碳离子束辐照拟南芥介导外源基因转移的研究

采用700keV或4.0MeV碳离子束辐照拟南芥种子,通过对样品的显微摄影,发现随着辐照剂量的增加,碳离子束对种子表面的损伤逐渐加剧,特别是在4.0MeV碳离子束辐照下,当剂量达到1×1014ions·cm-2后,种皮局部逐渐被刻蚀殆尽,甚至造成种皮局部破裂。对拟南芥种子进行台盼蓝染色后的显微观测显示,碳离子束辐照可以导致拟南芥种皮细胞着染,在剂量较大的情况下,部分皮下细胞也可着染,表明碳离子束可作用到皮下细胞,为外源基因提供导入的通道。GUS基因导入后的组织化学检测表明:质粒pCAMBIA1301能够进入4.0MeV碳离子束辐照后的拟南芥种子,并在种子和幼芽中获得瞬间表达。     

花粉介导的遗传工程方法的最初步骤

CIBA-GEIGY Agricultural Biotechnology的I.Dupuis和G.M.Pace报道,用单倍体的雄配子体作为外源DNA载体,可获得能直接发育成成熟转化植株的非嵌合性转化合子胚。作为这种花粉介导的遗传工程的最初步骤,Dupuis和Pace用微弹法将葡糖苷酸酶(GUS)基因和花色素苷标记基因导入     

用微弹轰击法将GUS基因导入小麦的完整细胞

微弹轰击法将外源DNA导入小麦(Triticum,aestivum)未成熟胚和悬浮细胞系的完整细胞。用pCI GUS作报告基因,质粒DNA涂于钨粉微弹表层,用基因枪轰击使微弹穿透细胞壁进入小麦细胞。处理2天后,用x—glucuronide染色,对GUS基因产物的活性进行鉴定,GUS基因表达的细胞呈深蓝色的小点。小麦幼胚表面的蓝点最多可达40～50个,悬浮细胞系中GUS基因表达的细胞较少。试验表明微弹轰击法是小麦组织水平上进行外源基因导入的一个有效途径。试验对微弹轰击的有关参数及条件进行了讨论。     

离子束介导GUS基因转化拟南芥种子的研究

以拟南芥种子为研究对象,研究离子束介导GUS基因导入拟南芥种子的转化效果。结果表明:直接注入干种子后进行转化,未得到瞬间表达的结果。而注入玻璃化保护后的2天龄幼苗,再进行转化,转化效果明显提高,得到了大约10%的瞬间表达。揭示对于像拟南芥这样的小粒种子,采用幼苗注入后转化,效果可能会更好一些。     

离子束介导大豆DNA转化小麦后代高蛋白株的RAPD标记分析

利用离子束介导法将大豆DNA导入小麦,经过连续4代田间筛选和蛋白含量测定,获得高蛋白变异株系.采用RAPD分析技术,用34条随机引物对供体大豆、受体小麦和3个高蛋白小麦变异株的基因组DNA进行扩增.有29个引物扩增出清晰稳定的条带,其中18个引物扩增出的条带有不同程度的差异.高蛋白小麦突变株与受体小麦(对照)相比出现了条带的增加、缺失、扩增带深浅等变化,也出现了与受体小麦不同而与供体大豆相同的扩增带.实验结果表明,外源大豆DNA导入受体小麦可以引起后代基因组DNA序列变化,扩大小麦遗传基础.     

利用精子介导法向蚕卵导入外源基因的研究

为建立家蚕转基因中切实可行、操作简便的外源基因导入方法,进行了精子介导法探索,以精子介导法的三种方式向家蚕导入所构建质粒pFbGFP,并通过PCR扩增和DNA印迹等手段,已连续两代从基因组DNA检测到导入外源基因GFP的存在,其中的一种导入方式到第二代阳性率约30% .结果表明该法可有效进行家蚕转基因的外源基因导入.     

利用激光微束将外源基因导入高等植物细胞的研究

本实验室已建立了激光微束向植物细胞导入外源基因的试验程序。用已建立的程序对多种经济作物和林木进行外源基因导入的研究,均得到了GUS基因瞬时表达的结果,正在继续研究GUS基因的整合表达。 广泛地选用了不同科属的植物及不同外植体进行外源基因导入的研究,所用材料如下:在被子植物中选用单子叶无性繁殖的兰科花卉兰花(Dendrobium)小圆球茎为外植体;以双子叶植物中的草本植物锦葵科的棉花(G.hirsulum L.)再生胚状体为外植体;十字花科油菜,选用     

