GFP基因转化藿香的研究

以藿香无菌苗幼嫩茎段为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染,通过共培养、选择培养后获得其抗性愈伤组织,对抗性愈伤组织的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织中强烈表达,证明GFP基因能够在藿香遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到藿香愈伤组织的基因组中。     

农杆菌介导的胀果甘草愈伤组织遗传转化

目的:旨在建立一个农杆菌介导的甘草愈伤组织遗传转化的可行方案,并对转化条件进行优化。方法:选择EHA105和LBA4404两种根癌农杆菌菌株,热激法转入含有绿色荧光蛋白GFP基因的植物表达载体pBI121-gfp,挑选转化的农杆菌用于侵染胀果甘草愈伤组织。设置不同培养时间的愈伤组织作为受体材料和农杆菌不同侵染时间两组条件,经共培养后的甘草愈伤组织进行荧光检测。结果:愈伤组织在含有100mg/l卡那霉素的继代培养基上进行筛选培养,得到了具有卡那霉素抗性的转化甘草愈伤组织,转化愈伤组织经继代培养基后在紫外光下仍可见绿色荧光,PCR检测转化愈伤组织基因组中含有gfp基因。结论:试验建立了农杆菌介导的甘草愈伤组织的遗传转化体系,为目的基因导入甘草细胞利用基因工程手段调控甘草次生代谢产物生物合成的研究奠定了基础。     

绿色荧光蛋白基因导入胡萝卜愈伤组织并获得表达

目的:将绿色荧光蛋白基因(green fluorescent protein,GFP)重组到胡萝卜愈伤组织细胞中,使其获得表达,为今后利用GFP基因作为植物报告基因提供条件。方法:通过冻融法将含有GFP基因的重组表达载体PBI1121转入到根癌农杆菌EHA105中,再利用根癌农杆菌介导的方法将GFP基因导入到胡萝卜愈伤组织细胞中,经过除菌和抗性筛选后观测转化结果。结果:荧光显微镜观测到被转化的愈伤组织在受蓝光激发后发出绿色荧光,利用PCR法扩增出约740bp的目的基因片断。结论:GFP基因在胡萝卜愈伤组织细胞中获得了表达。     

甘蔗转基因甘露糖筛选系统的建立

以甘露糖作为筛选底物,对甘蔗品种新台糖22号进行临界筛选浓度测定,获得愈伤组织的继代、分化、生根的临界筛选浓度。而后应用含有甘露糖筛选标记基因pmi及绿色荧光蛋白报告基因GFP的植物表达载体,通过农杆菌介导法对新台糖22号的愈伤组织进行遗传转化。用所测定的临界筛选浓度先后进行继代、分化、生根筛选培养,获得抗性植株。对获得的抗性植株分别进行pmi基因和GFP基因的PCR检测,以及GFP显微镜荧光检测,结果证实已成功建立了高效的甘蔗转基因甘露糖筛选系统。     

基因枪介导的大蒜遗传转化体系的建立

以大蒜无菌苗根诱导的愈伤组织为受体进行基因枪转化,根据愈伤组织的形态特征设计了6组基因枪转化参数进行试验。结果表明,其中4组转化试验都获得了转基因植株;轰击距离9 cm、压力1100 psi和轰击距离12.5 cm、压力1300 psi各轰击1次的轰击方式获得了最高的转化效率,为2.4%。对抗性植株进行了PCR和Southern blot检测,证明pCAMBIA1301载体上的潮霉素抗性选择标记基因和gus报告基因已整合到抗性植株的基因组中。对转化后的愈伤组织、胚状体、根、再生芽尖和叶片进行gus基因的表达检测,抗性材料显示出明显的蓝色反应。这些结果表明已初步建立了基因枪介导的大蒜遗传转化体系,将为大蒜的基因工程改良奠定基础。     

根癌农杆菌介导甘蔗遗传转化Bt(cry1Ab)基因北大核心CSCD

以自育甘蔗品种"川蔗23号"诱导的胚性愈伤为受体材料,首次用较低的根癌农杆菌侵染浓度(OD600=0.1左右)与较短的侵染时间(1.5 min)成功实现甘蔗遗传转化Bt(cry1Ab)基因。结果表明,胚性愈伤分化与小芽生根最适潮霉素筛选浓度分别为20 mg/L和30 mg/L;愈伤组织胚性的一致性是影响筛选阶段愈伤再生的重要因素,培养40 d的愈伤组织为转化最适受体;转化材料经连续的潮霉素抗性筛选后,获得65株抗性植株,通过特异性引物的PCR扩增检测,有2株获得阳性条带,初步表明目的基因已整合到甘蔗染色体基因组中。     

