基因枪在水稻遗传转化中的应用及其转化技术的优化

1983年Zambryski等人用根瘤农杆菌介导法进行烟草基因转移,获得了世界上首例转基因植株.随后,应用DNA直接导入技术如电击法(electroporation)和PEG介导法(PEG—mediated)成功地获得了转基因水稻植株.近年来,随着基因枪技术的建立和发展,水稻遗传转化成功的报道逐年增多.目前基因枪技术在植物遗传转化中的应用超过了根瘤农杆菌介导和其它转化方法的应用.这是因为基因枪转化技术不受植物种类的限制,不需要以原生质体作为转化的受体,可以将外源基因直接导入细胞、组织或器官,因而克服了根瘤农杆菌     

影响籼稻基因枪法遗传转化效果的几个因素的研究(简报)

基因枪转化技术在水稻的遗传转化上已被广泛地应用并获得显著的成效。与原生质体转化法相比较,基因枪法具有不受或少受基因型限制的优点且提高了转化效率,但对籼稻,仍有不少问题需要解决,转化系统尚须进一步完善。因此有必要对影响转化频率的因素进行深入的研究。我们在以barnase基因对籼稻的遗传转化以诱发工程雄性不育的研究中,特别注     

提高水稻基因枪转化效率的研究

在转化前将受体材料转移到高渗培养基上,即用含有一定浓度的甘露醇和山梨醇的培养基对受体材料进行处理。基因枪轰击后把受体材料转出,结果表明,这样做明显提高了瞬时表达（ＧＵＳ基因的表达）效果,用可筛选的标记基因（潮霉素磷酸转移酶基因,ＨＰＴ）试验表明,经高渗处理后,稳定转化的效率也有提高。用基因枪将带有ＨＴＰ基因的粒子轰击其盾片,经过筛选及植株再生,获得了转基因植株,ＰＣＲ扩增反应及Ｓｏｕｔｈｅｒｎ杂交证明了外源基因已整合入水稻的基因组     

基因枪轰击成熟花粉粒转化玉米的研究

利用基因枪轰击花粉粒再授粉的基因转化途径,将豇豆胰蛋白酶抑制剂基因（CpTI）成功导入玉米受体中。经卡那霉素筛选结果表明,非转化植株经1000ppm卡那霉素溶液处理后白化、死亡,余下大量健壮、可育的抗性植株,转化率约1．59％。通过对抗性植株进行PCR和PCR—Southern检测,初步确定CpTI基因已导入玉米基因组。饲虫实验结果表明转化植株具有较强的抗虫性。     

影响籼稻基因枪法遗传转化效果的几个因素的研究（简报）

Four factors influence on transformation of indica rice, which were high osmotic treatment; different explant as the target tissue; pressure of rupture disk and quantity of plasmid DNA, were investigated in this experiment. High osmotic treatment of target tissue prior to and after bombardment increased 3.2-fold for Gus transient expression than control. The best treatment of high osmotic was that the target tissues were kept in the target-bed medium which contained 0.4-0.6 mol/L sorbitol and manitol each for 4 h prior to bombardment and for 16 h after bombardment. Four explants: scutellum from mature seed, young panicle, embryogenic callus and suspension cells of indica rice were tested as target explant by particle bombardment. The results of Gus transient showed that the highest expression was scutellum and for other three explants, the order from high to low was young panicle, embryogenic callus and suspension cell. Transgenic plants were obtained from all of the explants except young panicle. For the pressure of rupture disk on transformation, 1100 psi or 1300 psi of the pressure of rupture disk were best one for the transformation and higher than 1300 psi could damage the target tissue which become black and died in the following culture duration. For the quantity of plasmid DNA, the results showed that 0.83 microgram of plasmid DNA per bombardment was preferred for the transformation of indica rice.     

通过基因枪提高根癌土壤杆菌转化水稻的效率

携带普通双元载体的根癌土壤杆菌（Ａgrobacterium tumefaciens (Smith et Townnsend)Conn)EHA105菌株对粳稻（Oryza sativa L.ssp.japonica)中花８号不敏感,转化效率较.在因枪将根癌土壤菌细胞直接轰击到水稻愈伤组织中可以显提高其转化效率。Ｓouthern检测证明转基因植物含有单拷贝的外源基因插入片段,转基因植物后代的ＧＵＳ基因表达呈３：１分离。     

