番茄ACC合酶反义基因对河套蜜瓜的转化

河套蜜瓜（CucumismeloLcvHetau）的子叶经预培养。芽诱导和生根培养,获得再生小植株,诱导率达58％。取带有番茄ACC合酶反义基因的双元载体pMQ6／JM109与农杆菌（Agrobacteriumtumefaciens）LBA4404经三亲融合后,与在MS0上萌发5d、并在MS＋1mg／LNAA培养基上预培养3d的子叶共培养48h,然后转入含50mg／L卡那霉素的MS＋6mg／LZT的芽诱导培养基中,1l个月后诱导生芽,待芽长1．5－2cm时转入生根培养基中,1－2周后可诱导产生大量的根,形成完整的转基因小植株。经PCR和分子杂交检测证明,目的基因已整合入河套蜜瓜的基因组中。     

在转基因烟草中表达番茄反义ACC合酶基因及其对芽再生的影响

利用从番茄(Lycopersicum esculentum Mill.)果实中分离到的ACC合酶cDNA,反向置于CaMV 35S启动子的控制之下,并转入烟草(Nicotiana tabacum L.)。PCR扩增证明此反义基因已整合到烟草的基因组上。Northern杂交及逆转录PCR分析表明,这种异源反义基因能在转基因烟草组织中表达,并抑制了烟草内源乙烯的合成,对乙烯合成的抑制在芽再生过程中更为明显,同时这也导致了转基因烟草在组织培养过程中芽再生能力的增强。这些结果从基因水平证明,乙烯在芽形成过程中具有重要的调控功能。     

ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg/L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9.5%,番茄的贮存期在50天以上。     

ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg／L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9．5％,番茄的贮存期在50天以上。     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

河套蜜瓜ACC合成酶cDNA片段的克隆和序列分析

1-氨基环丙烷-1-羧酸（ACC）合成酶是高等植物中乙烯生物合成的关键酶。以成熟河套蜜瓜（CucumismeloL．cvHetau）果实的RNA为模板,经反转录和PCR扩增得到预期大小的DNA片段,插入到pUC19的SmaⅠ位点后转化E.coliJM109,筛选出重组子pHMAS1。序列分析表明获得了长627bp的ACC合成酶cDNA片段。与已报道的ACC合成酶基因相应序列比较有很高的同源性．     

厚皮甜瓜（Cucumis melo var. reliculatus）的快速繁殖

以厚皮甜瓜（Cucumis melo var. reliculatus）西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明：在MS+BA 0.5～1.0 mg/L+IAA 0.1 mg/L 的培养基上利于诱导形成丛生芽, 芽的月增殖系数达到11以上; 在1/2 MS+IAA 0.5 mg/L培养基上并经暗处理3 d最易生根,生根率90%;在蛭石：草炭土=1：1(体积比)基质中移栽驯化效果好。试管植株定植大田后种性不变,生长和结果习性优于种子苗。     

Genetics of ethylene biosynthesis and restriction fragment length polymorphisms (RFLPs) of ACC oxidase and synthase genes in melon ( Cucumis melo L.)

We investigated the genetics of ethylene biosynthesis and its linkage to the RFLPs of the ACC oxidase and synthase genes in melon ( Cucumis melo L.). The results suggested that the A(0) and B(0) fragments of RFLP-MEL1 of the ACC oxidase gene were two alleles from a single locus, as were the B and C fragments of RFLP-MEACS1 of the ACC synthase gene. The B(0) allele seemed to be partially dominant over the A(0) allele, whereas B and C alleles appeared to map to quantitative trait loci (QTLs), which most likely contributed to ethylene production. Both RFLPs were linked to ethylene production rates, but they were not linked to each other. The interaction effects of the ACC oxidase and synthase genes on ethylene production were revealed by segregation of RFLP-MEL1 and RFLP-MEACS1. The results of single-copy-reconstruction assays suggested that the ACC oxidase gene is a single copy, whereas the ACC synthase gene is a component of a multigene family in the melon genome. The abscission phenotype appeared to be controlled by an independent locus, with the abscission (full-slip) allele dominant over the non-abscission (not full-slip) allele. These results may facilitate efforts toward mapping the quantitative trait loci (QTLs) of ethylene production. The RFLPs may be used in marker-assisted selection in developing melons with a more-desirable low ethylene production rate for enhancing postharvest storage life.     

