农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有１００ ％ 杀虫率。     

Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed

Here, we provide comprehensive, highly efficient protocols for Agrobacterium tumefaciens-mediated transformation of a wide range of rice genotypes. Methods that use either immature embryos (japonica and indica rice) or calli (japonica cultivars and the indica cultivar, Kasalath) as a starting material for inoculation with Agrobacterium are described. Immature embryos are pretreated with heat and centrifugal force, which significantly enhances the efficiency of gene transfer, and then infected with Agrobacterium. Callus is induced from mature seeds and infected. Transformed cells proliferated from these tissues are selected on the basis of hygromycin resistance, and transgenic plants are eventually regenerated. A single immature japonica or Kasalath embryo will produce between 10 and 18 independent transgenic plants; for other non-Kasalath indica varieties, the number of transgenic plants expected will be between 5 and 13. For japonica and Kasalath, transformants should be obtained from between 50 and 90% of calli. From inoculation with Agrobacterium to transplanting to soil will take 55 d for japonica and Kasalath, and 74 d for indica other than Kasalath using the immature embryo method, and 50 d for japonica and Kasalath using the callus method.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。     

提高农杆菌转化水稻频率的研究

以16种重要的籼稻和粳稻栽培品种为材料,研究了影响农杆菌转化水稻频率的有关因素,结果表明,CC培养基是绝大多数水稻全国组织的最适诱导与继代培养基;添加2．5－5mg/L ABA可以有效地改善水稻愈伤组织的质量,籼稻愈伤组织所需的筛选剂浓度低于粳稻愈伤组织所需的浓度,根癌农杆菌EHA105菌株对水稻的转化效果优于LBA4404和AGL1菌株的效果,头孢霉素对农杆菌的抑制效果优于羧苄青霉素的效果,共培养后进行适当的干燥处理既可增强脱菌效果,又可提高转化频率,应用我们所优化的农杆菌转化技术体系,获得了10个品种的水稻转基因植株。     

药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

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.     

Establishing a gene trap system mediated by T-DNA(GUS) in rice

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.     

籼稻基因枪转化的研究

对国内外１７个籼稻品种进行了基因枪转化,研究了有利于籼稻愈伤组织诱导和生长的培养条件和筛选程序,１５个品种获得了潮霉素抗性愈伤组织,５个品种再生了植株,包括当前国际上推广的ＩＲ系统及国内的优良品种和恢复系,分子生物学证据证明潮霉素基因已经整合入籼稻的基因组。最高植株转化频率接近一般粳稻水平,多数低于粳稻水平。这为建立籼稻的转化系统打下了基础。     

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts     

转基因培育抗除草剂水稻

以ｐＡＨＣ２０（含Ｂａｒ基因）和ｐＷＲＧ１５１５（含ＧＵＳ基因和潮霉素抗性基因）以及含Ｂａｒ基因和雪莲凝集素（ＧＮＡ）基因的ｐＣＡＭＢＩＡ３３００ ＲＧ为供体ＤＮＡ,选用水稻品系８７２０３、上农香糯及鄂宜１０５的成熟胚诱导出的愈伤组织及微不定芽为受体材料,分别采用基因枪和根癌农杆菌（ＬＢＡ４４０４,含ｐＡＬ４４０４）导入法进行基因转化;经抗性筛选、ＧＵＳ检测和ＰＣＲ分析。结果表明,外源基因已通过基     

Partial desiccation of mature embryo-derived calli,a simple treatment that dramatically enhances the regeneration ability of indica rice

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-Benzyl amino purine - DTT Dithiothreitol - EDTA Ethylene diamine tetra-acetic acid - MS Murashige and Skoog - NAA Naphtalene acetic acid - PAGE Polyacrylamide gel electrophoresis - PAR Photosynthetic active radiation - SDS Sodium dodecil sulfate     

高羊茅和黑麦草农杆菌介导转化体系的研究

利用C58C1农杆菌菌系（携带的表达载体上含GUS基因和nptII基因）感染4个草坪草品种追寻者、爱神特、腾跃和守门员成熟胚来源的愈伤组织,共培养后部分愈伤组织进行X-Gluc组织化学染色检测,其余愈伤组织在含G418 10-25 mg/L的MS改良培养上先后筛选抗性愈伤组织和分化抗性再生植株,对移栽成活的144棵抗性再生植株分别进行了ELISA检测、PCR检测和组织化学染色检测。愈伤组织阶段X-Gluc染色检测结果表明,4个草坪草品种GUS基因瞬间表达率8.6%～46.9%,爱神特愈伤组织对农杆菌侵染最为敏感,其次是腾跃和守门员,追寻者最不敏感;ELISA检测结果表明,45株呈现阳性,证明nptII基因已转入草坪草并已表达;PCR检测结果与ELISA检测结果一致,表明nptII基因确实已经整合到了草坪草基因组中,且没有发生沉默现象;转基因植株X-Gluc染色检测结果表明,GUS基因在43株中得到了稳定表达,在2株中发生了沉默现象。4个草坪草品种抗性再生植株分化率0～43.5%,转化率0～21.5 %。结果还表明,GUS基因瞬间表达率与稳定转化率在草坪草上很不一致,不能作为衡量基因型转化效果的指标。     

