双价抗虫基因陆地棉转化植株的获得

利用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ（ＳｍｉｔｈｅｔＴｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导法将含有豌豆外源凝集素（ｐｅａｌｅｃｔｉｎ,ＰＬｅｃ）基因和大豆Ｋｕｎｉｔｚ型胰蛋白酶抑制剂（ｓｏｙｂｅａｎＫｕｎｉｔｚｔｒｙｐｓｉｎｉｎｈｉｂｉｔｏｒ,ＳＫＴＩ）基因的双价抗虫基因植物表达载体ｐＢｉｎＬＫ用于陆地棉（ＧｏｓｙｐｉｕｍｈｉｒｓｕｔｕｍＬ．）栽培品种“新陆早１号”、“新陆中２号”、“冀合３２１”和“辽９”的转化。棉花无菌苗下胚轴经过与根癌农杆菌共培养、卡那霉素抗性愈伤组织的筛选、体细胞胚状体的诱导和植株再生等阶段成功地获得了双价抗虫基因陆地棉转化植株。ＮＰＴⅡ的ＥＬＩＳＡ检测、ＰＣＲ鉴定和ＰＣＲＳｏｕｔｈｅｒｎ检测证实,两个外源抗虫基因同时存在于再生植株基因组内。抗虫测试结果表明,转基因棉株对棉铃虫（ＨｅｌｉｏｔｈｉｓａｒｍｉｇｅｒａＨｕｂｎｅｒ）幼虫具有较强的抗性     

陆地棉栽培品种转复合启动子控制下的cry1Ac3基因获得高效抗虫植株(英文)

以根癌土壤杆菌 (Agrobacteriumtumefaciens)介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉(GossypiumhirsutumL .)栽培品种“新陆早 1号”、“晋棉 7号”、“晋棉 12号”和“冀合 32 1”。pBinMoBc携带有高效启动子复合OM启动子控制下的cry1Ac3基因 ,pBinoBc携带有 35S启动子控制下的cry1Ac3基因。经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导 ,得到了再生植株。对T2 代的PCR、Southernblotting、ELISA检测及Westernblotting证明cry1Ac3基因已整合入受体棉花基因组并得到表达。抗虫性检测表明转基因后代对棉铃虫 (Heliothisarmigera )具有良好的抗性 ,转pBinMoBcT2 代与转pBinoBcT2 代相比 ,对棉铃虫具有更快的致死速度。本研究建立了一套高效的陆地棉栽培品种转化体系 ;进一步的检测结果表明 ,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性。     

陆地棉栽培品种转复合启动子控制下的cry 1Ac3基因获得高效抗虫植株

以根癌土壤杆菌( Agrobacterium tumefaciens )介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉( Gossypium hirsutum L.)栽培品种"新陆早1号"、"晋棉7号"、"晋棉12号"和"冀合321".pBinMoBc携带有高效启动子复合OM启动子控制下的 cry 1Ac3基因,pBinoBc携带有35S启动子控制下的 cry 1Ac3基因.经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导,得到了再生植株.对T2代的PCR、Southern blotting、ELISA检测及Western blotting证明 cry 1Ac3基因已整合入受体棉花基因组并得到表达.抗虫性检测表明转基因后代对棉铃虫( Heliothis armigera ) 具有良好的抗性,转pBinMoBc T2代与转pBinoBc T2代相比,对棉铃虫具有更快的致死速度.本研究建立了一套高效的陆地棉栽培品种转化体系;进一步的检测结果表明,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性.     

农杆菌vir基因诱导因子研究进展

在众多遗传转化法中,农杆菌(Agrobacterium tumefaciens)介导法以易操作、低费用、插入片段明确、拷贝数低等独特优点成为植物遗传转化的首选。然而,至今仍有许多物种不能被农杆菌转化。研究表明,农杆菌的转化能力是由位于染色体基因组之外Ti质粒上的vir基因决定的。在所有vir基因中,除virA和virG组成型表达外,其它vir基因的表达均需酚类化合物的诱导;糖类物质可增强酚类化合物对vir基因的诱导;低磷酸和酸性pH环境也可促进vir基因的诱导表达。文章论述了酚类化合物、糖类物质、低磷酸、酸性pH和培养温度等因素对农杆菌vir基因诱导表达的影响,以期为更好地利用这一天然载体及为提高转化效率提供依据。     