石斛兰转ACS反义基因抗性原球茎及转化植株的筛选与鉴定

该试验就石斛兰转化ACS(1-氨基环丙烷-1-羧酸合成酶)反义基因的不同筛选方法和筛选处理对抗性原球茎筛选的影响,以及石斛兰转基因植株的再生与鉴定进行研究.结果表明:(1)石斛兰原球茎经带有gus报告基因和ACS反义基因的农杆菌LBA4404侵染共培养5d后除菌,采用逐渐提高选择压浓度的延迟筛选,并在低选择压浓度下切割而高选择压浓度下不切割的处理方式为抗性原球茎的最佳筛选途径,抗性原球茎获得率可达14.97％.(2)抗性原球茎繁殖时应逐渐降低选择压浓度,且在低选择压浓度下进行切割处理,繁殖倍数达到1.15倍,且原球茎生长势好.(3)抗性原球茎在1/2 MS+0.5 mg/L 6-BA培养基中的分化率达到73.85％;107株无根小苗培养于1/2 MS+1.0 mg/L NAA+50.0 mg/L Km(卡那霉素)+100.0 mg/L Cef(头孢霉素)培养基中进行生根培养,共获得了13株具有卡那霉素抗性的转化植株,转化效率达到12.15％.(4)转化植株经报告基因产物GUS组织化学检测和gus的PCR检测,证实带ACS反义基因的T-DNA已整合进石斛兰基因组中,且转基因植株在形态上与未转基因植株无明显差别,3株转基因植株移栽2个月后均已成活.     

草莓高频离体再生体系的研究

以6个草莓品种为试材,研究了影响草莓不定芽再生的各种因素,建立离体叶片高效再生系统。结果表明,外植体基因型、激素种类及配比、叶龄等是影响草莓再生的主要因子,其中‘鬼露甘’叶片最佳芽诱导培养基为MS 2.0 mg/L 6-BA 0.1 mg/L IBA,‘嫜姬’叶片愈伤组织的诱导以MS 3 mg/L 6-BA 0.2 mg/L 2,4-D较好,而且1周左右的暗培养可以防止外植体的褐化。芽伸长的最适培养基为MS 0.5 mg/L 6-BA 0.5 mg/L IBA,生根的最适培养基为MS 0.2 mg/L IBA,试管苗移栽后成活率为87%。     

Influence of genotype and explant source on the in vitro regeneration ability of different melon varieties

Nine genotypes of melon (Cucumis melo L.) were selected for the investigation of regeneration. Most of the tested varieties showed regeneration ability on medium containing 0.5 mg l?1 or 1 mg l?1 BA, but following the appearance of shoot buds, only six varieties produced leafy shoots. The effect of combinations of BA with different auxins (IAA, NA, 2,4-D) and ABA in the culture medium on shoot regeneration was tested on cotyledon explants of 'Hógolyó' and 'Hale's Best'. To establish optimal conditions for the adventitious shoot induction six types of seedling-derived explants were prepared from seedlings of four different ages. The best results for shoot forming capacity were achieved with cotyledons followed by decapitated seedlings and hypocotyls derived from 4-day-old seedlings. Cotyledon segments of 'Hógolyó' and 'Hale's Best' were also cultivated on media with different concentrations of IAA and BA supplemented with 0.26 mg l?1 ABA. The highest number of well-formed plantlets was counted for 'Hógolyó' on the medium supplemented with 0.9 mg l?1 BA+ 0.6 mg l?1 IAA+ 0.26 mg l?1 ABA. This is the first report on the in vitro regeneration of 'Hógolyó' from decapitated seedling and hypocotyl explants and of 'Javított Zentai', 'Muskotály', 'Hógolyó', 'Tétényi csereshéjú' and 'Magyar Kincs' from cotyledon explants.     

