基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

Oviposition induced volatiles in tomato plants

In response to attack by herbivorous insect, plants produce semiochemicals which act to attract their natural enemies. Recent work on plant volatile compounds has shown that they may play multiple roles as communication signals and defense agents. Here we measured the volatile profile of tomato plants with and without oviposition by the herbivore moth, Tuta absoluta. We used solid phase micro extraction (SPME) sample pre-concentration techniques combined with gas chromatography-mass spectrometry. We have found that different volatile profiles emitted from tomato plants with oviposition by T. absoluta compared to control plants. Hexanal, (Z)-3-hexen-1-ol and an unidentified compound were isolated only from tomato plants with T. absoluta eggs. (Z)-3-hexen-1-ol was identified only from tomato plants with T. absoluta eggs that were laid three days earlier. The percentage of sesquiterpenes (e.g. δ-elemene) has been found to increase in the headspace of tomato plants with oviposition. These results in respect to indirect defense of tomato plants to T. absoluta are discussed.     

Multiple inserts of gene of interest and selectable marker gene are co-integrated and stably transmitted as a single genetic locus in transgenic soybean plants

Particle bombardment has been used for soybean transformation for more than 20 yr, but the integration and segregation of transgene inserts in the soybean genome have not been clearly documented. Over the past 5 yr, we processed several hundred transgenic events. In each experiment, the expression cassettes of the gene of interest (GOI) and hygromycin selectable marker gene (SMG) were co-bombarded into soybean at a 1:1 molecular ratio. More than 75% of hygromycin-resistant events also carried the GOI. Molecular analysis of transgenic plants revealed that most events carried multiple inserts of the GOI and the SMG. The GOI and the SMG were linked in selfed T1 and T2 progeny. Segregation analysis of progeny indicated that, in over 98% of the transgenic events, the multiple inserts of the GOI were integrated into the same genetic locus resulting in a 3:1 segregation ratio. Furthermore, the multiple inserts of the GOI are transmitted into succeeding generations, and no recombinants were found. These data indicate that in soybean plants, co-bombarded genes are preferentially integrated and stably segregated as a single genetic locus.     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T(0) and T(1) transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.     

Heterologous expression of <Emphasis Type="Italic">P5CS</Emphasis> gene in chickpea enhances salt tolerance without affecting yield

Vigna Δ¹-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na⁺ was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.     

Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens

Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var. Shinano No. 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121). The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants). The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively. Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A. tumefaciens harboring a modified pBI121 for plasmid rescue. Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.     

Resistance performances of transgenic bt rice lines T(2A)-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T(2A)-1 and T1c-19 expressing Cry2A* and Cry1C* from 'Minhui 63' (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T(2A)-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT)50 of larvae fed with T(2A)-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T(2A)-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T(2A)-1 Bt rice lines.     

Inheritance of marker and target genes in seed and vegetative progenies of transgenic tobacco plants carrying the buckwheat serine protease inhibitor gene

The buckwheat serine protease inhibitor (BWI-1) target gene (ISP) was expressed under the control of the constitutive 35S promoter of the cauliflower mosaic virus was expressed in transgenic tobacco plants and conferred antibacterial resistance. A stable and linked inheritance and expression of the marker nptII and target genes were observed in a random sample of independent transgenic tobacco plants after longterm propagation by nodal segments or multiple (for 1.5 years) regenerations under nonselective conditions; the transgene insert was preserved in the T1 seed progeny. Transgenic plants displayed numerous alterations in microsporogenesis. A loss of kanamycin (Km) resistance was accompanied by a loss of antibacterial activity in two lines. Segregation was observed for Km resistance in line C7 and for seedling size in line C22.     

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.     

Effect of pRi T-DNA transformation on growth and gas exchange of transplanted tobacco regenerants of the T and T′ phenotypes

Five independent tobacco regenerant clones obtained after transformation withAgrobdcterium rhizogenes strain TR101 (four clones of the T phenotype, clone 5 a mixture of plants of the T and T′ phenotypes) and untransformed plants cultivatedin vitro were gradually acclimated to the atmosphere and transferred to the soil substrate in pots. During 28 d after transplanting the increase of the leaf area was in T phenotype plants greater than in untransformed plants, but 14 d later no significant differences were observed between T phenotype and untransformed plants in leaf area and dry matter. T′phenotype plants, however, had significantly lower values in all growth parameters than T phenotype and control plants. Abaxial net photosynthetic CO₂ uptake (PN) was in both T (clones 1–4) and T′ plants significantly lower than that in control and clone 5T plants. Nevertheless, due to the relatively higher adaxial PN in T plants, the total PN through both leaf surfaces was significantly lower only in clone 4 and clone 5′ plants. The tendency to higher transpiration rates (E) in clones 1 – 4 in comparison with the control resulted in significant differences in water use efficiency (W.U.E.). Relatively higher E in T plants of clones 1 – 4 was connected most probably with their more developed root system (greater root dry-matter) than in untransformed plants, because no influence of the differences in stornata density on E was found.     

Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach

Summary Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%–2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%–63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.     

Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Summary Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.     

美洲棉铃虫细胞中dsRNA介导的egfp基因沉默分析

为了分析在美洲棉铃虫细胞( HzAM1)内RNAi的效果,将egfp基因克隆到含有双向T7启动子/终止子的质粒载体中,在体外合成全长的增强型绿色荧光蛋白(egfp)基因dsRNA,将dsRNA和含有能在昆虫细胞内表达eGFP的质粒一起转染HzAM1细胞,分析dsRNA对eGFP表达的抑制作用.结果显示,由egfp基因转录的长dsRNA能有效抑制HzAM1细胞内eGFP的表达,而且该抑制作用表现为剂量依赖效应.但是抑制作用并不彻底,在高剂量的dsRNA处理下,仍有部分细胞内能观察到eGFP的表达.     

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.