基因枪法向小麦导入几丁质酶基因的研究

利用基因枪法,以菜豆几丁质酶基因转化小麦幼胚愈伤组织。在轰击压力1300psi,轰击距离6cm、100μg金粉/枪和轰击距离9cm、150μg金粉/枪的2种处理条件下,获得4株春小麦东农7742转化植株,转化频率分别为0.36％和0.56％。经PCR和PCR-Southern杂交分析,证实菜豆几丁质酶基因已整合到T0和T1小麦基因组中。采用氨基葡萄糖法测定几丁质酶活力,结果表明,转基因小麦的几丁质酶活力明显高于对照株;转基因植株对白粉病症状减缓,并获得一株赤霉菌接种未扩展的转基因T1植株。     

CHI-PAT双价基因遗传转化贵州禾来拢

以贵州禾来拢幼胚为转化受体,用农杆菌介导法将几丁质酶和抗除草剂抗性双价基因(CHI-PAT)导入来拢幼胚,筛选出抗性愈伤组织并获得抗性植株.抗性植株经GUS组织化学及PCR检测呈阳性,转基因植株对50 mg/L的Basta溶液有抗性.初步证明CHI和PAT基因已整合进了水稻基因组中.     

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。     

超声波辅助农杆菌介导CP基因转化番木瓜(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OD600nm=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15m1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.     

激光辐照葡萄愈伤组织的过氧化物酶同工酶分析

利用法国产ＤＡＴＡＣＨＲＯＭ—５０００型染料脉冲激光器处理葡萄“胜利”品种的愈伤组织,用聚丙烯酰胺凝胶电泳法（ＰＡＧＥ）分离过氧化物酶同工酶,研究了激光辐照对葡萄生长发育和过氧化物酶同工酶表达的影响,发现：适当剂量的激光辐照能使葡萄愈伤组织明显提前分化;激光能够影响葡萄过氧化物酶同工酶基因的调控,适当剂量的激光能够促进该基因的表达;激光处理的愈伤组织在不同的生长发育时期与对照相比,过氧化物酶同工酶在数目和活性上有明显增加。     

草木樨状黄芪抗甲硫氨酸变异系的离体筛选及鉴定

通过组织培养体系进行了草木樨状黄芪(Astragalus melilotoides Pall.)抗甲硫氨酸变异系的筛选。无菌苗幼茎切段诱导的愈伤组织经NaN3诱变后,经过筛选获得了抗14mmol/L甲硫氨酸的变异细胞系,并分化出大量植株。再生植株已经在大田中开花结实。经分析表明：抗性细胞系脱离选择压6个月后,放在含15mmol/L甲硫氨酸的培养基上培养25天后,其相对增长率是对照的10．2倍。抗性系再生植株游离甲硫氨酸是对照的2．12倍,并且天冬族氨基酸都有明显增加。SDS-PAGE及过氧化物酶同工酶分析表明：在蛋白质及同工酶酶谱上抗性系再生植株均出现与对照不同的差异。     

草木樨状黄芪抗甲硫氨酸变异系的离体筛选及鉴定

通过组织培养体系进行了草木樨状黄芪(Astragalus melilotoides Pall.)抗甲硫氨酸变异系的筛选。无菌苗幼茎切段诱导的愈伤组织经NaN_3诱变后,经过筛选获得了抗14mmol/L甲硫氨酸的变异细胞系,并分化出大量植株。再生植株已经在大田中开花结实。经分析表明:抗性细胞系脱离选择压6个月后,放在含15mmol/L甲硫氨酸的培养基上培养25天后,其相对增长率是对照的10.2倍。抗性系再生植株游离甲硫氨酸是对照的2.12倍,并且天冬族氨基酸都有明显增加。SDS-PAGE及过氧化物酶同工酶分析表明:在蛋白质及同工酶酶谱上抗性系再生植株均出现与对照不同的差异。     

超声波辅助农杆菌介导CP基因转化番木瓜（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(PT—NP—YKT)插入双元栽体质粒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(1／2MS 6％蔗糖 1％葡萄糖,pH5．7)调整至光密度OD600nm=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。     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

烟草组织培养过程中过氧化物同工酶的变化

在烟草的组织中,老叶的过氧化物酶,在迁移速率慢的酶区带对照植株比变异植株多二条酶带,但在幼龄叶片无差异。~(60)Coγ射线诱变的外植体与对照相比,酶谱有显著不同。在组织分化进程中,酶谱亦有显著变化。随着细胞分化进程,定期测定不同剂量γ射线处理的培养组织中酶谱的变化历程,观察到在分化前后酶谱无明显差异。只有在诱变植株生长状态表现明显差异时才见酶谱的变化。培养基中不同浓度组合的激动素、椰乳和2,4-D对细胞生长和分化有不同的调节作用,随着组织分化与脱分化历程,同工酶谱表现明显差异。在组织开始形成愈伤组织以后同工酶谱已有显著变化,但培养在分化培养基上的外植体,在分化前后,其同工酶谱未见明显变化。     

Agrobacterium-mediated transformation of indica rice with chitinase gene for enhanced sheath blight resistance

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.     

Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.     

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T₁ lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T₁ plants. Resistance to stripe rust in transgenic T₂ and T₃ XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27–36 %) was recorded for transgenic T₂ and T₃ XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat.     

小麦与多年生黑麦草原生质体的电融合及杂种愈伤组织的形成

多年生黑麦草(Lolium perenne L.)悬浮培养细胞来源的原生质体和小麦(Triticumaestivum L.)含叶绿体的悬浮培养细胞来源的原生质体间,用直流方波脉冲进行电融合,获得了体细胞杂种愈伤组织。小麦的原生质体经过碘乙酰胺失活。愈伤组织的形态和颜色被用作识别预期杂种的标记。为了对杂种愈伤组织进行同工酶分析,观察了亲本的9种同工酶谱,其中3种在亲本间表现出差异(ADH、GOT 和 SDH)。酒精脱氢酶(ADH)的分析结果表明,有6个细胞系表现出杂种带。这些细胞系经过其他两种同工酶分析和 rDNA 探针杂交试验表明,一个细胞系表现出基本完全的亲本基因组间的组合,其余5个细胞系是部分杂种。     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Summary Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10⁸ cells·ml^–1), cocultivated for 48–96 h and placed on Murashige and Skoog (MS) medium with 5.0 M each of 2,4-D and BA, 50 mg·l^–1 kanamycin and 500 mg·l^–1 carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 M 2,4-D/BA, 50 mg·l^–1 kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 M NAA/BA and 50 mg·l^–1 kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyl-aminopurine - CaMV cauliflower mosaic virus - NAA naphthaleneacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction     

几丁质酶基因表达载体构建及转化烟草的研究

构建了一个含有多个调控序列的带有几丁质酶基因的植物表达载体,通过发根农杆菌的Ri质粒介导转化烟草的三个品种：K326,RG11,VA116,在卡那霉素抗性培养基上筛选,获得了7株再生植株,以PCR检测、DNA斑点杂交证明表达载体构建成功,几丁质酶基因导入烟草并整合到烟草基因组中。