利用根癌农杆菌法获得转基因水稻植株及其后代

在100μmol／L乙酰丁香酮（AS）等vir基因诱导分子存在的情况下,用含双元载体pBYT2的根癌农杆菌菌株EHA101同水稻（OrizasativaL．）台北309悬浮培养细胞共培养3天。经过2个月的连续筛选,共从364颗同根癌农杆菌共培养的悬浮培养细胞团中得到17个具有稳定潮霉素抗性和GUS表达的愈伤组织。对从8个转化组织中得到的10株可能的R0代转基因植株及其后代进行外源基因的整合和表达分析,Southern分析表明外源基因已稳定地整合进水稻基因组中并实现了有性遗传传递。杂交结果显示在其中一个转化系的植株中有5个拷贝的T－DNA整合,而其余的转化系则只整合了1个拷贝。转基因水稻细胞及植株中GUS活性的组织化学染色观察和荧光分析表明玉米ubiquitin基因启动子在水稻细胞中能高效启动gus报告基因的表达。ndPAGE－X－Gluc法检测表明转基因水稻细胞中表达的GUS蛋白比Sigma公司的标准GUS蛋白（SigmaCo．G0786）要小,而与来自大肠杆菌HB101（pBI1121）中的GUS蛋白大小相同。结果表明,根癌农杆菌可有效且可靠地介导外源基因转化水稻。     

转三价抗虫基因彩色棉的获得

采用农杆菌介导法将外源三价抗虫基因Bt-CpTI-GNA导入彩色棉中,经分子检测已整合到受体植株体内,并已得到表达。目前已得到纯化株行。外源标记基因NPTII检测在T。代就有分离现象,各代检测淘汰3～4次才能基本清除分离株或非转化株。抗虫性检测表明转三价抗虫基因彩色棉对棉铃虫具有较高抗性,而对棉蚜则作用小大。     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Transformation of Populus tomentosa with Insecticidal Cowpea Proteinase Inhibitor Gene

Cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene, was introduced into poplar ( Populus tomentosa Carr. ) by gene transformation mediated by Agrobacterium tumefac/ens (Smith et Townsend) Conn. The influences on regeneration and transformation frequency of poplar by the concentration and addition of kanamycin were compared. Kanamycin resistant (Kmr) plantlets were obtained by 3 -4 cycles screening in selective condition. The ability of leaf regeneration and shoot subculture and rooting from the transformed and non-transformed plants in the presence of 50 mg/L kanamycin was examined. The presence of CpTI gene in the transgenic plants were confirmed by PCR and PCR-Southern blot. Assay on proteinase inhibition activity demonstrated that leaf protein extracts of the transgenic poplar showed higher inhibition activity against trypsin than that of control plants.     

转基因棉花外源基因的遗传

选用不同来源的转Bt基因抗虫棉中棉所30号和新棉33B、转tfdA基因抗除草剂棉花材料TFD,通过与不同主栽推广品种正反交,研究了外源Bt基因和tfdA基因在棉株体内的遗传和分离行为。结果表明,由于外源基因提供给转基因棉花的抗虫和抗除草剂性状均是由一对于基因控制的显性性状,且该性状不受细胞质效应的影响,符合孟德尔遗传规律。     

矮秆陆地棉品种陆矮1号的生长发育特性研究

本研究以矮秆陆地棉品种陆矮1号为材料,以株高正常品系97004H和陆地棉遗传标准系TM-1为对照,通过田间试验,比较研究了不同品种的生长发育规律。结果表明：陆矮1号苗期形态与对照品种无显著差异,随着生育进程株高差异逐渐增大,现蕾后主茎日增长量极显著低于对照品种,日增长高峰与对照品种一致,但高峰期的日增长量则不到对照品种的一半,盛花期以后主茎基本停止生长;最终株高只有对照品种的1／2左右。单株果枝数及主茎伸长节间数显著少于97004H和TM-1,每果枝的果节数及果枝长度与对照相比有极显著差异,主茎第12节以下节间长度与对照品种无显著差异,上部节间显著短于对照品种。株高差异主要发生在盛蕾期以后,由中上部节间缩短引起。陆矮1号生育期较短,结铃部位和成铃时间集中。     

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance. Received: 31 March 1996 / Revision received: 2 July 1997 / Accepted: 18 July 1997     

