Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the microparticle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

油菜农杆菌介导转化体系的建立

研究了抗生素浓度、菌液浓度、侵染时间以及共培养时间对转基因油菜中油821外植体分化的影响,并分别进行了分析,建立了高效的农杆菌介导的油菜遗传转化体系.实验结果表明：使用5 d苗龄的带柄子叶作为转化受体,转化率高;卡那霉素的筛选浓度对转化影响较大,最终确定11 mg/L为该实验筛选的临界浓度;侵染时间为5 min,菌液浓度OD600为0.4时,其外植体转化率最高;共培养时间为48 h时效果最好,不仅有利于外源基因的整合,也不会造成农杆菌的过度增殖;在培养基中分别加入200μmol/L乙酰丁香酮（AS）和5～10 mg/L的硝酸银,可明显提高转化率.     

根癌农杆菌介导苜蓿体胚转化及转基因植株再生

用含质粒载体pCAMBIA2301(带有受CaMV35S启动子调控的GUS基因和nptⅡ基因)的根癌农杆菌Agrobacterium tumefaciens转化晋南苜蓿Medicago sativa L.cv.Jinnan的体胚组织,发现负压处理有利于提高转化频率(可达35%),3批共158个体胚切块的转化实验共获得具有卡那霉素抗性的再生植株15株,经组织化学染色和分子检测,证实GUS基因已整合到转化植株基因组中,在芽、叶片、叶柄和根等组织中均有表达,并在土壤栽培过程中保持稳定的表达.     

生菜遗传转化受体系统的建立及鲑鱼降钙素基因的导入

以散叶生菜大速生(Lactuca sativa var.capatata L.)为试材，以MS为基本培养基。采用不同激素配比，确定生菜高效诱芽培养基为MS 0．1 mg／L6-BA 0．05mg／LNAA；抗生素敏感性试验表明，筛选培养基中适宜的卡那霉素选择压为75mg／L，抑菌剂羧苄青霉素的适宜质量浓度为300mg／L；通过根癌农杆菌介导的叶盘法将鲑鱼降钙素(sCT)基因转入大速生散叶生菜，PCR检测转化率达25．4％。     

根癌农杆菌介导的百脉根遗传转化体系的优化研究

百脉根(L otus corn icu la tus L.)是世界著名的多年生优良豆科牧草之一,也是常见的庭院观赏植物.本文通过比较不同根癌农杆菌转化百脉根的效率和优化百脉根子叶遗传转化的条件,建立了一套有效的遗传转化体系.实验结果表明,EHA 105作为宿主菌对百脉根进行转化较LBA 4404和GV 3101具有更高的转化率;5 d苗龄的子叶最适合作为转化的外植体;浸染过程中0.05 M Pa负压处理5 m im以及在共培养培养基中添加20 m g/L的乙酰丁香酮均可提高转化效率.卡那霉素抗性植株经3次选择继代培养后,PCR检测全部为阳性.对部分PCR阳性植株进行PCR-Sou thern杂交,证实PCR产物真实可靠,表明外源基因已整合进入百脉根基因组中.     

Studies of Improving the Frequency of Indica Rice Transformation by Biolistic Bombardment

In order to improve the frequency of indica rice transformation by biolistic bombardment,suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedling,and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars.The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly;The concentration of 30-40mg/L Hyg or 150-200mg/L G418 or 10-20mg/L Basta is suitable for selection of resistant calli;The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result;Keeping the target calli on osmotic medium containing 60g/L mannitol from 12-24h before bombardment to 24-48h after it can increase the efficiencies of transformation.Furthermore,some transgenic indica rice plants are obtained using this optimized transformation system.     