嫁接对网纹甜瓜果实乙烯生物合成及CmACO1基因表达的影响

以“新汉城翠蜜”网纹甜瓜为接穗,以“圣砧一号”南瓜为砧木进行嫁接,研究嫁接对目光温室网纹甜瓜果实乙烯生物合成及CmACO1基因转录水平表达的影响。结果表明：嫁接明显降低了网纹甜瓜果实ACC含量及乙烯释放量,在乙烯释放高峰时,嫁接的网纹甜瓜果实乙烯释放量比自根的降低了12．6％;嫁接降低了果实中ACC合成酶和ACC氧化酶活性,其最大值分别比自根网纹甜瓜降低了9．0％和6．8％;嫁接抑制了CmACO1的表达,其相对表达量最大值比自根网纹甜瓜降低了39．0％;嫁接网纹甜瓜果实中乙烯释放高峰、ACC合成酶和ACC氧化酶活性峰值及CmACO1相对表达量峰值出现的时间均比自根的延迟了3d。     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

Efficient Parthenogenesis Induction and In Vitro Haploid Plant Regeneration in Cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) Using Putrescine,Spermidine, and Cycocel

In this study, the effect of spraying mother plants with various levels of putrescine, spermidine, and cycocel (each at 0, 50, 500, and 5000 mg/l) were assessed on the frequency of haploid embryos produced from unfertilized ovaries and subsequent regeneration of derived embryos. Significantly higher haploid embryos were obtained when mother plants were sprayed with putrescine at 500 mg/l (5.2 embryos/fruit), spermidine at 50 mg/l (4.8 embryos/fruit), and cycocel at 50 mg/l (5.2 embryos/fruit) as compared to the control (without spraying, 3.2 embryos/fruit). However, embryogenesis induction was decreased drastically as the concentration of all the three compounds tested was increased and the lowest haploid embryos were observed when 5000 mg/l of spermidine (0.4 embryos/fruit) or cycocel (2.0 embryos/fruit) were applied. Only spermidine at 50 mg/l led to 100% regeneration into fully developed plantlets. The seed setting and size of fruits were also affected by polyamines and cycocel applications. Ploidy analysis using a flow cytometer indicated that all regenerated plantlets contain the gametic chromosome number (n?=?x?=?7) of parental plants and the results of chromosome counting also confirmed the haploid nature of regenerated plantlets. It can be concluded that the induction of haploid embryogenesis from unfertilized ovaries after pollination with irradiated pollen and subsequent conversion of derived embryos into the plantlets could be improved in Cucumis sativus L. by applying appropriate levels of putrescine, spermidine, and cycocel.     

The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers

We investigated the function of the tomato (Lycopersicon esculentum) E8 gene. Previous experiments in which antisense suppression of E8 was used suggested that the E8 protein has a negative effect on ethylene evolution in fruit. E8 is expressed in flowers as well as in fruit, and its expression is high in anthers. We introduced a cauliflower mosaic virus 35S-E8 gene into tomato plants and obtained plants with overexpression of E8 and plants in which E8 expression was suppressed due to co-suppression. Overexpression of E8 in unripe fruit did not affect the level of ethylene evolution during fruit ripening; however, reduction of E8 protein by cosuppression did lead to elevated levels during ripening. Levels for ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC oxidase mRNA were increased approximately 7-fold in fruit of plants with reduced E8 protein. Levels of ACC synthase 2 mRNA were increased 2.5-fold, and ACC synthase 4 mRNA was not affected. Reduction of E8 protein in anthers did not affect the accumulation of ACC or of mRNAs encoding enzymes involved in ethylene biosynthesis. Our results suggest that the product of the E8 reaction participates in feedback regulation of ethylene biosynthesis during fruit ripening.     

Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants

Summary Transgenic cucumber (Cucumis sativus L.) plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with NOS-nptII, CaMV 35S-I-gus and CaMV 35S-hph genes. Acetosyringone enhanced the efficiency of transformation at the cut surface cells of hypocotyl explants during five days of co-cultivation. Transformed cells were more effectively selected using 20–30 mg/l hygromycin B than using 50–100 mg/l kanamycin. Shoot regeneration occurred within 4–6 wks, and 12 of 21 regenerated plantlets displayed strong GUS expression in the very young leaves. All of 8 GUS-positive R0 plants examined showed single or a few positive bands by Southern blot analysis. The expression of the CaMV 35S-I-gus gene was observed in various tissues and organs of R0 and R1 transgenic cucumber plants.     

