Cry1Ah蛋白标准物质候选物的制备与纯化

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白 (排阻色谱纯度：99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

Cry1Ah蛋白标准物质候选物的制备与纯化（英文）

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白(排阻色谱纯度:99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析

对利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)强启动子——cry1Ac基因启动子p1Ac指导cry基因在大肠杆菌中的表达进行了研究。结果显示,大肠杆菌中由启动子p1Ac指导表达的Cry1Ac蛋白与苏云金芽胞杆菌来源的Cry1Ac蛋白在碱溶性、胰蛋白酶活化、杀虫活性等方面有较好的一致性,从而解决了目前商业化载体大肠杆菌表达cry基因时形成不易溶解的包涵体问题。同时,还对p1Ac指导的cry1Ac基因在大肠杆菌中表达的发酵条件进行了初步探索。     

cry1A基因在转基因玉米中的遗传与表达

通过Southern杂交、ELISA分析等方法, 研究了cry1A基因在转基因玉米中的遗传与表达.结果表明,cry1A基因在转基因玉米中呈单位点显性基因遗传.cry1A杀虫蛋白在转基因玉米不同株系中的表达量存在显著差异;在转基因玉米同一植株不同组织中的表达量也明显不同,在叶片、苞叶等绿色组织中的表达量显著高于在髓、花丝等非绿色组织中的表达量;在玉米叶片中的表达量随着发育期的推进有上升的趋势;在研究的3个转基因玉米株系中,cry1A杀虫蛋白的表达量在R2、R3、R4代之间无显著差异.     

cry1A基因在转基因玉米中的遗传与表达(英文)

通过Southern杂交、ELISA分析等方法 ,研究了cry1A基因在转基因玉米中的遗传与表达。结果表明 ,cry1A基因在转基因玉米中呈单位点显性基因遗传。cry1A杀虫蛋白在转基因玉米不同株系中的表达量存在显著差异 ;在转基因玉米同一植株不同组织中的表达量也明显不同 ,在叶片、苞叶等绿色组织中的表达量显著高于在髓、花丝等非绿色组织中的表达量 ;在玉米叶片中的表达量随着发育期的推进有上升的趋势 ;在研究的 3个转基因玉米株系中 ,cry1A杀虫蛋白的表达量在R2 、R3 、R4代之间无显著差异。     

苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis,Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因(去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆,将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315,分别构建重组质粒pHUAccB6、pHUAccB7,在大肠杆菌中扩增后,将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中,获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后,将晶体和上清液分别进行SDS-PAGE分析,双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较,几丁质酶活性提高5.2倍,双价重组蛋白表达量显著提高,主要产生130kDa蛋白条带。经定量分析:双价重组目的晶体蛋白占总蛋白量的61.38%;Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60%硫酸铵沉淀,显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察,表达后的重组蛋白呈菱形或椭原形晶体,其规格约为1.5×3.0μm;经生测分析,重组菌株HAccB6和HAccB7毒力相近,与HAc菌株比较毒力提高4.5倍,对棉铃虫(Helicourpa armigora)具有高效杀虫活性,其3dLC50值分别为9.1μg/mL和11.34μg/mL。研究结果表明,烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。     

一种新的Bt杀虫基因HJC原核表达蛋白活性的鉴定

HJC基因是由2个Bt基因(cry1Ab和vip3)经过人工融合而成,具有更广谱的杀虫活性,可延缓害虫产生交互抗性的时间。将已构建好的携带HJC基因的重组质粒pET28a-HJC转化到大肠杆菌BL21中诱导表达。该HJC融合蛋白主要以包涵体形式存在,变性条件下使用镍亲和层析柱对其进行纯化,并经尿素梯度透析复性后,进行免疫反应活性及美国白蛾杀虫活性测定。Western blot结果显示,该原核表达蛋白与转HJC基因水稻中的HJC蛋白有相同的免疫反应性,对美国白蛾也有一定的杀虫活性,可以替代植物外源蛋白进行转HJC基因产品的食用安全性评价。     

