Studies of Transgenic Hybrid Poplar 741 Carrying Two Insect-resistant Genes

Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.

Research on Frequency of Exogenous Gene Flow from Marber-free Insect-resistant Transgenic Rice to Conventional Rice Varieties

[Objective] This study aimed to investigate the frequency of exogenous gene flow to non-transgenic conventional rice cultivars and assess the potential risks of marker-free of insect-resistant transgenic rice to agricultural ecological environment. [Method] Insect-resistant transgenic rice variety HUAHUI No.1 was planted as the experimental material and surrounded by several non-transgenic conventional rice cultivars. F1 non-transgenic rice seeds were collected according to different distances and identified by using PCR technology, the frequency of exogenous gene flow from insect-resistant transgenic rice to non-transgenic conventional rice cultivars was counted and analyzed. [Result] The average frequency of exogenous Bt gene flow to P13381 and CHUNJIANG063 was 0. Transgene flow occurred to varying degrees from insect-resistant transgenic rice HUAHUI No.1 to several non-transgenic rice lines including HEX122-2, TIANXlANG, MINGHUI63 and Pl157, with the maximum average gene flow frequency of 0.875%. The frequency of gene flow was gradually reduced with the increase of distance, and the average transgene flow frequency de- creased to 0 in all the sampling points 7 m away from transgenic rice material. [Conclusion] This study revealed that the exogenous gene flow frequency of insect-re- sistant transgenic rice variety HUAHUI No.1 was very low, leading to very small risk to the eco-environment. Rational distribution in the field for physical isolation, keeping the appropriate distance and scientific farming arrangement to avoid the synchronization of flowering can effectively control the exogenous gene flow from transgenic rice and reduce he ecological risks caused by transgene escape.

Genetic Transformation of NGc Insect-resistant Gene in Hi-Ⅱ Maize

In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with CryNGc insect-resistant genes were cultured by explant infection,co-culture and differentiation screening to study the genetic expression and resistance of exogenous genes in the offspring.The results showed that the infection effect was the best when the size of young maize embryo was 1.2-1.8 mm.Ten positive transformed plants with CryNGc insect-resistant genes were successfully obtained,and the transformation efficiency was 1.428‰.

Cotton Plants Transformed with the Activated Chimeric Cry1Ac and API-B Genes

A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.

Indication of Genetic Linkage Map for Sunflower(Helianthus annuus L.) by SSR Markers

In order to investigate the possibility and efficiency of exogenous gene spread in nature and potential ecological risk of transgenic rice, as well as analyze the effect of exogenous Bt gene insertion on ecological fitness of transgenic rice plants, a experiment was carried out with three insect-resistant Bt transgenic rice cultivars Bt63, R1 and R2 and one conventional rice line 11-838 as experimental materials, the insect-resistant transgenic and non-transgenic rice plants were inter- cropped pair-wisely under high and low insect-infestation pressures, and the vegeta- tive growth, seed-setting and the resistance to rice stem borers were compared be- tween transgenic and non-transgenic lines. According to the experimental results, both the tiller number and fresh weight of Bt transgenic rice plants under low insect- infestation pressure showed no significant differences compared with the control, but the plant height, spike length and spike weight were all lower than those of non- transgenic rice plant, and Bt63 and R2 were significantly different compared with the control. On the contrary, under high insect-infestation pressure, the tiller number, spike length and spike weight of three Bt transgenic rice cultivars were significantly higher than those of the control, while the plant height showed different fitness ef- fects among various transgenic rice cultivars, which might be related to the charac- teristics of the receptive cultivars. The individual filled grain number and 1 000-grain weight of three transgenic rice cultivars showed no significant difference compared with the control under two different insect-infestation pressures, suggesting that the effect of exogenous Bt gene on seed setting was not significant. Under insect-infes- tation pressure, the resistance of three Bt transgenic rice cultivars against rice stem borer was significantly superior to non-transgenic rice, indicating that the effect of exogenous Bt gene on insect resistance of receptive plants was distinctly. Further- more, experimental results showed that the fitness cost of Bt transgenic rice was rel- atively low, which implied that exogenous Bt gene in insect-resistant transgenic rice might escape under certain environmental conditions, but this risk was very low.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