基因枪转化获得的转基因水稻中外源基因整合与表达规律研究

通过基因枪法将cecropin B和bar基因共转化水稻,获得多个水稻优良品系的转化材料,对转基因的结构和表达的系统分析,发现外源基因的整合模式多种多样,有简单也有复杂,其中插入位点数的变化范围为1－7个,拷贝数的变化范围为1－10个,转基因拷贝数的多少与其表达和沉默不存在必然的联系,相同整合模式的转基因事件中基因的表达存在较大的差异,选择标记基因bar比非选择标记基因cecropin B的表达框的完整性和转录概率要高,但是发现大部分表达框完整的bar基因发生基因沉默,而在终止子发生序列丢失的cecropin B基因的表达则明显提高。     

基因枪介导的籼稻遗传转化及外源基因在受体中的遗传研究

由来自水稻成熟胚的愈伤组织或由此而建立的悬浮细胞系作基因枪转化的靶材料,将质粒ｐＩＬＴＡＢ２２７（含ｈｐｈ基因和ｇｕｓ基因）导入籼稻,在Ｂａｓｍａｔｉ－１、青油粘、新山粘２９、胜优２号,明恢６３等品种获得了转基因植株。Ｔ０转化体ｇｕｓ基因组织化学染色和ＤＮＡ分子杂交（Ｓｏｕｔｈｅｒｎ ｂｌｏｔ）证实,ｇｕｓ和ｈｐｈ基因已整合到上述品种的基因组中。本实验仔细研究了ｈｐｈ基因在Ｂａｓｍａｔｉ－１的Ｔ１     

基因枪介导的籼稻遗传转化及外源基因在受体中的遗传研究

由来自水稻成熟胚的愈伤组织或由此而建立的悬浮细胞系作基因枪转化的靶材料 ,将质粒 p ILTAB2 2 7(含 hph基因和 gus基因 )导入籼稻 ,在 Basmati- 1、青油粘、新山粘 2 9、胜优 2号 ,明恢 63等品种获得了转基因植株。 T0 转化体 gus基因组织化学染色和 DNA分子杂交 ( Southern blot)证实 ,gus和 hph基因已整合到上述品种的基因组中。本实验仔细研究了hph基因在 Basmati- 1的 T1、T2 和 T3等各个世代的遗传及表达。对 T1家系中各个体核 DNA的分子检测表明 ,hph基因普遍呈 3∶ 1的分离 (转基因阳性∶阴性 ) ,表明 hph基因为单一位点的整合 ,其遗传符合单 (显性 )基因控制的孟德尔遗传规律。还发现一些株系表现 1∶ 1的不正常的分离 (阳性个体较预期的少 ) ,推测这是由于雌或雄配子之一方的 hph基因受到抑制而产生基因沉默的结果。在 T2 、T3世代获得了具 hph基因的纯合体。笔者还探讨了外源基因在核基因组中的遗传稳定性及基因的沉默与活化问题     

应用多重PCR技术检测性器官异常患者的性别决定基因

采用针对人SRY基因及X与Y染色体同源序列的两对引物进行多重聚合酶链反应技术检测204例新生儿脐血DNA,男性均显示590bp、 355bp及280bp 3条扩增带,女性只有590bp1条扩增带,性别鉴定准确率为100%。又检测47例性器官异常患,13例社会性别为女性者只出现590bp扩增带,与核型性别结果一致。 34例社会性别为男性者中,有2例SRY基因检测结果阴性,只出现590bp带,核型为46,XX,证明这两例患者社会性别不相符,经病理证实后应诊断为女性假两性畸形。 Abstract:This investigation adapted multiplex PCR technique with two pairs of primer to determine sex according to the human SRY gene and X,Y chromosome analogical sequence.We detected bellybutton blood DNA from 204 newborns.Male revealed three bands:590bp,355bp and 280bp,and females only had one band which was 590bp,the accuracy of sex determination was 100%.47 sexual abnormal patients were tested.The results showed that 13 cases,whose social sex were female,had one 590bp band which consistent with their chromosome type sex.In the rest 34 cases,whose social sex were male,2cases showed,on the contrary,only one 590bp band and their chromosome type were 46,XX.This proved that the two patients did not consistent with their social sex,and pathological analysis showed that they were female pseud-hermaphroditism.     

Biolistic mediated site-specific integration in rice

Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.     

Genetic Transformation of Rice

Rice was the first major monocot crop species to be transformed and regenerated. Initially, rice transformation was limited to japonica cultivars. Subsequently, a number of indica and javanica cultivars have also been transformed and regenerated into fertile transgenic plants. Most transformation studies in rice have used direct DNA uptake into protoplasts, induced by polyethylene glycol treatment or electroporation. Recently, other transformation methods have been developed that are less genotype dependent, such as microprojectile bombardment of cell suspensions and immature embryos. This review summarizes progress in both protoplast-based and other transformation methods.     

