloroplast genome of sugar beet though the sequence ofchloroplast genome are unknown. We constructed sugar beet chloroplastmulticistron site integration expression vector. The atpB and rbcL genes clonedfrom sugar beet chloroplast genome is homologous fragments and Bt genecry1Ac acted on lepidopteran insects is target gene, and aadA gene is selectivemarker. After transformation with particle-bombardment, screening of putativeplants was antibiotic, molecular detection and bioassay, finally transgenic sugarbeet plants and the T1seeds were obtained which have resistance to pests. Thegenetic stability of transgenic plants were studied and the mechanism ofchloroplast transformation was researched. The sugar beet chloroplasttransformation system was established.
The culture medium component were confirmed after the experiment whichwere designed based on the different combination of genotype, explant type andplant growth regulators. Optimize sugar beet regeneration system was established.It provided a good foundation for regeneration of transgenic sugar beet.Afterselected with5antibiotics, kanamycin and spectinomycin were chosen as theselective agent, and their respective concentration were50mg/L and30mg/L. TheaadA gene was chosen as selectable marker in chloroplast transformation system.
Because the feature of sugar beet which contain much sugar, the existingmethods of chloroplast DNA extraction methods are not applicable for sugar beet.Therefore, according to the advantages of existing methods and the feature ofsugar beet,a new method of sugar beet chloroplast DNA extraction was used in the research. The chloroplast DNA prepared with such method had not only high-yield more three times, but also high-quality, which could be directly used forgenome sequencing.
The sequence of sugar beet chloroplast genome is unknown, so it is difficultto clone homologous fragments. In this research, we analyzed the completesequences chloroplast genome of higher plants, such as tobacco, rice, spinach andmaize, by referring to the conservation status of plant chloroplast genome formedthrough evolution. With the primers that designed according to the nucleotidesequences of atpB/rbcL and rps7/ndhB genes, we cloned the four significantfunctional genes from sugar beet chloroplast genome successfully by PCRreaction. The sequencing results showed no difference with expectancies and theatpB/rbcL genes had been submissioned to Genbank, the Accession No. areDQ067451and DQ067450. The atpB and rbcL genes cloning would alsocontribute to studying the photosynthesis mechanisms of sugar beet.
A sugar beet chloroplast transformation vector was constructed, whichcontained homologous recombinant fragments atpB and rbcL, insect-resistant Btgene cry1Ac expression cassette（Prrn-cry1Ac-psbA3’）and selective markergene aadA expression cassette（Prrn-aadA-TpsbA）. After transformation withparticle-bombardment and screening several times, some transgenic sugar beetwere obtained. The chloroplast transformation system of sugar beet wasestablished, and it would be a good foundation for further research.
After PCR identification, Southern blot, Northern blot and Western blot, theresults indicated that insect-resistant Bt gene cry1Ac had integrated andexpressed in sugar beet chloroplast genome. We detected the insecticidal activityof cloning recombinant strain and transgenic plants with the second instar larvaesto Barathra brassicae L. The results indicated that the Bt toxin protein of cloningrecombinant strain had high insecticidal activity and the mortality of larvaes was90%; the mortality was33.3%~66.7%when the larvaes weve bred with leavesfrom transgenic plants. The difference of mortality is relevant to the differenthomoplasmic level. As we know, there are hundreds of chloroplasts in onemesophyll cell. When the numbers of chloroplasts integrated with target gene islarger, the homoplasmic level of transgenic plants and the expression level oftarget gene will be higher, and the insecticidal activity will be higher too. Secondary regeneration and many times of subculture on medium with antibioticswere carried out to filtrate homoplasmic plants. The results indicated that twoadditional rounds of regeneration and were subcultured for many times on MSmedium containing spectinomycin in the stem sections, increased thehomoplasmic level of transgenic plants markedly. This study of increasinghomoplasmic level of transgenic plants will extremely contribute to the obtainingof homopladmic pure line. The genetic stability of transgenic plants were studiedand it showed that the Bt gene could express steadily and expressed insecticidalactivity in the T1generation. The mortality of larvaes was33.3%-55.6%.
The research also used the bar gene and aadA gene as a selective marker forcontrast, and the result showed that different selective marker had different effecton screening of transgenic plants. Meanwhile, the research used rps7rps12/ndhBwhich were also cloned from sugar beet chloroplast genome as homologousfragments, and the result showed that the position of the homologous fragmentshad no effect on tansformation. Those study may contribute to chloroplasttransformation on other crops.