农杆菌介导的杜氏盐藻Dscbr基因转化紫花苜蓿的初步研究

利用农杆菌介导法进行紫花苜蓿遗传转化研究．将从盐生杜氏藻(Dunaliella samlina)中克隆到的抗逆基因Dscbr(编码一种早期光诱导蛋白)导入紫花苜蓿栽培种“中苜一号”,获得52个转化株系．经过卡那霉素抗性鉴定和PCR分子检测,获得6株阳性苗,PCR阳性率为11．54％．结果表明Dscbr基因已整合到苜蓿的基因组中．     

p21HBsAg/HBsAg和P21HBX/HBX转基因小鼠不同月龄血液生理指标的测定

目的检测p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠的血液生理指标，分析p21^HBsAg/HBsAg和p21^HBX/HBX基因引入小鼠基因组p21位点中对其血液指标的影响。方法用日本光电MEK-5126K血球计数仪（动物芯片）及配套试剂盒对四个月龄不同基因型小鼠的血液生理指标进行测定，并且与野生型对照组进行比较。结果多数指标雌雄之间差异显著，并且两种基因型小鼠及野生型在不同年龄、不同指标的动态变化不同。结论p21^HBsAg/HBsAg和p21^HBX/HBX基因转入小鼠基因组p21位点对其血液生理指标具有一定的影响。     

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit （HMW-GS）genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of To generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20--150 mg/L PPT.Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1DylO genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1DylO. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared,but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2149.0 mL) was remarkably improved,59.6%---64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3--7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.     

Transformation of the salt-tolerant gene of<Emphasis Type="Italic">Avicennia marina</Emphasis> into tobacco plants and cultivation of salt-tolerant lines

Salt-tolerant gene, CSRG1, which was isolated from a kind of salt-tolerant mangroves, Avicennia marina, constructed the transgenic plasmid, pGAM189/CSRG1. CSRG1, GUS, Kmr and Hyg^r could be transferred into tobacco genome by the ameliorated leaf discs method of agro-bacterium-mediate transformation. Thirteen stable resistant lines were obtained when fifty transgenic explants were selected through 50 mg/L hygromycin and 150 mg/L kanamycin. Assessments of PCR amplification, Southern blot analysis and GUS histochemical staining showed that CSRG1 has been integrated into the genome of the eleven transgenic lines (frequency of transformation was 22%). Northern bolt analysis revealed that CSRG1 had expressed in transgenic lines. The assessments of salt-tolerant ability and photosyn-thetic rates indicated that the survival rate of the transgenic lines is 80%—90% and the transgenic lines could increase by 30%—40% in plant height, even when they were cultivated in MS medium containing 2% NaCl and the total seawater (salinity 24). It is supposed that the special physiologic metabolic pathway formed by the products of CSRG1 can really endow the tobacco plants with the high salt-tolerant ability, not only to Na^ stress, but also to the comprehensive stress of various ions.     

根癌农杆菌介导的异角状拟盘多毛孢菌转化系统研究

以携带潮霉素B磷酸转移酶抗性基因(hph)的pBHt1作为转化载体,根癌农杆菌AGL-1作为转化介体,实施转化异角状拟盘多毛孢菌。研究发现,异角状拟盘多毛孢菌的最优转化体系为:异角状拟盘多毛孢菌分生孢子悬浮液浓度为1×106个/mL,根癌农杆菌OD600为0.3,共培养时间48 h,共培养温度为25℃,诱导培养基中乙酰丁香酮(AS)浓度为200μmol/L,选择培养基添加250μg/mL潮霉素B、250μg/mL头孢噻肟钠。1×106个异角状拟盘多毛孢菌分生孢子可以产生200～300个转化子,随机挑选10个转化子进行PCR检测,均能扩增出预期条带,且在不含潮霉素B的PDA培养基平板上转化子连续培养5代后,hph基因仍能稳定遗传,这表明T-DNA已经插入到异角状拟盘多毛孢菌的基因组中。此次建立的异角状拟盘多毛孢菌的转化体系可为该病菌的功能基因研究和寄主与病原菌的互作研究提供有效的工具。     

