樱桃矮化砧木''吉塞拉6号''基因转化体系的建立

采用'吉塞拉6号'甜樱桃矮化砧木(Prunus cerasus×P. canescens)离体叶片外植体,在再生培养基附加生长素的条件下通过根癌农杆菌(Agrobacterium tumefaciens)EHA105(p35SGUS intron)介导研究了β-葡萄糖醛酸酶基因(GUS)的瞬时表达、稳定表达和转基因植株再生,证明了培养基中生长素(IBA或NAA)的存在可促进基因转化,转化效率比对照提高2倍以上.将500个叶片外植体与EHA105(p35SGUS intron)株系在含有生长素的培养基中共培养,获得了11个转基因株系.采用PCR分析和Southern Blotting核酸杂交,确定GUS基因已整合到矮化砧木'吉塞拉6号'植株的染色体上.组织化学染色确定了GUS基因在植株体内的表达.     

草莓主栽品种再生和转化的研究

 建立草莓主栽品种高效、稳定的离体再生体系和遗传转化体系, 获得了转基因植株。‘弗吉尼亚’和‘森嘎拉’的叶片离体再生芽频率达到100 %。试管苗叶片与农杆菌菌株EHA105 共培养3 d。共培养后的叶片在附加卡那霉素40 mg/L 的再生培养基上选择培养4周后, 外植体再生出转化芽, 弗吉尼亚的转化芽再生频率可达6. 8 %。采用组织化学染色法对随机选取的10 个GUS 基因转化植株进行基因表达测定, 结果5 个植株强烈表达GUS 活性。转bar 基因植株在附加除草剂草丁膦10 mg/L 的培养基上能够正常分化, 在田间对草丁膦表现出强烈抗性。转基因植株开花、结果正常。     

大白菜转修饰豇豆胰蛋白酶抑制剂基因获得抗虫植株

 以大白菜3 d 苗龄带柄子叶为外植体, 经根癌农杆菌(Agrobacterium tumef aciens) 介导, 将修饰的豇豆胰蛋白酶抑制剂基因(sck) 导入大白菜自交系‘GP-11’和杂交种‘中白4号’, 并获得了卡那霉素抗性植株。PCR 检测和Southern blot 杂交证实, sck 基因已整合进大白菜基因组中; 豇豆胰蛋白酶抑制剂活性检测表明, 大部分转基因植株都对牛胰蛋白酶有一定的抑制活性, 对照未转化植株抑制活性很低。室内离体叶片饲虫和田间自然抗虫性鉴定进一步证明, 转基因植株对菜青虫(Pieris rapae L. ) 具有一定抗性。     

梨糖转运相关基因PbTMT4启动子克隆及功能分析

以‘砀山酥梨’(Pyrus bretschneideri‘Dangshan Suli’)为材料,利用梨基因组数据库,通过PCR获得了糖转运相关基因PbTMT4(Pbr032130.1)2 211 bp的CDS序列及其编码起始位点上游长度为1 220 bp的启动子序列。使用农杆菌介导法将PbTMT4导入拟南芥,与野生型对照植株相比,转基因拟南芥植株的生长速度更快,抽薹、开花时间更早,叶片糖积累量更高。生物信息学分析表明,该启动子中含有多个与逆境应答、激素信号和光信号相关的顺式作用元件。为进一步分析PbTMT4启动子功能,构建了该启动子与GUS基因融合的植物表达载体并转化拟南芥。对T_3代转基因拟南芥各组织进行GUS活性染色和半定量分析,发现GUS基因在根、茎、叶、花和果荚中均有表达,NaCl、干旱、GA_3、MeJA以及光照处理均能一定程度上提高转基因拟南芥中GUS基因的转录水平。逆境处理发现,PbTMT4转基因株系较野生型植株受到的伤害小。研究结果初步表明,PbTMT4可促进转基因拟南芥发育并提高糖积累量,可能在抗非生物胁迫中起重要的调控作用。     