31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究

利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明：25株含cry1基因,表达蛋白130～150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明：cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94 μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/cry1Ac基因玉米ZZM030心叶中高表达的Cry1Ab/Cry1Ac融合蛋白对亚洲玉米螟初孵幼虫具有极高的杀虫效果。     

转修饰cry1Ac基因籼稻明恢81经花药培养获得抗虫DH系

对基因枪法获得的明恢81转修饰的cry1Ac基因当代植株进行花药培养,共接种花药2600枚,获得83份花培植株,其中双倍体植株43份,单倍体植株40份,PCR结果表明含有cry1Ac基因的植株55份,花培植株群体中转基因与非转基因植株的比值为2：1（55／28）,进一步结合Southern blot和ELISA分析,于花培植株当代筛选到转基因纯合株系36份,外源蛋白表达量上,花药来源于同一克隆的DH系的不同植株之间基本一致,最高的Cry1Ac含量达0．25％,田间抗虫性试验表明,经花药培养纯合获得的部分转基因纯合系植株对二化螟（chilo suppressalis)表现出高抗,而且主要农艺性状保持不变,以上结果表明水稻花药培养可以加速转基因的纯合与良种利用。     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

转cry1Ab基因水稻对二化螟幼虫血细胞的影响

研究了转cry1Ab基因水稻“克螟稻1号”对二化螟Chilo suppressalis幼虫细胞免疫系统的影响。结果表明,转cry1Ab基因水稻对二化螟幼虫的血细胞影响明显,取食转cry1Ab基因水稻后,二化螟幼虫各类血细胞都明显低于取食非转基因水稻“秀水11”的对照组（原血细胞和囊血细胞在取食初期例外）,随取食时间延长,各类血细胞数量及血细胞总数均呈递减的趋势。从各类血细胞所占血细胞总数的百分比来看,原血细胞在取食36 h后锐减,而浆血细胞和粒血细胞则比例增加,其余珠血细胞、囊血细胞的变化不明显。另外,血细胞还出现空泡化、肿胀等病态变化,致使血细胞快速破裂。由此推测转cry1Ab基因水稻自身表达的毒蛋白能严重干扰靶标昆虫二化螟幼虫的细胞免疫系统。     

Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera

A cry1Ia5 insecticidal toxin coding gene has been cloned from an Indian isolate of Bacillus thuringiensis. Sequence analyses of the cry1Ia5 gene revealed the absence of potential polyadenylation signal sequences thus making it a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cry1Ia5 gene into a plant expression vector and then transferring it to Nicotiana tabacum through Agrobacterium-mediated transformation. Our results demonstrate that N. tabacum with a stably integrated native cry1Ia5 gene afforded complete protection against predation by Heliothis armigera. Forty three percent of the transgenic plants displayed a high level of protection against insect predation. The protection obtained in transgenic plants with the cry1Ia5 gene was comparable to that obtained with the synthetically modified cry1A(b) or cry1A(c) genes. The results demonstrate that novel insecticidal genes already exist in nature that do not require extensive modifications for efficient expression in plants.     

分子改造cryNAc基因的转基因水稻创制及其功能评价

利用蛋白质工程技术对Cry蛋白进行改造是创制新Bt蛋白的主要途径之一。Cry蛋白改造涉及结构域交换、定点突变、蛋白截断等多种方法。利用结构域交换、密码子优化方法对Bt基因进行合理化设计改造,获得新型Bt蛋白编码基因cryNAc,进一步利用农杆菌介导法转入吉林省水稻主栽品种吉粳88中,并开展了转基因后代的分子鉴定和抗虫性功能评价相关研究工作。分子检测结果表明cryNAc基因成功整合进入吉粳88基因组中,且稳定表达;CryNAc蛋白在各个发育时期根、茎、叶中的表达存在显著差异,灌浆期水稻叶片中蛋白表达量最高（2 959.73 ng/g）,分蘖期茎中蛋白表达量最低（150.9 ng/g）;田间接虫试验表明cryNAc转基因水稻抗二化螟的能力显著。上述结果表明cryNAc基因可作为新的cry基因用于作物遗传改良。     

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae)

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future.