一种改进的水稻成熟胚愈伤组织高效基因转化系统

以成熟胚愈伤组织为材料的农杆菌介导水稻转化法虽已建立, 但转化频率仍有待提高。本文以粳稻(Oryz a sativa)品种(中花10号和中花11号)的成熟胚诱导的愈伤组织为受体材料, 对组织培养体系及影响遗传转化的因素进行优化, 建立了一套改进的农杆菌介导的水稻高效遗传转化系统。农杆菌菌株为EHA105, 质粒载体是pUN1301/ OsRAA1, 其中含有标记基因GUS 和筛选基因HPT。愈伤组织诱导培养基为NBD2 (NB+2 mg·L-12,4-D), 继代培养基为NBD0.5, 预分化与分化培养基为RE1 (MS+1 mg·L-16-BA + 0.25 mg·L-1 NAA + 0.5 mg·L-1 KT + 0.2 mg·L-1 ZT)和RE2 (MS+ 1 mg·L-1 6-BA + 0.5 mg·L-1 NAA+ 0.5 mg·L-1 KT + 0.2 mg·L-1 ZT)。另外, 还分析了影响T-DNA转移的多种因素, 如外植体种类、愈伤组织预培养基和愈伤组织继代次数等。采用优化的转化程序, 水稻愈伤组织转化率和植株转化率可达70%以上。     

Reasons for lower transformation efficiency in indica rice using Agrobacterium tumefaciens-mediated transformation: lessons from transformation assays and genome-wide expression profiling

Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In this study, transient and stable transformation assays using different japonica and indica cultivars showed that the lower transformation efficiency in indica rice was mainly due to the low efficiency in T-DNA integration into the plant genome. Analyses of the global gene expression patterns across the transformation process in different varieties revealed major differences in the expression of genes responding to Agrobacterium within the first 6 h after infection and more differentially expressed genes were observed in the indica cultivar Zhenshan 97 (ZS), with a number of genes repressed early during infection. Microarray analysis revealed an important effect of plant defense response on Agrobacterium-mediated transformation. It has been shown that some genes which may be necessary for the transformation process were down-regulated in the indica cultivar ZS. This dataset provided a versatile resource for plant genomic research to understand the regulatory network of transformation process, and showed great promise for improving indica rice transformation using genetic manipulation of the rice genome.     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。     

几种影响籼稻成熟胚愈伤组织诱导及再生的因素

建立了适合5个籼稻品种成熟胚籼稻遗传转化的高效植株再生体系.N6基本培养基有利于籼稻愈伤组织的诱导和继代培养;N6大量元素和MS微量元素有利于愈伤组织的分化.降低分化培养基中蔗糖含量,加入适量山梨醇、Cu2 、Ag 和玉米素(2T)均可明显提高水稻愈伤组织的再生植株频率,5个品种分化频率均达到75%以上.     

Microsatellite diversity within Oryza sativa with emphasis on indica-japonica divergence

The molecular evolution of cultivated rice Oryza sativa L. has long been a subject of rice evolutionists. To investigate genetic diversity within and differentiation between the indica and japonica subspecies, 22 accessions of indica and 35 of japonica rice were examined by five microsatellite loci from each chromosome totalling 60 loci. Mean gene diversity value in the indica rice (H=0.678) was 1.18 times larger than in the japonica rice (H=0.574). Taking the sampling effect into consideration, average allele number in the indica rice was 1.40 times higher than that in the japonica rice (14.6 vs 10.4 per variety). Chromosome-based comparisons revealed that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. An overall estimate of F(ST) was 0.084-0.158, indicating that the differentiation is moderate and 8.4-15.8% of the total genetic variation resided between the indica and japonica groups. Our chromosome-based comparisons further suggested that the extent of the indica-japonica differentiation varied substantially, ranging from 7.62% in chromosome 3 to 28.72% in chromosome 1. Cluster analyses found that most varieties formed merely two clusters for the indica and japonica varieties, in which two japonica varieties and five indica varieties were included in the counterpart clusters, respectively. The 12 chromosome-based trees further showed that 57 rice varieties cannot be clearly clustered together into either the indica or japonica groups, but displayed relatively different clustering patterns. The results suggest that the process of indica japonica differentiation may have proceeded through an extensive contribution by the alleles of the majority in the rice genome.     

水稻幼穗培养高效再生系统的建立

本文测试了不同基因型幼穗取材时期、消毒方式、4℃下处理时间、愈伤组织诱导、分化及生根条件等对水稻幼穗培养再生成株的影响,实验结果表明,在优化条件下,籼稻的绿苗分化率在85%以上,成苗率115%左右;粳稻的绿苗分化率在90%以上,成苗率130%左右;粳型广亲和的绿苗分化率、成苗率分别达到85%和115%以上。由此建立了一套高效、可靠、重复性好的水稻组织培养再生系统,为水稻遗传转化的顺利进行奠定了基础。     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。