农杆菌介导的苜蓿次级体细胞胚的遗传转化

采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。     

The expression of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> HAL1 gene increases salt tolerance in transgenic watermelon [<Emphasis Type="Italic">Citrullus lanatus</Emphasis> (Thunb.) Matsun. &; Nakai.]

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Expression of anthocyanins and proanthocyanidins after transformation of alfalfa with maize Lc

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5'-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.     

Introduction and expression of an insect proteinase inhibitor in alfalfa Medicago sativa L.

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 g/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.Abbreviations Km kanamycin - PI protease inhibitor - SDS Sodium dodecyl sulfate - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - SH Shenk and Hildebrandt (1972) medium     

The role of cuscutain-propeptide inhibitor in haustoria parasitism and enhanced resistance to dodder in transgenic alfalfa expressing this propeptide

Cuscutain is a cysteine protease produced by dodder (the most important weeds of alfalfa) that is essential for the development and penetration of the haustoria in host. The propeptide subunit of cuscutain has a specific inhibitory function and inhibits the enzymatic activity of the cuscutain. In this study, we introduced the gene encoding the propeptide segment of the cuscutain (signal peptide-less inhibitor) into alfalfa and investigated its roles in parasitism and the alfalfa resistance to C. reflexa. Results demonstrated that cuscutain is mainly expressed in haustoria and the expression of propeptide in transgenic alfalfa plants effectively inhibited cuscutain enzyme activity and consequently interrupted haustoria development at the pathogenic stage. Digitate cells of haustoria could not differentiate into the xylem and phloem hyphae in dodder grown on transgenic alfalfa. Dodder development on transgenic alfalfa lines showed an overall reduction in fecundity and vigor due to imperfect attachment of haustoria. Morphology, nodule development and biomass of transgenic plants indicate that the inhibitory transgene exhibits exquisite specificity for cuscutain enzyme and by expression of the inhibitor in transgenic plants, there was no obvious adverse effect on them. The increased development and growth of dodder-challenged alfalfa transgenic plants compared to controls, showed the efficacy of propeptide in dodder control.     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.     

生长素结合蛋白基因在黄瓜果实发育中的功能研究

应用逆转录-聚合酶链式反应(RT—PCR),从黄瓜子房(幼果)中扩增出生长素结合蛋白ABP1)cDNA片段。该基因在开花前1天的子房中表达信号较弱,在授粉后2、4和6天的幼果中表达较强;在开花后2天有单性结实能力的子房中表达信号较强,不能形成果实的子房中信号较弱,所以ABP1基因可能参与黄瓜果实的生长发育过程。将拟南芥ABP1基因转入黄瓜中,转基因黄瓜的单性结实率平均为31.7％,高于对照(19．9％)。由于黄瓜的单性结实主要与生长素有关,所以,转基因植株单性结实率的提高可能是由于子房增强了对自身所含生长素的敏感性所致,说明生长素结合蛋白参与生长素在黄瓜果实生长发育中的生理作用。     

苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

Isolation and characterization of a harvest-inducible gene <Emphasis Type="Italic">hi11</Emphasis> and its promoter from alfalfa

The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. To investigate gene expression in harvested alfalfa, cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11 gene, which has high homology to a putative protein of unknown function in Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a wounding treatment. Northern blot analysis confirmed that the expression patterns of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In addition, genomic walking was performed to isolate the 5′ flanking region of the hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants of M. truncatula and tobacco, GUS gene expression was observed in harvested tissue, especially in the transgenic tobacco plants, but not in the non-harvested control tissue.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.