提高苹果基因转化效率的研究

采用根瘤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）强株系ＥＨＡ１０５（ｐ３５ｓＧＵＳ－ｉｎｔｒｏｎ）研究了影响‘皇家嘎拉’苹果（Ｍａｉｎｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ．）外植体的β-葡萄糖醛酸酶（ＧＵＳ）基因的瞬时、稳定表达水平和转基因植株的再生。证明在培养基生长素（ＩＢＡ、ＮＡＡ）存在的条件下,外植体的ＧＵＳ基因的瞬时表达水平提高了３－４倍,而共培养两周后稳定表达水平提高２倍以上,产生９．８个ＧＵＳ愈伤组织表达区域。白化处理促进外植体的基因转化,白化处理的新梢顶端第一节间外植体ＧＵＳ表达区域比常用的叶片外植体高４倍。在生长素存在的条件下 ２％外植体获得了转基因植株。Ｓｏｕｔｈｅｒｎ　ＢｌｏｔＤＮＡ杂交和组织化学染色分析证明ＧＵＳ基因已整合到苹果的染色体上,并得以表达。     

大白菜的马铃薯蛋白酶抑制剂基因转化及抗菜青虫性的鉴定

 以大白菜(B．campestris ssp．pekinensis)‘北京80号’生长3 d无菌苗的带柄子叶为外植体，通过根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘法导人马铃薯蛋白酶抑制剂基因(pinⅡ)。通过抗性株的真叶在不定芽诱导培养基上的再生， 证实了嵌合体的存在，并通过此方法来消除嵌合体。获得的转基因植株经PCR、PCR-Southern杂交以及植物基因组Southern杂交证实，目的基因已经整合入植物基因组中。对菜青虫(Pieris rapae L．) 进行了转基因大白菜叶片的连续离体饲喂，结果表明：受试的转基因大白菜对菜青虫的生长发育有较明显的抑制作用。     

苹果MdFT基因对番茄的遗传转化

 通过RT2PCR扩增, 从苹果叶片cDNA中克隆了^FT基因的同源基因^MdFT, 构建了花椰菜病毒35S启动子驱动的MdFT植物表达载体35S: : MdFT, 并利用根癌农杆菌介导法将其导入番茄栽培品种‘中蔬四号’; 同时转化拟南芥A tFT基因作为阳性对照。从添加卡那霉素的筛选培养基上再生了抗性植株,PCR扩增证明, 外源基因MdFT和AtFT已经整合到转基因番茄的基因组, 半定量RT-PCR则证明它们已经在转基因番茄中得到异位过量表达。形态鉴定发现, 转基因番茄植株比非转基因对照植株开花早, 表明成功地从苹果中克隆了成花素基因MdFT, 该基因具有通过转基因缩短苹果树童期的潜在价值。     

露地菊离体再生体系建立及BrDFR基因遗传转化

以露地菊(Chrysanthemum morifolium)品种‘神韵’无菌苗叶片为外植体材料,研究植物生长调节剂配比对其愈伤组织诱导再生芽的影响,以建立离体再生体系.研究结果表明,露地菊‘神韵’在含NAA 1.0 mg·L-1+BA 1.0 mg· L-1的MS培养基上获得了最高的再生芽分化率.利用已经克隆的‘津田’芜菁BrDFR基因构建非抗生素筛选的表达载体.以露地菊‘神韵’的无菌苗叶片为受体,通过农杆菌介导进行BrDFR基因的遗传转化.以载体的PMI基因为筛选标记,通过甘露糖筛选获得了‘神韵’的遗传转化再生植株.经过PCR验证和Southern杂交检测证实BrDFR基因已经整合到‘神韵’基因组中.     