建立低能离子束介导小麦转基因方法并获得转GUS基因植株

研究了注入离子种类、能量、剂量等参数对于低能离子束介导的遗传转化的影响,建立了适于小麦成熟胚转化的组培条件和筛选程序。以携带ＧＵＳ基因的质粒为供体,进行了报告基因转化研究。分子生物学证据表明ＧＵＳ基因已整合到小麦基因组中。３个小麦品种的抗性愈伤转化率分别为９．５％、１０．８％、１１．２％,再生植株转化率分别为１．４％、３．４％、１．７％,首次证明了离子束介导小麦遗传转化是可行的,为离子束介导小麦遗     

脉冲电泳不同参数对介导GFP基因转化玉米种胚效果的研究

利用脉冲电泳介导绿色荧光蛋白(GFP)基因导入玉米种胚;以GFP基因在种胚中瞬时表达作为外源基因导人种胚细胞的标记,分析了外源．DNA浓度、电泳时间、电压、电流转换时间等脉冲电泳转化参数对种胚发芽率和外源基因导入率的影响。结果表明：脉冲电泳时间对种胚发芽率和外源GFP基因导入率影响最大;通过脉冲电泳可将外源基因导入胚芽细胞,其GFP基因导人种子的频率与各电泳参数均呈二次曲线关系,300μg／ml外源DNA浓度、120min电泳时间、5V电压、2s电流转换时间可作为脉冲电泳介导玉米种胚转化较适宜的参数。     

低能离子束介导的遗传转化研究进展

低能离子束注入对细胞的刻蚀作用提供了外源遗传物质进入细胞的途径,离子束介导的遗传转化技术已在水稻,小麦,烟草,棉花等多种植物上取得成功,对离子束介导遗传转化的原理和最新进展进行了评述。．     

通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍     

The Expression of GUS Gene Driven by T-cyt Promoter in Transgenic Tobacco andPotato

The location of GUS gene expression under control of T-cyt gene (gene 4 of T- DNA coding isopenteryl transferase) 5′ region in transgenic tobacco (Nicotiana tabacum cv. W38) and potato (Solanum tuberosum L, cv. Desiree) plants was examined with biochemical assays. The results showed differential distribution in various organs and different cell types. The highest levels of GUS activity were found in tobacco stem where axillary bud was initiated and potato buds on tubers. Moreover, the expression of T-cyt promoter/GUS was found to be inducible in transgenic tobacco stem with cytokinin rather than auxin treatment. Additionally, the level of expression was high in the wounded leaf of transgenic potato. It was suggested that T-cyt promoter may be selectively induced by some exogenous plant hormones.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Characterization of an expansin gene and its ripening-specific promoter fragments from sour cherry (<Emphasis Type="Italic">Prunus cerasus</Emphasis> L.) cultivars

The presence of expansins was investigated in various developmental and ripening stages of cherry fruits by SDS-PAGE and immunoblotting. An expansin gene and three fragments (242, 607 and 929 bp) of its promoter region were cloned. The genomic clone of the expansin gene contained three introns, two exons spanning a 1.6 and a 1.0 kb upstream region. Semi-quantitative PCR analysis showed that this gene was ripening specific. Chimeric promoter—GUS constructs were made and truncated forms of the expansin promoter were introduced into tomatoes by agroinjection and fruits were analyzed for GUS expression by histochemical GUS staining and enzyme activity assays. The 0.60 kb expansin promoter efficiently induced GUS expression in transgenic tomatoes, whereas constructs with the 0.25 kb promoter did not display significant GUS staining. The highest GUS activity was detected in tomatoes containing the 1.0 kb promoter construct. Both large base pair promoter constructs drove the expression of the GUS gene at an equal or higher rate than the tomato E8 promoter.     

Functional analysis of the ver gene using antisense transgenic wheat

The function of ver203 , a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion‐expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S‐promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen‐tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double‐ and single‐stranded probes, and detection of GUS activity by X‐gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.     

Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F₁ progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and β-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F₁ generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F₁ progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.     

Expression of foreign genes in transgenic yellow-poplar plants

Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves.     

根癌农杆菌介导的玫瑰茄愈伤组织的遗传转化

以根癌农杆菌LBA4404和EHA105为供体菌株,对玫瑰茄愈伤组织进行了转化条件的研究,建立了一套玫瑰茄愈伤组织遗传转化体系。利用该转化体系获得了2个稳定表达新霉素磷酸转移酶活性的玫瑰茄转化细胞系。GUS活性组织化学检测和PCR扩增鉴定的结果表明,愈伤组织的转化率为4%。说明采用农杆菌介导法将外源基因经愈伤组织导入玫瑰茄细胞是可行的。