水稻基因枪法多基因转化研究

为探索多基因转化系统,利用基因枪法进行水稻3个质粒的共转化研究,结果从25个转基因植株中获得3个三转化植株N12、K4-39和K1-1-66,成功地将分别位于不同质粒载体上的GUS,hpt和RTBV CP基因同时导入到水稻中。3个三转化植株中N12、K4-39正常可育。对N12的后代遗传分析表明,该转基因植株中3个载体所携外源基因均呈孟德尔式分离(3:1),3个外源基因分别整合到水稻的两条染色体上。GUS基因与hpt连锁,RTBV CP位于另一条染色体上。     

兰州百合基因枪转化方法的研究

以兰州百合试管苗鳞片叶为受体,通过基因枪转化法将脱氢抗坏血酸还原酶基因(DHAR)转入兰州百合,并用GUS染色、PCR扩增和Southern杂交等技术进行转基因株系的检测.结果表明:以兰州百合鳞片叶为受体材料,在轰击压力1 100 psi、轰击距离6 cm时,抗性率高达14%,转化率为1.5%;10 mg/L的潮霉素对鳞片叶具有很好筛选作用.GUS分析发现,转化株系叶片为蓝色;PCR检测和Southern杂交进一步证实抗性株系为转基因植株,且为单拷贝.     

中国畲族群体D17S30位点遗传多态性研究

采用聚合酶链反应 (PCR)技术对中国畲族群体270名无关个体D17S30位点的VNTR进行了研究,共检出12个等位基因,片段大小为168bp-1008bp,基因频率为0.0056-0.3259,杂合度为79.10%,父权排除率为0.4358。家系分析表明,扩增片段按孟德尔方式遗传。统计学分析证明,本资料符合Hardy-W einberg平衡定律(x2=79.97,v=66,P=0.1158>0.05)。 Abstract:The variable number of tandem repeat(VNTR)of D17S30 locus in 270 unrelated individuals of She ethnic group was studied by polymerase chain reaction(PCR).The results showed that 12 alleles were detected and their sizes ranged from 168bp to 1 008bp.The allele frequencies were 0.0056~0.3259,the heterozygosity of this locus was 79.10% and the paternity exclusion probability was 0.4358.The number of D17S30 VNTR allele and allele frequency of She were different from those of Han ethnic group in Mendelian Law.Statistical analysis demonstrated that their geneotypes were consistent with Hardy-Weinberg equilibrium(x2=79.97,v=66,P=0.1158>0.05).     

Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane

 Embryogenic calli of the Brazilian sugarcane (Saccharum officinarum L.) genotype SP80–180 were transformed with two plasmids containing genes coding for neomycin phosphotransferase (neo) and phosphinotrycin acetyltransferase (bar), by particle bombardment using an apparatus developed at Copersucar Technology Center. Transformed plants were initially selected on culture medium containing Geneticin, and resistance was confirmed by localized application of a kanamycin solution to leaves of hardened plants at the nursery. A commercial formulation of ammonium gluphosinate was sprayed twice on these antibiotic-resistant plants. The resistant plants were considered co-transformed, and Southern analysis confirmed stable integration of both bar and neo genes. In addition, phosphinotrycin acetyltransferase expression was supported by RT-PCR analysis and neomycin phosphotransferase presence was demonstrated by western blotting. Similar analyses were also performed with micropropagated transformants after three cycles of subculture. Received: 15 December 1999 / Revision received: 15 April 2000 / Accepted: 1 June 2000     

Agrobacterium -mediated Transformation of Rice with Help of Bombardment

Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Agrobacterium mediated transformation with help of bombardment. The transformation efficiency can be raised greatly. Single copy of gene insertion in the genome of transgenic rice plants was proved by Southern analysis and the expression of GUS gene was observed. GUS gene and hygromycin-resistant gene show 3∶1 segregation in progenies of the transgenic rice plants.     

利用热稳定蛋白特异条带鉴别籼粳稻的方法研究

以85份栽培稻为材料,利用SDS-聚丙烯酰胺凝胶电泳和蛋白免疫印迹技术分析籼稻和粳稻热稳定蛋白的表达差异.探讨利用特异蛋白来建立籼粳稻的鉴别方法.结果表明,在籼粳稻品种之间存在热稳定蛋白的差异表达,尤其在分子量约42～47kD范围.其中45.2kD条带(Band I)和46.5 kD(Band Ⅱ)条带为典型粳稻特异蛋白(Os03g0168100)的标志带,42.0kD条带(Band Ⅲ)为典型籼稻特异蛋白(OsI_10172)的标志带.以这3条带作为鏊别方法,并与程氏指数法进行比较,典型籼稻和粳稻的一致度分别为80.0%和86.4%,表明热稳定蛋白标志带法在一定程度上可用于鉴别典型的籼稻和粳稻.