通过对甜菜不同基因型、外植体及培养基激素组合等再生影响因素的筛选，优化了各类培养基配方，建立了以甜菜叶柄为外植体的高效不定芽直接再生体系，为转基因再生植株的获得打下了基础。通过抗性筛选试验确定了甜菜遗传转化中适宜的筛选剂（壮观霉素和卡那霉素）及其使用浓度（30mg/L和50mg/L），确定了甜菜叶绿体转化的筛选标记基因为aadA基因。
通过比较试验，借鉴已有植物叶绿体DNA提取方法优点，并结合甜菜特性（糖分含量较高），研究建立了甜菜叶绿体DNA分离纯化方法。试验证明：利用该方法获得的甜菜叶绿体DNA不仅纯度高，得率提高3倍以上，可直接用于分子生物学研究及基因组测序。
由于甜菜叶绿体基因组全序列未知，本研究首先利用高等植物叶绿体基因组在进化过程中的高度保守的特性，借助生物信息学软件分析了烟草、水稻、菠菜、玉米等高等植物的叶绿体基因组全序列资料，筛选到紧密连锁的atpB基因与rbcL基因、rps7基因与ndhB基因两个位点，并参照烟草、水稻和菠菜叶绿体基因组全序列资料中相应区段核苷酸序列，分别设计引物，PCR方法成功克隆了甜菜叶绿体中与光合作用有关的重要功能基因atpB和rbcL及rps7和ndhB的完整基因，测序、序列分析及同源性比较证实：得到的结果与预期相符。其中，atpB和rbcL基因序列已提交到GenBank，登录号分别为DQ067451、DQ067450，atpB和rbcL基因的克隆同时也为研究甜菜光合作用机理提供依据。
采用分子生物学手段成功构建了抗虫Bt基因cry1Ac甜菜叶绿体转化载体，该载体包含有同源片段atpB/rbcL、Bt基因cry1Ac表达盒（Prrn-cry1Ac-psbA3’）、筛选标记基因aadA表达盒（Prrn-aadA-TpsbA），采用基因枪法转化甜菜，通过多次继代及2轮分化与多次继代结合的抗性筛选，获得一批转基因抗性植株。建立了甜菜叶绿体遗传转化体系，为今后继续开展甜菜叶绿体遗传转化研究提供了依据。
转基因再生植株外源基因的PCR检测、Southern blot、Northern blot、Western blot，结果表明：抗虫Bt基因cry1Ac已定点整合到甜菜叶绿体基因组中，并得到了表达。以二龄末甘蓝夜蛾为试虫，通过人工饲虫法，分别检测克隆工程菌及转基因再生植株的杀虫性，结果表明：以克隆菌菌体蛋白涂抹甜菜叶片饲喂的二龄末幼虫，幼虫平均死亡率达到90%；以不同转基因再生植株叶片饲喂二龄末幼虫，幼虫死亡率达33.3%~66.7%。之所以出现差异，可能与转化体的同质化程度不同有关。众所周知：每个叶肉细胞含有数十个甚至数百个叶绿体，被整合外源基因的叶绿体数量越多，即转化体同质化程度越高，其外源基因表达量越高，杀虫性也越好。采用在抗性培养基上2轮筛选及多次继代方法对转基因植株进行同质化筛选研究，结果表明：转基因植株的同质化程度明显提高，加快了获得同质化纯系的进程。转基因甜菜后代抗虫基因的遗传稳定性和抗虫性分析表明，外源基因在转基因后代中得到稳定遗传并表达稳定的杀虫活性,幼虫死亡率仍达33.3%~55.6%。
为了使叶绿体转化技术能广泛应用，本论文对其机理进行了研究。以除草剂抗性基因bar为筛选标记基因，比较研究bar基因和aadA基因的筛选效率，结果表明：筛选标记基因是影响目的基因转化效率的因素；以本研究克隆的位于叶绿体基因组中反向重复序列上的rps7-rps12/ndhB基因为同源片段，比较研究同源片段位置与目的基因转化效率的关系，结果表明：同源片段的位置对目的基因转化效率影响差异不大。此研究结果为其他植物开展叶绿体转化研究提供理论依据。
Sugar beet is a important sugar crops in China, but it’s always infested bymany insect pests, especially Barathra brassicae L. The chemical insecticidescann’t exterminate the insects effectively and also have bad effect on theentironment. With the emphasis of ecological and environmental protection,ecological agriculture has become a significant demand in the future and insect-resistant genetic engineering has been an effective method for breeding insect-resistant sugar beet. In this research, insect-resistant gene was transformed andexpressed into the chloroplast genome of sugar beet though the sequence ofchloroplast genome are unknown. We constructed sugar beet chloroplastmulticistron site integration expression vector. The atpB and rbcL genes clonedfrom sugar beet chloroplast genome is homologous fragments and Bt genecry1Ac acted on lepidopteran insects is target gene, and aadA gene is selectivemarker. After transformation with particle-bombardment, screening of putativeplants was antibiotic, molecular detection and bioassay, finally transgenic sugarbeet plants and the T1seeds were obtained which have resistance to pests. Thegenetic stability of transgenic plants were studied and the mechanism ofchloroplast transformation was researched. The sugar beet chloroplasttransformation system was established.