根癌农杆菌介导Cry1Ac基因转化‘雪青’梨获得转基因植株

 以‘雪青’梨叶片愈伤组织为外植体, 经根癌农杆菌介导将CaMV35S启动子调控下的Cry1Ac基因导入‘雪青’梨。698块愈伤组织和231个Kan^r芽与携带表达载体质粒pBX203的根癌农杆菌菌株LBA4404共培养3 d后, 转入含50 mg·L^-1 Kan的筛选培养基培养30 d, 15 d转接1次。结果表明,在MS + 5 mg·L ^-1 6-BA + 0.1 mg·L^-1 IAA筛选培养基中, Kan^r芽率为34.24% , 在1/2MS + 2 mg·L^-1 IBA+ 0.5 mg·L^-1 6-BA + 500 mg·L^-1AC筛选培养基中, 15.58% Kan^r芽生根。共获得再生植株32株, 经PCR和Southern-blot分析证明, 其中4株的基因组已成功导入和整合Cry1Ac基因。     

华东葡萄泛素连接酶基因VpUIRP2和VpUIRP3的抗白粉病特性分析

【目的】分析华东葡萄抗白粉病株系‘白河-35-1’的Ring型泛素连接酶基因VpUIRP2和VpUIRP3的转基因植株对葡萄白粉病的抗性,研究这2个基因的抗白粉病特性,为改良欧洲葡萄抗病性提供可用基因。【方法】利用同源克隆的方法获得中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2和VpUIRP3的编码区序列(CDS);通过实时荧光定量PCR方法检测其在白粉菌诱导和水杨酸处理后的葡萄叶片中的表达;使用聚乙二醇(PEG)介导法转化拟南芥原生质体进行亚细胞定位;通过农杆菌介导法获得转基因‘无核白’和‘红地球’植株;经过苯胺蓝染色,利用血球计数板计数分析白粉菌在转基因植株叶片上的生长情况,鉴定转基因株系的抗病性。【结果】中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2和VpUIRP3响应白粉菌和水杨酸处理,表达模式明显区别于感病对照‘赤霞珠’。中国野生华东葡萄‘白河-35-1’VpUIRP2蛋白定位在细胞质,VpUIRP3蛋白定位无特异性。通过农杆菌介导的遗传转化获得4个VpUIRP2的转基因‘红地球’株系和1个‘无核白’株系,转基因株系对白粉病表现出抗性。【结论】中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2能增强葡萄对白粉病的抗性,可以作为抗病基因用于改良欧洲葡萄的抗病性。     

甜蛋白基因MBLII 对莴苣的遗传转化

 以4 日龄莴苣(Lactua sativa L.) 无菌苗子叶为外植体, 通过根癌农杆菌介导,成功地进行了马槟榔甜蛋白基因MBLII 对莴苣的遗传转化。抗生素浓度和子叶外植体与农杆菌的共培养时间是影响转化的重要因素。附加卡那霉素( Kan) 50 mg/ L 的诱芽培养基MS I(MS+ NAA 0. 1 mg/ L+ 6􀀁BA 0. 1 mg/ L+ 羧卞青霉素500 mg/ L) 最适于侵染后子叶外植体的诱芽培养。在外植体与农杆菌共培养0~ 7 d 的范围内, 以共培养3 d 最佳( 生芽率58. 3%, 白化率29%) 。1~ 2 cm 再生芽移入诱根培养基MS II (MS+ NAA 0. 05 mg/ L+ Kan 50 mg/ L+ 羧苄青霉素300 mg/ L) 中, 诱根率可达100%。获得的抗性植株经组织化学及PCR 特异扩增鉴定和统计, 7. 6%阳性。Southern blot 结果证明MBL II 基因已整合到莴苣基因组中。     

