抗冷冻蛋白基因(afp)转化大豆的研究

目的:通过农杆菌介导法遗传转化大豆。方法:通过热激法将质粒pCAAFP66导入根癌农杆菌菌株EHA105中获得含有抗冷冻蛋白基因(afp)及除草剂抗性筛选标记基因(bar)的农杆菌工程菌株;以大豆品种华春6号和马祖1号种子的下胚轴为外植体,经过农杆菌介导将抗冷冻蛋白基因导入大豆基因组中,在含有除草剂草丁膦(PPT)的培养基中筛选、并经过PCR鉴定获得大豆转化植株。结果:PPT的最佳筛选浓度为1.0mg/L,华春6号和马祖1号的阳性植株数分别为6株和2株,转化效率分别为3.70%和0.94%。结论:不同基因型大豆的转化率存在差异,抗冷冻蛋白基因成功遗传转化进大豆细胞中。     

影响基因枪法遗传转化的因素

通过对影响基因枪法遗传转化的因素探讨,以期对提高基因枪法的转化效率提供参考。     

甜蛋白基因MBLII对番茄的遗传转化

以5～7d龄的 丽春 番茄无菌苗子叶作为外植体,研究了子叶外植体对抗生素卡那霉素的敏感性,抗生素卡那霉素对番茄筛选的适宜浓度为70mg/L.通过根癌农杆菌 Agrobacteriumtumefaciens 介导,成功地进行了马槟榔甜蛋白基因MBLII对番茄的遗传转化,获得转化番茄抗性植株,组织化学法有阳性表现、PCR特异扩增及Southern杂交检测出现特异条带,表明MBLII基因已顺利整合到转基因番茄植株的基因组.     

甜蛋白基因MBLⅡ对番茄的遗传转化

以5～7d龄的“丽春”番茄无菌苗子叶作为外植体,研究了子叶外植体对抗生素卡那霉素的敏感性,抗生素卡那霉素对番茄筛选的适宜浓度为70mg／L。通过根癌农杆菌(Agrobacterium tumefaciens)介导,成功地进行了马槟榔甜蛋白基因MBLⅡ对番茄的遗传转化,获得转化番茄抗性植株,组织化学法有阳性表现、PCR特异扩增及Southern杂交检测出现特异条带,表明MBLⅡ基因已顺利整合到转基因番茄植株的基因组。     

小麦遗传转化技术研究进展

植物基因工程研究兴起于本世纪八十年代中期。自其诞生至今,如何将这项技术应用于小麦、水稻、玉米等农作物的遗传改良研究便始终是人们努力的重要目标之一。要实现这一目标,首先需要建立一套高效、可靠、重复性好的基因转化系统。而在植物基因转化系统研究方面,禾谷类相对于其他双、单子叶植物而言,具     

天花粉蛋白基因转化番茄的研究

核糖体失活蛋白（ｒｉｂｏｓｏｍｅｉｎａｃｔｉｖａｔｉｎｇｐｒｏｔｅｉｎ,ＲＩＰ）是一类作用于核糖体,抑制蛋白质合成的蛋白毒素,它具有广谱抗植物病毒和动物病毒的活性,在异种植物中的抗性效应尤其明显。迄今为止,人们已经从５０多种植物中分离出６０多种不...     

草菇低温诱导蛋白研究初探

对低温处理的草菇Ｖｏｌｖａｒｉｅｌｌａ ｖｏｌｖａｃｅａ菌丝体的可溶性蛋白进行分析,发现草菇菌丝在低温协迫中有新的可溶性蛋白产生,应用电泳技术分离纯化了草菇菌丝体中的一个低温诱导蛋白,经等电聚焦分析,该蛋白质的等电点６．７９,ＳＤＳ－聚丙烯酰胺凝胶电泳分析该蛋白质是由分子量为７０ｋＤ和４８ｋＤ的两条多肽所组成。     

高粱遗传转化研究进展

高粱是世界上仅次于小麦、水稻、玉米和大豆的重要作物之一,然而由于其高效、稳定的遗传转化体系的建立较难,限制了其转基因研究进程.近年来,随着转基因技术的不断发展和完善,高粱转基因研究也取得了飞速的发展.从高粱遗传转化再生系统中外植体的选择、转化方法、影响转化和基因表达效率的因素等几方面进行了综述,并总结转基因高粱研究进展.     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