The effects of the herbicide quinclorac on shoot growth in tomato is alleviated by inhibitors of ethylene biosynthesis and by the presence of an antisense construct to the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene in transgenic plants

Reduction of shoot growth, leaf epinasty and chlorosis in young tomato plants (Lycopersicon esculentum Mill. cv. Hellfrucht/Frühstamm) treated hydroponically with 10^-7 M of the herbicide quinclorac were partially compensated when the plants were simultaneously sprayed with salicyclic acid or the oxime ether derivative PACME. Since salicyclic acid and PACME are known inhibitors of ethylene biosynthesis, it is suggested that this pathway is implicated in quinclorac action. Further support for this hypothesis was obtained in experiments with transgenic tomato plants containing an antisense gene to 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis. When quinclorac was applied via the root antisense plants showed reduced phenotypical alterations compared to those of wild-type plants.     

Production of the polyketide 6-MSA in yeast engineered for increased malonyl-CoA supply

The heterologous production of fungal polyketides was investigated using 6-methylsalicylic acid synthase (6-MSAS) as a model polyketide synthase and Saccharomyces cerevisiae as a host. In order to improve the production of 6-MSA by enhancing the supply of precursors, the promoter of the gene (ACC1) encoding acetyl-CoA carboxylase, which catalyzes the conversion of acetyl-CoA to malonyl-CoA, was replaced with a strong, constitutive promoter (TEF1p) in a strain harboring two plasmids carrying the genes encoding 6-MSAS from Penicillium patulum and PPTase from Aspergillus nidulans, respectively. The strain was characterized in batch cultivations with a glucose minimal media (20 g/L), and a 60% increase in 6-MSA titer was observed compared to a strain having the native promoter in front of ACC1. The production of 6-MSA was scaled up by the cultivation in minimal media containing 50 g/L of glucose, and hereby a final titer of 554+/-26 mg/L of 6-MSA was obtained.     

徐长卿子叶组织培养及再生体系的建立

以徐长卿子叶为材料,进行愈伤组织诱导、愈伤组织和不定芽分化、试管苗生根、移栽及移植等研究。结果表明,MS+6-BA 0.3 mg/L+NAA 0.2 mg/L+2,4-D 0.4 mg/L是子叶愈伤组织诱导和继代培养的理想培养基;MS+6-BA 0.8 mg/L+NAA 0.2 mg/L是愈伤组织分化培养的理想培养基;MS+6-BA 0.6 mg/L+NAA 0.2 mg/L是不定芽分化继代培养的理想培养基;炉灰渣是试管苗移栽的理想基质,移栽成活率可达97%。试管苗移植后长势旺盛,根系发达,当年开花。根据本试验结果可建立徐长卿子叶诱导再生体系。     

Plant Regeneration from Protoplasts of Talinum paniculatum

The protoplasts of Talinum paniculaturn (Jaeq.) Gaertn. were isolated from leaves and calli. The mesophyll protoplasts did not undergo normal division and lived one week at the longest in culture. However, the callus protoplasts, cultured in P4 medium (K8p+2, 4-D 0.2 mg/L, NAA 1.0 mg/L, ZT 0.5 mg/L, coconut milk 50 mL/L, glucose 0.5 mol/L), underwent first division after 3 d of culture. The division frequency was 36.7 % after 7 d of culture. The regeneration frequencies of callus were 0.31% in liquid culture and 0.34% in double-layer culture. Shoots differentiated on regeneration media and rooted on R3 and R7 media. Mature plants were obtained 2～3 months after transplanting the protoplast-derived plantlets into flower pot or successive subculturing in test tubes. The results also indicated that: (1) Too long a period of callus culture in liquid medium or in solid proliferation medium was unfavorable to differentiation. (2) Low concentration of 6-BA in medium was suitable for callus differentiation. (3) GA3 promoted development of young adventitious bud. (4) Multi-effect triazole significantly strengthened sprout and root development in test tube cultures.