The culture medium component were confirmed after the experiment whichwere designed based on the different combination of genotype, explant type andplant growth regulators. Optimize sugar beet regeneration system was established.It provided a good foundation for regeneration of transgenic sugar beet.Afterselected with5antibiotics, kanamycin and spectinomycin were chosen as theselective agent, and their respective concentration were50mg/L and30mg/L. TheaadA gene was chosen as selectable marker in chloroplast transformation system.
Because the feature of sugar beet which contain much sugar, the existingmethods of chloroplast DNA extraction methods are not applicable for sugar beet.Therefore, according to the advantages of existing methods and the feature ofsugar beet,a new method of sugar beet chloroplast DNA extraction was used in the research. The chloroplast DNA prepared with such method had not only high-yield more three times, but also high-quality, which could be directly used forgenome sequencing.
The sequence of sugar beet chloroplast genome is unknown, so it is difficultto clone homologous fragments. In this research, we analyzed the completesequences chloroplast genome of higher plants, such as tobacco, rice, spinach andmaize, by referring to the conservation status of plant chloroplast genome formedthrough evolution. With the primers that designed according to the nucleotidesequences of atpB/rbcL and rps7/ndhB genes, we cloned the four significantfunctional genes from sugar beet chloroplast genome successfully by PCRreaction. The sequencing results showed no difference with expectancies and theatpB/rbcL genes had been submissioned to Genbank, the Accession No. areDQ067451and DQ067450. The atpB and rbcL genes cloning would alsocontribute to studying the photosynthesis mechanisms of sugar beet.
A sugar beet chloroplast transformation vector was constructed, whichcontained homologous recombinant fragments atpB and rbcL, insect-resistant Btgene cry1Ac expression cassette（Prrn-cry1Ac-psbA3’）and selective markergene aadA expression cassette（Prrn-aadA-TpsbA）. After transformation withparticle-bombardment and screening several times, some transgenic sugar beetwere obtained. The chloroplast transformation system of sugar beet wasestablished, and it would be a good foundation for further research.
After PCR identification, Southern blot, Northern blot and Western blot, theresults indicated that insect-resistant Bt gene cry1Ac had integrated andexpressed in sugar beet chloroplast genome. We detected the insecticidal activityof cloning recombinant strain and transgenic plants with the second instar larvaesto Barathra brassicae L. The results indicated that the Bt toxin protein of cloningrecombinant strain had high insecticidal activity and the mortality of larvaes was90%; the mortality was33.3%~66.7%when the larvaes weve bred with leavesfrom transgenic plants. The difference of mortality is relevant to the differenthomoplasmic level. As we know, there are hundreds of chloroplasts in onemesophyll cell. When the numbers of chloroplasts integrated with target gene islarger, the homoplasmic level of transgenic plants and the expression level oftarget gene will be higher, and the insecticidal activity will be higher too. Secondary regeneration and many times of subculture on medium with antibioticswere carried out to filtrate homoplasmic plants. The results indicated that twoadditional rounds of regeneration and were subcultured for many times on MSmedium containing spectinomycin in the stem sections, increased thehomoplasmic level of transgenic plants markedly. This study of increasinghomoplasmic level of transgenic plants will extremely contribute to the obtainingof homopladmic pure line. The genetic stability of transgenic plants were studiedand it showed that the Bt gene could express steadily and expressed insecticidalactivity in the T1generation. The mortality of larvaes was33.3%-55.6%.
The research also used the bar gene and aadA gene as a selective marker forcontrast, and the result showed that different selective marker had different effecton screening of transgenic plants. Meanwhile, the research used rps7rps12/ndhBwhich were also cloned from sugar beet chloroplast genome as homologousfragments, and the result showed that the position of the homologous fragmentshad no effect on tansformation. Those study may contribute to chloroplasttransformation on other crops.
引文
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