苦参凝集素蛋白基因转化水稻的研究

以水稻杂交品种‘云资粳41号’为受体材料,通过农杆菌介导法将苦参凝集素蛋白基因(SFL)导入水稻细胞,采用氯酚红法和PCR检测外源基因是否整合到水稻基因组中。结果显示:外源基因成功转入水稻基因组,并获得一批转基因水稻植株;转基因植株叶片离体接种稻瘟病菌的检测结果显示,转基因植株与对照(非转基因植株)相比有明显的抗性,证明SFL基因在水稻中得到表达。研究表明,基于SFL基因所具备的广谱抗菌作用,可以预期所得转基因水稻植株很可能对水稻的多种病原菌具有良好的抗性,为选育新的抗稻瘟病水稻新品种以及拓宽栽培稻抗病遗传基础增加抗稻瘟病基因奠定了基础。     

Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment

Uracil auxotroph of Pleurotus ostreatus was transformed to prototrophy by means of particle bombardment. Five transformants were obtained under three conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant per microg of DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of P. ostreatus by particle bombardment.     

Genetic transformation of Cymbidium orchid by particle bombardment

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse. Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Transformation of <Emphasis Type="Italic">Lyophyllum decastes</Emphasis> by particle bombardment

The basidiomycete Lyophyllum decastes was transformed by means of particle bombardment. We isolated five transformants under twelve conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant/μg DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of L. decastes by particle bombardment.     

草菇GH61家族基因的生物信息学分析及金属离子对其表达水平的影响

在前期工作中发现草菇含有30个GH61家族基因同源物（Vv_gh61_1至Vv_gh61_30）,进一步分析了这些基因的结构特点以及其编码蛋白基本性质和系统进化关系,并研究了Cu2+和Mn2+对基因表达水平的影响。分析表明有17个草菇GH61基因串联成6个基因簇,存在明显的串联重复现象,系统发育树与基因外显子位置分析表明串联重复基因分布在同一进化分枝上并具有相似的基因结构,串联重复基因编码序列一致性在71%–94%之间。草菇GH61编码蛋白的氨基酸数目在217–442aa之间,分子量和等电点分别介于22.4–45.4kDa和5.2–9.3之间,绝大多数都含有信号肽并定位在细胞外,都含有Glyco_hydro_61功能域以及CBM1、peroxidase等多样化的功能域,系统进化树表明草菇GH61家族基因具有3个主要进化分枝,与灰盖鬼伞等的GH61基因有较近的进化关系。金属离子诱导作用显示,Mn2+对草菇GH61家族基因的表达水平存在诱导作用而Cu2+的诱导作用不明显。     

草菇Vvrin1基因RNAi转化子的筛选及其转录组分析

草菇味道鲜美,食用价值高,是我国主要栽培的商业食用菌之一.MADS-box转录因子对真核生物的生长发育和信号传导具有关键性的调控作用.本研究通过农杆菌介导的方法获得5个草菇MADS-box转录因子Vvrin1基因的RNA干扰转化子,并进一步对转化子的表型进行分析,发现5个转化子菌丝在PDA固体培养基和栽培料中的生长速度...     

草菇基因组中11个漆酶同源基因的生物信息学分析及铜离子对其表达的影响

通过分析草菇基因组中11个漆酶同源基因所编码的蛋白的性质、转录调控元件和测定铜离子存在条件下的草菇漆酶活性及11个漆酶基因的转录水平,揭示了草菇漆酶基因的各自特性、差异以及基因功能与进化机制。分析表明,这11个漆酶同源基因编码的蛋白具有508–562aa个氨基酸,分子量和理论等电点分别为56.25–60.75kDa和4.51–6.18（未经翻译后修饰）,且都具有真菌漆酶铜离子结合区域的特征序列、4个能够结合催化底物的环形结构以及信号肽序列,都属于分泌性的胞外蛋白,但其底物结合位点数目、loop序列的一致性、跨膜区域数目和位置以及信号肽位置等存在较大差异。草菇11个漆酶起始密码子上游2 000bp的序列中含有真核生物的基本转录调控元件（TATA-box,CAAT-box及GC-box）和多个潜在的调控元件（MRE、XRE、STRE、HSE、ARE、TRE、NIT元件等）,但每个基因所含调控元件数目及种类各有不同。在液体培养条件下,铜离子能够诱导除vv-lac2、vv-lac3和vv-lac7之外的其余8个草菇漆酶基因的表达,且适宜浓度的铜离子有助于草菇漆酶活性的增加。     

Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997