Integration and inheritance stability of foreign Bt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.

mtlD/gutD双价基因转化美洲黑杨×青杨的研究

利用叶盘法开展了mtlD gutD双价基因转化美洲黑杨×青杨的研究 ,经诱导不定芽及诱导生根阶段卡那霉素 (选择性抗生素 )连续筛选 ,获得了 38株卡那霉素抗性植株。抗性植株经PCR检测 ,2 8株呈阳性。对其中 5株PCR阳性植株进行Southern及Western杂交 ,有 4株二者均呈阳性 ,证明双价基因成功整合到美洲黑杨×青杨基因组中并得到表达。耐盐实验表明这

转双抗虫基因741杨的抗虫效应及其规律研究

通过室内饲虫试验 ,应用昆虫的总死亡率、各龄期死亡率、各龄历期及发育速率、昆虫体重增长速率等指标 ,测定了转双抗虫基因 74 1杨的抗虫性。研究结果表明 ,转基因抗虫杨不同株系对昆虫的致死效应有显著差异 ;不同昆虫种类和同一昆虫幼虫的不同龄级对转基因杨株系的敏感性有明显差异 ,4种测试昆虫的敏感性强弱依次为 :杨小舟蛾 >美国白蛾 >杨扇舟蛾 >舞毒蛾 ,随着昆虫幼虫龄级的增加 ,昆虫对转基因杨株系的敏感性逐渐降低 ;高抗虫株系主要表现为对低龄幼虫的强致死作用 ,中等抗虫株系表现为对各龄幼虫稍低的但是持续的致死效应 ;各转基因杨株系对测试昆虫的生长和发育都有明显抑制作用 ,可导致昆虫的发育历期延长、发育速率和体重增长速率降低。

青杨双价抗虫基因转化系统研究

利用农杆菌介导法,对青杨进行含部分改造的BtCryAc基因(Bt基因)和慈姑蛋白酶抑制剂(API)基因A的双价抗虫基因转化,确定了适于青杨的遗传转化系统。经含Km40mg/L的生根培养基的筛选,获得了青杨转基因植株。

健脾生血片益气生血的作用机制研究

目的:考察健脾生血片对低铁贫血模型大鼠的作用机制。方法:SD大鼠50只随机分为5组,分别为正常组、硫酸亚铁组、健脾生血片低、中、高剂量(0.65,1.3,2.6 g·kg^(-1))组,建立低铁贫血模型,健脾生血片各组及硫酸亚铁组水溶液灌胃,给药2周后,检测大鼠肠黏膜二价金属转运体(DMT-1)表达量及肝脏Hepcidin mRNA表达量。结果:健脾生血片中、高剂量组能显著提高DMT-1蛋白表达量,硫酸亚铁组DMT-1蛋白表达下调。肝脏中Hepcidin mRNA表达量随着给药剂量的增加而增加。结论:健脾生血片中药成分对上调DMT-1起到重要作用,通过改善Hepcidin mRNA的表达以达到补铁作用。

二价核酶基因构建及对PLRV负链靶序列的体外切割研究

根据锤头状核酶的作用模式,设计、合成并克隆了特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)中国分离株(PLRV-Ch)复制酶基因负链RNA的二价核酶序列。将该核酶基因克隆到pGEM-4Z中,构建成体外转录载体pGEM-4ZDR;同时将包含核酶切割识别位点的PLRV-Ch复制酶基因cDNA近5'端的874 bp片段反向插入到pGEM-4Z中,构建了体外转录载体pGEM-4ZR5。以线性化的重组质粒pGEM-4ZDR和pGEM-4ZR5为模板,在T7 RNA聚合酶的作用下,分别转录获得核酶RNA和PLRV-Ch复制酶基因5'端负链RNA底物片段。将以上2种RNA转录物混合并经37℃保温后,检测结果表明,所得核酶RNA对PLRV-Ch复制酶基因负链RNA在体外具有较强的特异切割活性。