含编码类铁氧还双亲性蛋白ap1基因的转基因水稻植株的获得

利用基因枪法将含有潮霉素抗性基因（hpt),gusA报告基因和ap1基因的2个质粒（pJIMB15和pBiSAP1)共同转化同转化由粳稻品种鄂宜105号种 子在胚诱导的愈伤组织（2－3周龄）。ap1基因编码一种双亲性的蛋白。该蛋白能延缓因假单孢菌感染所引起的非寄主植物中的过敏反应。经过2轮潮霉素（30mg/L)筛选,抗性愈伤组织被转入含30mg/L潮霉素的再生培养基中再生植株。从轰击的186块愈伤组织中共再生出32株独立的转基因水稻植株（转化率为17．2％）,PCR／Southern blot分析显示84％的转基因植株含有所有3个基因。     

抗盐基因Gm01g04890大豆子叶节遗传转化研究

利用根癌农杆菌介导的大豆子叶节转化方法,将转录因子HD-Zip家族基因Gm01g04890分别导入测序品种Willimas 82(W82)和小粒豆品种东农50(DN50)两种大豆品种的子叶节中.探讨了种子萌发时间、农杆菌菌液浓度、侵染条件、共培养时间和乙酰丁香酮(AS)浓度等因素对大豆子叶节形成不定芽的影响,优化了大豆子叶节遗传转化体系.T0代再生植株经草铵膦筛选以及RT-PCR检测,Gm01g04890基因已成功转入并整合于大豆基因组,获得了转Gm01g04890基因的DN50大豆抗性植株.     

拟南芥多效性基因CPR5转化水稻中花11的研究

 利用根癌农杆菌介导法将拟南芥多效性基因CPR5转化水稻中花11,同时优化了其再生体系和遗传转化体系。结果表明：相比27 ℃、暗培养,32 ℃、持续光照培养可使诱导率提高到100 %,且缩短了诱导周期;〖JP2〗预分化培养后,在附加5 mg/L 6 BA和1 mg/L NAA的分化培养基上分化率和再生率可分别达到100 %和90.50 %。同时探索了较高转化频率的条件为 A600≈ 0.05~0.1的农杆菌菌液浸染5 min,共培养时间为3 d,同时添加1张用AAI培养液浸湿的无菌滤纸。对部分转化植株进行PCR、Southern blot和半定量RT PCR检测,结果表明AtCPR5基因已经整合到水稻基因组中,在所试验的转化植株中均为单拷贝,但表达程度存在差异。     

转乙肝病毒表面抗原基因烟草发根的获得

构建了乙肝病毒表面抗原基因（HBsAg）植物表达载体,通过冻融法将HBsAg 基因转入到发根农杆菌LBA1314中,采用叶盘法将HBsAg基因导入到烟草中,获得了转基因烟草发根.对转基因烟草发根的GUS检测结果表明：转HBsAg基因烟草发根可以染成蓝色,而非转基因烟草的根没有染色反应.这说明gus基因在转HBsAg基因烟草发根获得了表达.     

棕榈花苞营养成分分析

【目的】研究棕榈花苞的营养价值,为其生物活性功能成分的提纯和活性探究提供基础数据,并为棕榈花苞资源的综合开发利用提供参考依据。【方法】以蓓蕾期棕榈花苞为原料,对棕榈花苞的基本营养成分及功能性成分进行测定,并同常见的可食用树花(桂花、槐花)的营养成分进行对比分析和评价。【结果】新鲜棕榈花苞中蛋白质含量达0.050 8 g/g,含粗纤维0.011 6 g/g、粗脂肪0.003 7 g/g、维生素C(VC)0.18 mg/g等,同时富含钙、钾等矿物质元素成分及17种氨基酸,其中人体必需氨基酸占全部氨基酸的25.82%,增香型氨基酸占38.49%,功能成分中含总酚12.664 mg/g、总黄酮11.032 mg/g、粗多糖4.605 mg/g。【结论】与其他森林蔬菜对比分析显示,棕榈花苞营养成分十分丰富,具有高蛋白、低脂肪的营养优点,功效成分含量很高,是一种宝贵的可食用森林资源,符合现代健康食品的要求,可进一步开发其营养保健价值。     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS} alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin a from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.