"全明星"草莓叶片遗传转化体系的建立

采用根癌农杆菌介导法进行nptⅡ基因对全明星草莓叶片的遗传转化研究,对转化过程中的一些因素进行了探讨：全明星叶片对卡那霉素的敏感实验,得出最适筛选浓度为30mg／L,羧苄青霉素的最适抑菌浓度为450mg／L;50μmol／L乙酰丁香酮(AS)的加入可提高GUS的瞬时表达率;10～15min是最适合叶片侵染的时间;共培养3d对叶片转化较适宜,得到抗卡那霉素的抗性芽的频率为1．1％,初步鉴定是转化芽。为草莓的遗传转化奠定基础。     

农杆菌介导佛手遗传转化主要影响因素的研究

采用根癌农杆菌介导的佛手叶盘转化法,在建立转海藻糖合酶基因(TPS)佛手体系过程中,对影响农杆菌转化频率的各种因素进行了研究。结果表明,佛手叶盘需在黑暗条件下MT培养基上预培养2-3d,与农杆菌共培养3d较合适;农杆菌菌液浓度OD600约为0.6-0.8,感染时间20min;抑制农杆菌生长的抗生素浓度以头孢霉素(Cef)250mgL-1和羧苄青霉素(Cb)250mgL-1且延迟筛选时间4d最好;共培养基中添加100μmol/L乙酰丁香酮(AS)和400mgL-1半胱氨酸(L-Cys)对佛手遗传转化有明显的促进作用。经GUS报告基因和PCR技术检测,初步证实TPS基因已整合到佛手基因组中,且GUS报告基因瞬时表达率为5.9%。     

根癌农杆菌介导的乙肝表面抗原基因对烟草的转化

构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于农杆菌的转化;通过冻融法将重组质粒pBRSAg转入根癌农杆菌LBA4404中,利用农杆菌介导法转化烟草叶盘,经筛选培养获得烟草植株。抗性植株经GUS染色和PCR检测为阳性,初步表明乙肝表面抗原基因在烟草中得到表达。     

红树林耐盐相关基因转化水稻的研究

运用农杆菌介导法将红树林耐盐相关基因mangrin转入粳稻品种‘日本晴’中,通过GUS基因检测愈伤组织转化率,确定农杆菌菌液浓度OD600为0.5,浸染时间30min,共培养时间3d为最佳转化体系;经潮霉菌筛选,获得抗性再生植株。通过PCR扩增检测、Southern blot分析和GUS基因活性检测,结果表明,mangrin基因整合到再生水稻的染色体DNA上。耐盐性测定结果表明,转基因植株在200mmol/L NaCl胁迫下,成活率保持在83.3%,株高增长20%~40%,mangrin基因能提高转基因水稻对盐胁迫的抗性。     

EuFPS基因表达载体构建及对杜仲遗传转化的研究

本实验用EcoRⅠ和BamHⅠ双酶切植物表达载体pSH737和含有目的基因的pUC-FPS,定向连接得到重组质粒pSH-FPS,将其导入农杆菌EHA105.采用农杆菌介导法对杜仲进行遗传转化,研究了卡那霉素(kanamycin,Km)浓度、预培养时间、菌液浓度及侵染时间、乙酰丁香酮(acetosyringone,AS)浓度、共培养时间等对杜仲遗传转化效率的影响.结果表明,选择无菌苗苗龄15 d的杜仲下胚轴,卡那霉素浓度50mg/L,农杆菌浓度OD600值0.3-0.6,侵染时间8 min,侵染时菌体重悬液中添加50 μmol/L乙酰丁香酮,共培养时间3 d,抗性芽的获得率最高.对再生植株进行GUS检测发现有45%的植株呈阳性.     