育龄妇女DMT1基因多态性与缺铁性贫血关系

目的 探讨二价金属离子转运蛋白1（DMT1）的基因变异与缺铁性贫血的关系。方法 收集北京大学第三医院非孕18～45岁缺铁性贫血（IDA）女性患者82人作为病例组，选取北京市顺义区和河北省廓坊市94名同龄健康育龄妇女作为正常对照，应用聚合酶联反应-限制性酶切片段长度多态性（PCR—RFLP）技术，分析DMT1基因的IVS4＋44位点多态性。结果 本研究人群中存在DMT1基因的IVS4＋44位点杂合突变（＋／-）和纯合突变（＋／＋）基因型。对照组（＋／-）基因型和（＋）等位基因的频率分别为28．7％和14．4％；IDA组（＋／＋）、（＋／-）基因型和（＋）等位基因的频率分别为2．4％。19．5％，12．2％。与对照组相比。基因型构成差异无统计学意义。（＋／-）＆（＋／＋）基因型患IDA的比值比（OR）为0．70，95％CI：0．35～1．39；IDA组及对照组不同基因型血清铁蛋白（SF）、血红蛋白（Hb）水平差异均无统计学意义。结论 DMT1基因的IVS4＋44位点变异与IDA的发生无明显关联。

幼畜腹泻双价基因工程苗对牛和羊的攻毒保护试验

用幼畜腹泻双价基因工程苗以两种剂量（正常免疫剂量和1／2免疫剂量）分别免疫接种怀孕母牛和怀孕母羊，待其分娩并保证其所产幼畜（犊牛、羔羊）采食初乳后，经口灌服致幼畜腹泻的强毒活菌。结果，正常免疫剂量组，犊牛腹泻发病率为14．3％，死亡率为零；羔羊腹泻发病率为15．8％，死亡率为2．7％。1／2免疫剂量组，犊牛腹泻发病率为30．7％，死亡率为10．3％；羔羊腹泻发病率为24．4％，死亡率为12．2％。未接种疫苗的对照组，犊牛腹泻发病率为83．3％，死亡率为44．4％；；，羔羊腹泻发病率为78．9％，死亡率为57．8％。经统计学处理，三组之间犊牛和羔羊的发病率和死亡率均是极显著差异（P＜0．01）。

二价金属离子转运蛋白1基因多态性与原发性帕金森病患者中脑黑质铁沉积的关系

目的:探讨二价金属离子转运蛋白1(DMT1)的基因多态性与原发性帕金森病患者中脑黑质铁沉积的关系。方法:收集行经颅超声检查的原发性帕金森病患者共120例,根据结果将患者分为超声阳性组(有铁沉积,n=67)和超声阴性组(无铁沉积,n=53)。采用聚合酶链反应-限制性酶切片段长度多态性(PCR-RFLP)技术,比较2组患者DMT1基因rs6580779位点的多态性。结果:2组之间的基因型、最小等位基因频率分布的差异皆无统计学意义,超声阳性组和阴性组的基因型AA、AC频率分别为98.5%、1.5%和94.3%、5.7%(P=0.20),2组的最小等位基因频率分别为0.7%和2.8%(P=0.24)。晚发病型患者在超声阳性组与阴性组之间没有显著差异。结论:DMT1基因多态性位点rs6580779的分布在原发性帕金森病患者超声阳性与阴性组中无统计学差异,其变异与原发性帕金森病患者中脑黑质铁沉积无明显关联。

Transgenic Tobacco Plants With Efficient Insect Resistance

Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of B.t.toxin gene.Leave stripes of tobacco plant var.NC89 widelygrown in China were transformed with A.tumefaciens LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were regenerated.Insect test with tobacco budwormH.assulta showed that insect-resistant transform.ants could be obtained from the regenerated plantstransformed with B.t.genes of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin gene.Genetic,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that B.t.toxin gene and the character of insect resistance have been inherited in the pro-genies.Insect-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.