根癌农杆菌介导蓝猪耳转化系统的建立

 通过对影响根癌农杆菌介导蓝猪耳转化效果各种因素的研究, 建立了蓝猪耳高效稳定的遗传转化系统。研究结果表明, 农杆菌侵染叶片外植体效率最高; 外植体预培养不利于农杆菌的侵染; 乙酰丁香酮能大大提高根癌农杆菌转化蓝猪耳的效率。另外, 菌液浓度、侵染和共培养的时间及再生的途径等均对转化效率有一定的影响。叶片外植体与农杆菌共培养后, 经过抗性芽的诱导、根的再生, 70 d左右就可以获得抗性苗。抗性植株经GUS染色、PCR分析和Southern blot检测, 外源基因已经整合到蓝猪耳基因组中, 转化频率为7%～8%。     

根癌农杆菌介导荔枝遗传转化研究

利用带有内含子的GUS基因(uidA)的瞬时表达,研究影响根癌农杆菌介导荔枝胚性愈伤组织遗传转化的若干因素。结果表明:在AGL-1、LBA4404和EHA105三种菌株中,EHA105对荔枝胚性愈伤组织的侵染力最强;采用2d的共培养时间既有较高的瞬时表达率,又避免了农杆菌的过度生长;0.5×10~8个细胞/mL的菌液密度为最佳侵染浓度;继代培养15d的胚性愈伤组织处于最旺盛分裂的时期,是转化的合适受体;愈伤组织转化前干燥处理对uidA瞬时表达率的提高有一定的促进作用。使用优化的农杆菌侵染条件获得了稳定表达uidA基因的荔枝抗性愈伤组织。     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

An efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L^?1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).     

小西葫芦黄花叶病毒外壳蛋白基因导入西瓜的遗传转化

研究了西瓜品种ZKM的再生体系和农杆菌介导小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)的外壳蛋白(cp)基因导入西瓜的遗传转化体系。结果表明,质量分数0.6%琼脂是适宜种子萌发的基本培养基,切取5d龄的西瓜无菌苗子叶为外植体,接种在MS+BA2.0mg/L诱导培养基上,诱导不定芽效率最高。选用75mg/L卡那霉素筛选西瓜抗性芽较为合适,外植体经预培养2～3d,菌液浓度以OD600nm值为0.4,共侵染10min有利于提高西瓜转化的效率。经PCR检测,ZYMVcp基因已经导入西瓜植株,转化频率为0.15%。     

In vitro regeneration and Agrobacterium mediated transformation in gladiolus

Summary

In vitro regeneration and transformation studies were conducted on two cultivars of gladiolus. Cormels of 1.0 to 1.5 cm diameter cut into 2–3 mm thick slices of top, middle and bottom, and in vitro derived bisected shoot tips were used as explants on MS medium supplemented with 18.6 μM kinetin for multiple shoot induction. Amongst the cormel slices, the top slice gave better shoot induction response of 89% with an average of 2.4 shoots per explant over both cultivars. In vitro derived bisected shoot tips were inoculated on the medium oriented cut-side up, cut-side down and vertically both with and without the cormel base attached. Bisected shoot tips without attached cormel base and inoculated in the cut-side down orientation showed an average of 90% shooting response. In vitro derived shoot tips were used as explants for transformation. Explants were wounded by scalpel and particle bombardment with 1.6 μm naked gold particles by the biolistic delivery system. The wounded explants, after 3 d of recovery period, were co-cultivated with Agrobacterium strain LBA4404 harbouring the binary vectors pBI141 and pTOK233 which contained gus reporter gene with rice actin and 35S promoters respectively. GUS expression frequencies of 5.3% and 23% was obtained from scalpel and particle bombardment wounded explants, respectively. Particle wounded explants showed an average of 63 and 103 GUS spots when co-cultivated with pBI141 and pTOK233 binary vectors respectively. Explants co-cultivated with pBI141, after three weeks of selection on antibiotic containing medium showed blue streaks of GUS expression. It was concluded that Agrobacterium could infect the monocot gladiolus and transform the tissue eficiently when tissues were prewounded with naked gold particles delivered by particle gun.     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.