根癌农杆菌对巴戟天遗传转化的影响因素

以巴戟天带节茎为材料,研究了根癌农杆菌对巴戟天遗传转化的影响因素。结果表明:外植体感染前先进行2 d预培养,对转化有一定促进作用;外植体与农杆菌共培养时间以3 d为宜;乙酰丁香酮能提高转化效率,抗性芽分化率可达18.0％;外植体与农杆菌共培养后延迟4 d选择,抗性芽分化率有所提高;硝酸银能抑制外植体表面农杆菌的生长,提高GUS阳性芽的比例,硝酸银浓度2 mg/L时,GUS阳性芽比例最高(42.9％)。     

农杆菌介导的紫茎泽兰遗传转化研究

以紫茎泽兰为受体材料,GUS基因为报告基因,对农杆菌介导的遗传转化条件及影响因素进行研究,建立了农杆菌介导的紫茎泽兰遗传转化体系。结果表明,将未经过预培养的幼叶外植体在OD600为0.4的稀释菌液中浸泡12min,共培养3d后,转移到加有卡那霉素50mg/L(筛选压)和羧苄青霉素250mg/L的分化培养基上,经过20d外植体直接分化出不定芽,诱导生根成苗。经PCR和组织化学染色鉴定,可稳定获得较高的阳性转化率。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率,将一个植物的内含子插入卡那霉素抗性基因编码区的N端。合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达载体pYP1202并转化烟草,受外植体在含卡那霉素50－200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比较仅有26.7％,在200mg/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

受MeJA诱导的烟草GTs基因启动子的克隆及转化

中南民族大学生命科学学院生物技术国家民委重点实验室袁磊。刘学群。王春台利用已克隆的1个甲基茉莉酸（MeJA）诱导的糖基转移酶（GTs）基因,以PCR扩增得到该基因启动子序列,其长度约为1．4kb。用改造后的双元载体pCAMBIA1301与GTs基因启动子连接,构建了含目的启动子与GUS基因的融合基因质粒pCAP—GUS,通过根癌农杆菌介导的烟草叶盘转化法转化烟草W38后,     

农杆菌介导红江橙遗传转化的研究初报

用红江橙实生苗的上胚轴为材料 ,初步研究以根癌农杆菌介导的 GUS基因转化。结果表明 :以卡那霉素作为选择试剂进行选择培养时 ,Km浓度为 5 0 mg/L;外植体以平放为好 ;抑菌剂选择头孢霉素较好。GUS基因瞬时表达检测 ,70 .4%的外植体呈阳性反应 ;GUS基因稳定表达检测 ,在获得的 1 2株抗性植株中 ,GUS反应呈阳性所占比例为 1 6.7%。     

秦美猕猴桃叶片最佳再生系统的建立

利用正交等试验,系统开展了秦美猕猴桃叶片再生系统建立研究,确定了秦美猕猴桃叶片不定芽诱导最佳培养基及激素配比为MS＋6－BA 5mg/L NAA 0.1mg/L,不定芽诱导生根最佳培养基及激素配比为1／2MS＋NAA 0．01mg/L IBA0.5mg/L GA 1mg/L,该实验结果为通过叶盘法开展农杆菌介导的猕猴桃遗传转化奠定了良好基础。     

PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-ipt嵌合基因导入马铃薯栽培品种,对影响马铃薯遗传转化的多种因素进行系统研究.结果表明:马铃薯茎段分化效率高于叶片,马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/L,添加1%Na2SO3能有效防止褐化;茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12和ipt双重PCR检测再生植株,阳性转化率为65.8%.Southern blotting结果表明,转基因植株多以单拷贝形式整合进马铃薯基因组中.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia crassicarpa</Emphasis> via organogenesis

A protocol was developed for Agroacterium-mediated genetic transformation of Acacia crassicarpa via organogenesis by using in vitro phyllode (leaf) as the explant. Phyllode (leaf) explants were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI101 (harboring antisense Pt4CL1 with respect to the Pt4CL1P promoter). The selection for transgenic shoots was performed through two consecutive steps on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators and antibiotics in the following order: 0.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 300 mg/l carbenicillin (Car) and 20 mg/l kanamycin (Km) for 10 days; 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days; 0.5 mg/l indole-3-butyric acid (IBA), 100 mg/l Car and 20 mg/l Km 50 days. 21.7% of nodules produced multiple adventitious shoot buds, of which 27.7% survived in initial selection. The shoot buds were subjected to repeated selection on MS medium supplemented with 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.5 mg/l IBA, 100 mg/l Car 20 mg/l Km 50 days. Genomic PCR analysis confirmed the incorporation of the antisense Pt4CL1 with respect to the Pt4CL1P promoter fragment into the host genome.     