根癌农杆菌介导的高羊茅遗传转化研究

采用携带卡那霉素抗性基因nptⅡ和Na^ ／H^ 反向运输AtNHX1基因的表达载体pROK2／AtNHX1(带有35S启动子)和pROK2U／AtNHX1(带有ubi1启动子)的根癌农杆菌AGL1和GV3101,对4个品种高羊茅下胚轴来源的胚性愈伤组织进行了遗传转化。胚性愈伤组织经根癌农杆菌感染和共培养后,用50～150mg／L巴龙霉素筛选抗性愈伤组织,获得1126棵再生植株,用10～20mg／L卡那霉素进一步筛选再生植株,总共得到525棵绿色抗性植株。抗性植株的总DNA用AtNHX1基因的特异引物进行PCR检测,其中21棵为PCR阳性,最高转化频率为1．77％。Southern杂交结果证实,外源基因以低拷贝整合到高羊茅的基因组中,实验发现,在不同品种之间转化效率有所差异。     

茉莉茎段遗传转化体系初步研究

以双瓣茉莉(Jasminum sambac)一年生茎段为外植体,探索茉莉花遗传转化体系。结果显示,预培养7 d后进行农杆菌侵染,然后置于含卡那霉素(Kan) 80～100 mg·L-1的筛选培养基上培养可获得成活率较高的抗性茎段。GFP转化预培养7 d的茉莉茎段再培养3 d及30 d后,以及转化预培养20 d茉莉茎段愈伤再培养14 d后愈伤组织中均能检测到GFP荧光。说明利用该转化系统进行茉莉遗传转化是可行的。     

超声波辅助农杆菌介导CP基因转化番小瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV 外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV 菌株、美国夏威夷PRSV 菌株、泰国PRSV 菌株及日本PLDMV 菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset 的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS 6%蔗糖 1%葡萄糖,pH 5.7)调整至光密度OD_(600(?)m)=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml 无菌离心管装载胚性愈伤材料进行15s 的超声波处理,在80块被转化的胚性愈伤中获得21个CP 基因G 转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s 的超声波处理48块被转化的胚性愈伤中获得8个CP 基因B 转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP 基因转化上均表现出相似的效果。试验还表明:120mg/L 是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR 和Southern 印迹杂交进行了鉴定。     

根癌农杆菌介导转化川草二号老芒麦胚性愈伤组织

以川草二号老芒麦成熟种子为外植体,经过对培养基的筛选和培养条件优化,建立了愈伤组织再生系统。转化载体为pCAMBIA1304质粒,其T—DNA上携有潮霉素抗性基因（hptII）和类产碱假单胞菌杀虫蛋白基因（ppIP）,经根癌农杆菌EHA105介导转化结构致窑、颗粒状、黄白色的胚性愈伤组织。通过潮霉素筛选和对抗性植株进行分子检测,获得了转基因植株。同时优化了农杆菌遗传基因转化的参数,建立了农杆菌介导的川草二号老芒麦程序化转基因方案。     

Identification of marker compounds for discriminating between embryogenic and nonembryogenic calluses of higher plants using pyrolysis gas chromatography mass spectrometry and genetic programming

When whole cells are subjected to pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) analysis, it provides biochemical profiles containing overlapping signals of the majority of compounds. To determine marker compounds that discriminate embryogenic calluses from nonembryogenic calluses, samples of embryogenic and nonembryogenic calluses of five higher plant species were subjected to Py-GC/MS. Genetic programming of Py-GC/MS data was able to discriminate embryogenic calluses from nonembryogenic calluses. The content ratio of 5-meyhyl-2-furancarboxaldehyde and 5-(hydroxymethyl)-2-furancarboxaldehyde was greater in nonembryogenic calluses than in embryogenic calluses. However, the content ratio of phenol, p-cresol, and¹H-indole in embryogenic calluses was 1.2 to 2.4 times greater than the ratio in nonembryogenic calluses. These pyrolysates seem to be derived from the components of the cell walls, which suggests that differences in cell wall components or changes in the architecture of the cell wall play a crucial role in determining the embryogenic competence of calluses.     

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea)

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.     

Nitrogen compounds in embryogenic and non-embryogenic calluses of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Nitrogen (N) metabolism during embryogenesis may be fundamental in the embryogenic response. We used different explants of Medicago arborea L. subsp. arborea seedlings: cotyledons, petioles and leaves, which form calluses with different embryogenic responses. The endogenous contents of total nitrogen, nitrate, nitrite and ammonia and nitrate reductase activity were determined in embryogenic and non-embryogenic calluses induced from the different explants. The endogenous total N content decreased in the calluses as the culture time progressed, this decrease being more pronounced in the more embryogenic calluses obtained from petioles with the H8 and F0 media. Inorganic N decreased during embryogenesis, coinciding with an increase in organic N. Thus, N metabolism somehow seems to be essential in embryogenesis. The N detected in calluses, at the start of culture, was mainly metabolised to nitrite. This metabolism was very pronounced; especially in embryogenic calluses obtained from cotyledons and petioles. That is, the metabolism of N seemed to be more marked in the calluses in which embryogenesis was greater. The nitrite content decreased in all the calluses, the contents being lower, especially in the last months of culture, in the more embryogenic calluses obtained from petioles. In many calluses, ammonia levels did not follow any general pattern. Neither was it possible to detect changes in ammonia levels between the embryogenic and non-embryogenic calluses. Regarding nitrate reductase activity, no clear differences between embryogenic and non-embryogenic calluses were found.     

