含ipt基因生长素调控基因根癌农杆菌转化牵牛萌动种胚同工酶分析

以牵牛萌动种胚为受体,用含有３５Ｓ启动子－Ｇｕｓ基因－ｉｐｔ基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４３及含有３５Ｓ启动子－Ｇｕｓ基因－生长素调控基因－Ｎｏｓ基因的根癌农杆菌ＬＢＡ４４０４进行转化。通过对转化及对照植株苗期叶的同工酶分析,发现转化植株叶的过氧化物酶同工酶及酯酶同工酶谱带来对照植株有明显差异。转化频率较高。间接验证了外源ＤＮＡ的导入,说明同工酶分析方法可做为转基因植株早期筛选的方法之     

沙田柚遗传转化的探讨

采用含ｎｏｓ基因和人工合成柞蚕抗菌肽Ｄ基因ＡＰ－Ｄ基因的根癌农杆菌对沙田柚外植体进行转基因研究,与根癌农杆菌共培养产生的芽,苗经Ｋｍ敏感性筛选,获得了若干转基因植株,并对这些植株进行了报告基因ｎｏｓ表达的电泳检测,同位素标记、ＡＰ－Ｄ基因为探针的点印迹杂交,外源ＡＰ－Ｄ基因的ＰＣＲ扩增和Ｓｏｕｔｈｅｎ杂交。结果显示,报告基因ｎｏｓ、抗菌肽Ｄ基因已转入沙田柚植株细胞中。     

黑核桃体细胞胚状体发生及其基因转化系统

以我国种植的优良品种黑核桃（Ｊｕｇｌａｎｓ ｎｉｇｉａ Ｌ．）幼胚和幼叶为外植体诱导出体细胞胚状体，通过根癌农杆菌介导将ｎｐｔⅡ和ｇｕｓ基因导入细胞胚，同时研究了激素、体细胞胚的发育时期及预培养等对转化率的影响，建立了黑核桃体细胞胚基因转化系统，还分析了体细胞胚胎转化系统的特点和潜力。     

提高苹果基因转化效率的研究

采用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ）强株系ＥＨＡ１０５（ｐ３５ｓＧＵＳ－ｉｎｔｒｏｎ）研究了影响‘皇家嘎拉’苹果（Ｍａｌｕｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ．）外植体的β－葡萄糖醛酸酶（ＧＵＳ）基因的瞬时、稳定表达水科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     

超声波辅助农杆菌介导八棱海棠转rolC基因

应用超声波辅助农杆菌介导法,对八棱海棠进行rolC基因转化,以期提高转化效率并获得转基因植株。利用gus基因瞬间表达的方法研究了超声波处理时间、处理时期和农杆菌悬浮液中乙酰丁香酮(As)浓度对rolC基因转化率的影响。结果表明,叶盘在D600nm为0．6且含有75 mg／L As的农杆菌悬浮液中侵染2 min后,用功率为100 W的超声波处理30 s,再浸泡2．5 min,然后放到再生培养基上共培养3 d,能获得最佳的gus基因瞬间表达率。最佳处理条件下转化683枚八棱海棠叶片,共得到138个抗性愈伤组织和15株抗性苗,转化率为2．2％。GUS染色、PCR及Southern blotting检测结果显示,有12个八棱海棠株系的基因组中整合了完整的外源rolC基因。     

青花菜农杆菌遗传转化影响因素研究

以青花菜“绿秀”为试材,以带柄子叶为外植体,采用根癌农杆菌LBA4404菌株进行农杆菌遗传转化,研究了影响青花菜遗传转化的主要因素.结果表明:乙酰丁香酮浓度、OD600值、侵染时间等对瞬间表达均有一定的影响.其中,OD600值影响最大,AS浓度影响次之,再次是侵染时间.最适宜的试验条件为:乙酰丁香酮浓度为100μmol/L、OD600值为0.3、侵染时间为5min.抗性植株经PCR检测、Southern杂交和GUS组织化学染色法,表明目的基因已整合到青花菜基因组中并得到了很好的表达.     

苜蓿花叶病毒侵染对菜豆胁迫响应基因表达的影响

苜蓿花叶病毒（ＡｌｆａｌｆａＭｏｓａｉｃＶｉｒｕｓＡＭＶ）侵染菜豆（ＰｈａｓｅｏｌｕｓｖｕｌｇａｒｉｓＬ．）叶片后,能强烈地诱导病程相关蛋白ＰＲ２基因的表达,促进富含脯氨酸细胞壁蛋白、ｄｅｈｙｄｒｉｎ、ｐｏｌｙｕｂｉｑｕｉｔｉｎ和ＤａｎＪｌｉｋｅ蛋白等基因的转录水平。这些防御蛋白共同作用对于保护细胞免受病毒伤害和维持细胞的正常代谢有重要作用。     

