Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l⁻¹ kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.     

APETALA1基因启动子的克隆与功能分析

为了分析AP1的表达调控模式,本研究克隆了拟南芥花异常株系AFDL的AP1启动子,启动子元件预测结果表明:AP1启动子中含有3个结合MADS调控因子的CArG box(从5'依次编号为CArG1、CArG2、CArG3),通过删减AP1启动子长度以及改变CArGbox数量构建了5个GUS表达载体并转化野生型拟南芥.测序结果显示:AFDL的AP1启动子在核苷酸序列上与野生型拟南芥完全一致,这表明AP1在AFDL中的表达显著降低并不是启动子序列突变引起的;转基因植株的GUS表达模式说明了CArG1在花发育早期及后期激活基因的表达,CArG2在整个后期都对基因的表达有抑制作用,而CArG3在花发育初期就能抑制基因的表达,并且在中后期仍然保持了对下游基因的抑制作用,CArG box1、2、3对AP1的表达有显著但非决定性的影响.此外,还推测在AP1启动子0～-3 579 bp范围之外存在影响AP1在第4轮花器官表达的调控元件,-3 579 bp至-1 752 bp区域可促进AP1的表达,而AP1启动子-1759 bp至-1359 bp区域除CArG2外的其它元件对调节其表达无明显作用.     

LAT52启动子在甘蓝花药中的表达模式

[目的]验证花药特异性启动子LAT52能否在甘蓝花药中发挥功能。[方法]从番茄中扩增得到LAT52核心序列608 bp,进行序列分析,连接LAT52启动子到双元表达载体p BI121,转化农杆菌GV3101,用农杆菌浸染法侵染甘蓝幼苗下胚轴,获得2株阳性植株,对转基因甘蓝植株的花蕾、花药及花粉进行GUS染色。[结果]LAT52核心序列608 bp具多个与花粉特异性表达相关的元件,浸染的LAT52启动子仅在花药及花粉中表达出蓝色,其他部位未染上蓝色。[结论]LAT52启动子仅在甘蓝花药及花粉中特异性表达。     

青蒿非分泌型腺毛特异TPS7基因启动子的克隆及功能分析

研究运用基因组步移法克隆了长度为1 167bp的青蒿启动子proTPS7,经生物信息学分析发现了proTPS7中的多个顺式调控元件。构建启动子与GUS融合载体,稳定转化青蒿并对转基因植株进行GUS染色实验,结果表明该启动子能驱动报告基因在非分泌型腺毛中的特异表达。对转基因青蒿喷洒甲基茉莉酸和脱落酸,3h后GUS基因表达水平有所上调,说明proTPS7能受到上述2种激素的诱导。     

柑橘转基因胚性愈伤组织的筛选与再生

转化子的高效筛选与再生是获得转基因植株的关键。为快速有效地筛选出转化子,并尽可能在较短时间内获得纯合的转基因植株,开展了柑橘胚性愈伤组织的转化研究。以椪柑、冰糖橙、默科特橘橙等胚性愈伤组织为外植体,采用根癌农杆菌介导法进行转化试验,根据愈伤组织再生过程中不同发育阶段的组织对抗生素的敏感程度及在不同筛选压下抗性组织的生长情况,提出了＂5步筛选再生法＂,获得了大量的再生细胞系,并经GUS检测证实为转化子。     

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L. ssp. chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi (Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis) and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.     

影响农杆菌介导甜瓜子叶遗传转化的因素

以甜瓜子叶作为根癌农杆菌介导转化的受体,通过GUS基因瞬时表达率的分析,研究此转化体系的最佳实验参数.实验结果表明,预培养时间、预培养后对外植体进行处理、感染时间、共培养时间、农杆菌工程菌的浓度等对转化效率都有一定的影响,但是根癌农杆菌诱导物AS并不能大幅度提高转化效果.对外植体进行重新处理,预培养2 d,用OD560为0.3的农杆菌工程菌感染15～25 min,共培养3～4 d的理想条件下,GUS瞬时表达率可达85%.     

农杆菌介导的半夏GUS基因瞬时表达

采用根癌农杆菌感染半夏无菌叶片、叶柄和愈伤组织,对GUS基因的瞬间表达进行研究,并分析了不同转化受体、感染时间、菌液浓度、共培养、预培养时间、菌种对GUS基因的瞬间表达的影响。结果显示,以半夏的无菌苗叶柄和愈伤组织,在OD值为0.2的EHA101或EHA105农杆菌液中感染15 min,叶柄暗处共培养3 d,愈伤组织2 d,能够得到较高的GUS基因瞬间表达。     

影响麻竹基因转化的几个因子研究

对影响麻竹转基因过程中的几个关键性因素展开研究。卡那霉素和潮霉素都可以用于麻竹抗性愈伤组织筛选,25mg/L的潮霉素基本上可以排除非转化体：共培养时间对抗性愈伤组织和不定芽诱导再分化有明显影响,考虑到抑制农杆菌过度生长需要,选择共培养时间为3d。通过研究,初步建立了麻竹转基因技术体系．为丛生竹类转基因育种奠定了基础。     

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.