Development of blunt Tilletia caries (D.C.) Tul. pathogen on embryogenic wheat callus

The development of Tilletia caries on embryogenic and morphogenic wheat calluses from germination of spores up to sorus formation have been investigated. Pathogene growth was similar in variants with wheat calluses of susceptible species Triticum aestivum and resistant species T. timopheevii, but intensity of its development of embryogenic calluses of both species was lower, than on morphogenic. Necrosis of parenchyma-like cells was more distinct in the embryogenic than in morphogenic calluses. It was combined with low accumulation rate of infected cells in embryogenic calluses. Necrosis reduced the healthy cell reproduction in meristem-like cells of morphogenic calluses and thereby additionally decreased resistance of morphogenic calluses to pathogene.     

Feeding stimulant in Cinnamomum camphora for the common bluebottle, Graphium sarpedon nipponum (Lepidoptera: Papilionidae)

The acceptance of camphor tree (Cinnamomum camphora) as a host plant for the larvae of common bluebottle (Graphium sarpedon nipponum) was explained by the presence of feeding stimulants in the leaves. When the active methanol extract of C. camphora leaves was separated into hexane and water layers, both layers showed high feeding activities for the larvae of G. sarpedon nipponum. Bioassay-guided fractionation of the hexane layer resulted in the isolation of a highly active compound, which was identified as a-linolenic acid by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry.     

新丰毒蛋白——一种新的具有活性小A链的核糖体失活蛋白

辛纳毒蛋白是从香樟种子中分离的一种Ⅱ核糖体失活蛋白.最近,从香樟种子中还分离到另一种微型双链核糖体失活蛋白,命名为新丰毒蛋白.还原的新丰毒蛋白表现出与还原的辛纳毒蛋白同样的RNA N-糖苷酶和体外对抑制蛋白质翻译的活力.新丰毒蛋白的B链与辛纳毒蛋白的B链具有同样的分子质量和相同的N端10个氨基酸序列.它的A链N端10个氨基酸序列也与辛纳毒蛋白的A链完全一致,并且C端与辛纳毒蛋白的A链一样具有半胱氨酸,但是它的分子质量却只有辛纳毒蛋白A链的一半.RT-PCR和RNA印迹结果表明体内不存在新丰毒蛋白的mRNA.推测新丰毒蛋白是从辛纳毒蛋白通过蛋白质剪接而产生的,是一种研究蛋白质剪接的好材料.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

Somatic embryogenesis in wild cherry (Prunus avium)

Indirect somatic embryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic. Embryos at different stages of development were produced. Among cotyledonary-stage embryos, 50% had two cotyledons and a distinct hypocotyl, 43% had one or more than 2 cotyledons and 7% had fused cotyledons. Most of the embryos were translucent and conversion into plantlets was very rare. Secondary embryos could be observed to occur with low frequency from cultured somatic embryos and from embryos emerging from calluses. Indirect somatic embryogenesis was also induced from immature zygotic embryos. From one donor tree, 51% of the explants were embryogenic when cultured on a medium containing 0.9 μM kinetin, 0.9 μM BA and 0.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

樟树内生细菌EBS05发酵条件的研究

枯草芽胞杆菌EBS05是从樟树中分离的1株对多种植物病原真菌具有拮抗作用的内生细菌。以小麦纹枯病菌为靶标菌,通过单因素试验和正交设计试验对其发酵条件进行了优化。结果表明,内生细菌EBS05适宜的发酵培养基主要营养成分的组成和配比分别为可溶性淀粉3%、蛋白胨2%、NaCl 0.25%。最佳发酵条件为:初始pH5~9,最适温度34℃,装液量25 mL/250 mL三角瓶,接种量3%,发酵时间72 h。     

Discrimination of higher plant calluses based on embryogenic capacity and taxonomic classification by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum, and has been widely applied to the discrimination of closely related microbial strains. Minimally prepared samples of embryogenic and non-embryogenic calluses derived from various higher plants (sweet potato, morning glory, Korean ginseng, Siberian ginseng, and balloon flower) were subjected to PyMS for spectral fingerprinting. A dendrogram based on the unweighted pair group method, with arithmetic mean of pyrolysis mass spectra, divided the calluses into Siberian ginseng embryogenic callus and the others, which were subsequently divided into embryogenic and non-embryogenic callus groups, regardless of plant species from which the calluses were derived. In the non-embryogenic callus group, the dendrogram was in agreement with the known taxonomy of the plants. These results indicate that PyMS analysis could be applied for discriminating plant calluses based on embryogenic capacity and taxonomic classification.