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。     

Ri质粒介导TMV和CMV外壳蛋白基因转化甜椒研究

利用发根农杆菌（Ａｇｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）的Ｒｉ质粒介导的二元载体,将ＰＢＴＣ－８质粒上的ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒中,用共培养的方法,诱导愈伤组织,经卡那霉素的抗性筛选,进一步的培养获得再生植株,经ＰＣＲ和ＥＬＩＳＡ的检测呈阳性反应,转化植株田间人工接种为抗性,证明ＴＭＶ－ｃｐ和ＣＭＶ－ｃｐ基因导入甜椒植株并已表达。     

发根农杆菌介导西瓜转基因过表达体系的建立

【目的】建立发根农杆菌介导的西瓜转基因不定根过表达体系。【方法】探索利用野生型发根农杆菌K599侵染西瓜材料'Sugar Baby',诱导西瓜产生不定根的实验方法。借助该方法,将含有GUS基因和EGFP基因的过表达载体pCAMBIA3301和pCAMBIA1300-ProSuper导入发根农杆菌K599,侵染西瓜材料'Sugar Baby',通过常规PCR、qRTPCR、GUS染色和激光共聚焦显微镜检测,评价该过表达体系的效果。【结果】利用野生型和导入外源质粒的发根农杆菌均能成功诱导西瓜'Sugar Baby'产生不定根。PCR检测结果显示,携带pCAMBIA3301和pCAMBIA1300-ProSuper载体的发根农杆菌K599诱导产生的不定根阳性率达到100%。qRT-PCR、GUS染色和激光共聚焦显微镜检测均能成功检测到GUS基因和EGFP基因的过量表达。【结论】利用发根农杆菌K599诱导西瓜产生不定根介导基因过表达的方法,具有周期短,操作方便,转化率高的特点,可实现基因功能的快速验证,为西瓜根系相关基因的功能研究提供技术支持。     

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。     

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.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.     

根癌农杆菌介导的韭菜基因转化体系的建立

 以GUS 基因为报告基因, 建立了农杆菌介导的韭菜基因转化体系。研究结果表明, 以预培养4～6 d 的根尖为转化受体, 用OD₆₀₀0. 6 ～0. 8 的LBA4404 浸染2～5 min , 共培养2 d , 然后在MS + NAA1 mg/L + BA 2 mg/L + Km 25 mg/L + Timentin 400 mg/L + As 100 mg/L 培养基上培养40 d , 后有愈伤组织产生,转入光下培养20 d 后有少量绿色不定芽产生。经X-Gluc 染色、PCR 分析和Southern blot 检验, GUS 基因已经整合到韭菜基因组染色体上。     

甘蓝Ogura 细胞质雄性不育相关基因BoMF1启动子的克隆及功能分析

 通过TAIL-PCR 染色体步移技术,从甘蓝（Brassica oleracea L. var. capitata L.）基因组中克 隆到胞质雄性不育（OguCMS）相关基因BoMF1 翻译起始位点上游521 bp 的启动子序列。软件分析预测 表明,该启动子序列中存在多个顺式作用元件,包括TATA-box、CAAT-box、MYB 结合位点、植物激 素响应单元等。为了研究该启动子的表达特性,亚克隆了BoMF1 转录起始位点上游521 bp 序列,将其置 换pBI121 中的CaMV35S 启动子,驱动其下游的GUS 基因,构建植物表达载体pBI121-BoMF1P,以pBI121 空载体作为阳性对照,通过农杆菌（LBA4404）介导法转入拟南芥。结果表明,甘蓝BoMF1 启动子序列 能驱动GUS 基因在拟南芥花药发育晚期的花药和花粉中特异表达,表达具有组织特异性。     

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%).     

根癌农杆菌介导的‘魏可’葡萄遗传转化体系的优化

以‘魏可’葡萄离体叶片为外植体,利用植物表达载体上含有卡那霉素抗性基因(nptⅡ)的根癌农杆菌LBA4404(pCAMBIA2301)对影响‘魏可’葡萄遗传转化效率的因素进行系统研究。结果表明,最佳农杆菌介导‘魏可’葡萄遗传转化体系为农杆菌侵染4 min,共培养3 d,卡那霉素质量浓度5 mg·L-1,羧苄青霉素质量浓度200 mg·L-1。采用此体系对‘魏可’葡萄进行转化,共获得9株抗性苗。利用GUS组织化学染色、PCR扩增的方法检测,结果表明,有4株通过了PCR检测,1株通过了RT-PCR检测,初步证明nptⅡ基因已经整合进入‘魏可’葡萄基因组中。将PCR产物回收,经测序验证nptⅡ基因完全整合进入‘魏可’葡萄基因组中。对CK及经PCR检测呈阳性的株系进行卡那霉素抗性鉴定,发现PCR呈阳性的株系均比CK抗Kan的能力明显增强。     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

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。