Plant Regeneration and Transformation of Sweet Papper (Capsicum frutescens)

Three strains of sweet pepper, (Capsicum frutescens) “Shuang Feng”, “Zhong Jiao No. 2” and “Zhong Jiao No.3” were screened out of six Chinese cuhivars for their high capacity of regeneration. The normal flowering and fertile regenerated plants have been obtained from cotyledons of seedlings from 10 to 16 days old by a four-step culture procedure; short induction, shoot elongation, rooting of excised shoot and transplanting into soil. MS was the basal medium in all steps, supplemented with different kind and different concentration Of phytohomores. Optimal shoot ioduction medium is MS +4–6mg/LBA+0.5mg/LIAA which gives rise a shoot regeneration frequency of 100%. 35% of the induced shoots elongated on the medium of MS+2mg/L Zeatin or 2mg/L. BA+l–3mg/L GA, and subsequently rooted on MS medium or in addition of 0.1–0.5mg/LNAA. The regenerants were transplanted into soil and developed into normal plants. In the transformation of sweet pepper using the leaf disc method, two kinds of wild type Agrobacterium tumefaciens, C58 and GV3111, have been screened out in regard to their high infection capacity. The transient expression of GUS gene was detected and Kanamycin-resistant shoots from infected cotyledons have been obtained. Further assay and transfering the TMV-resistant and CMV-resistant genes into sweet pepper are in progress.     

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

Summary An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog basal medium - LSD least significant difference - NOS nopaline synthase promoter - NPTII neomycin phosphotransferase (EC 2.7.1.95) - CaMV35S cauliflower mosaic virus promoter - GUS beta-glucuronidase (EC 3.2.1.31) - LB Luria Broth base - CTAB hexadecil trimethyl ammonium bromide - PCR polymerase chain reaction - X-gluc 5-bromo-4-chloro-3-indolyl-glucuronide     

一种快速有效的741杨离体叶片再生芽方法

在对杂交品种741杨(Populus alba(P.davidiana×P.simonii)×P.tomentosa)进行农杆菌介导法转基因的试验中发现了一种快速有效的叶片再生芽的方法.首先叶片外植体在培养基Ⅰ(MS培养基添加0.5 mg/L BA和1.0 mg/L 2,4-D)上培养2～3 d,再转移到培养基SH(MSmedium containing 2.0 mg/L of BA and 0.1 mg/L of NAA)上培养10 d,然后再转移到培养基Ⅱ(MSmedium with 0.5 mg/L of BA)上,培养大约5 d之后86.7%的叶片外植体产生的芽,每片叶片外植体(1 cm×1 cm)可产生40～50个芽.但是,如果叶片外植体在培养基Ⅰ上培养的时间长于5 d,再依次转移到培养基SH和Ⅱ上,则叶片会产生大量根.     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.)

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.     

银柴胡离体培养与毛状根诱导技术初步研究

以银柴胡茎段为外植体,经消毒获得无菌再生材料后,筛选发根农杆菌介导毛状根诱导产生的最适条件。结果显示：最适的无菌消毒方法为：70%酒精浸5 s,0.1%升汞消毒3 min,获得了银柴胡离体培养材料;以发根农杆菌A4菌株介导的银柴胡毛状根诱导过程中,与叶片和不带腋芽茎段相比,带腋芽茎段为最适转化外植体,用OD600=0.8的菌液侵染茎段15 min,共培养3 d,800 mg/L头孢噻肟钠除菌,其诱导率及诱导密度最高,分别为100%和4.7,为最适诱导条件。研究结果说明在适合条件下,银柴胡带腋芽茎段适于诱导毛状根。