通过构建三段T-DNA载体高频率获得无标记转基因大豆植株的研究

高频率获得无选择标记转基因植株有利于转基因植物的环境释放和安全性生产,农杆菌介导的共转化法是获得无标记转基因植株的方法之一。含二段T-DNA载体的共转化法已被人们成功应用,而二段以上T-DNA载体的共转化法还未见报道。基于这一目的,通过几个中间质粒构建了含有三段T-DNA的双元表达载体pNB35SVIP1,其中包含1个拷贝bar基因选择标记基因表达盒和2个拷贝VIP1目的基因表达盒。利用EHA101农杆菌菌系介导法转化大豆子叶节,经过在含3~5mg/Lglufosinate培养基上多次筛选,获得了一定数量抗性再生植株,然后对抗性再生植株进行叶片涂抹除草剂、Southernblot和Northernblot检测,共鉴定出51棵T0代转基因植株,转化频率0·83%~3·16%,二个基因的共转化频率为86·4%。在对T1代群体进行叶片涂抹除草剂检测的基础上,不抗除草剂植株进行PCR、Southernblot和Northernblot检测,共鉴定出41棵无选择标记转基因植株,无标记植株获得率为7·6%。检测结果还表明,T1代群体中22·7%的株系发生了基因丢失现象,27·3%的株系发生了bar基因沉默现象,目的基因在37·1%的无标记植株中发生了沉默现象。三段T-DNA的双元表达载体是获得无标记转基因植株的理想途径。     

棉花高品质纤维性状QTLs的分子标记筛选及其定位

利用7235、TM-1亲本(P1、P2),以及(7235×TM-1)F1、F2(南京和美国2个环境)与F23(南京和海南2个环境)家系群体,根据F2与F23的纤维品质性状表现,构建了纤维强度、细度与长度的极值DNA混合池,通过221对SSR引物、1840个RAPD引物对亲本和极值DNA混合池筛选,共得到了13个多态性标记,其中8个标记可能与高强有关,1个标记与低强有关;3个标记与麦克隆值有关;1个与绒长有关.进一步通过F2分离群体检测,连锁分析表明与高强有关的8个标记(2个SSR标记和6个RAPD标记)紧密连锁,覆盖15.5cM.这一高强纤维的QTL,4个环境中均以FSR1933为最近,相距不超过0.6cM,能解释35%的F2变异,53.8%的F23的表型变异,是目前纤维强度单个QTL效应最大的,多个环境下稳定,可以直接用于标记辅助育种.单体测验表明,该在棉花的第10染色体上.麦克隆值的一个主效QTL标记FMR1603,在F2中能解释7.8%的变异,在F23中能解释25.4%的变异,同样表现环境稳定.纤维长度的一个标记FLR11550,在3个环境中预测到,最大能解释9.5%     

农杆菌介导籼稻优良恢复系bar基因的遗传转化研究

应用农杆菌介导转化体系,成功地将含有CaMv35s启动子启动的bar基因导入籼稻幼胚来源的愈伤组织,获得籼稻优良恢复系T461、R402和752三个品种（系）共47个抗除草剂Basta的转基因株系,Southem分析结果表明,转基因植株基因组中检测到bar基因的整合,转基因植株自交后代Basta除草剂抗性鉴定表现出分离,且大多数为1-2个整合位点的孟德尔方式遗传。结果表明,根癌农杆菌介导法可以有效且可靠地转化籼稻。     

转SAMS基因玉米自交系获得及抗旱性分析

以玉米自交系‘掖478’的茎尖为受体,采用农杆菌介导法将小麦抗旱SAMS基因转入玉米中,用PCR和RT-PCR法对转化玉米进行检测,并以18%PEG-6000模拟水分胁迫对T1代转基因玉米和非转基因玉米进行抗旱性分析。结果显示:(1)共转化518个玉米芽,得到453株转化苗。获得T0代阳性植株16株,转化率为3.53%,有14株自交结实,经RT-PCR检测,T1代转基因玉米有9个株系为稳定遗传阳性株系。(2)在同一水分胁迫时间下,转基因玉米的叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性均高于非转基因玉米,而转基因玉米叶片的电导率、MDA含量均低于非转基因玉米。在60 h水分胁迫处理下,转基因玉米叶片相对含水量、叶绿素含量、脯氨酸含量和SOD活性比非转基因玉米上升8.16%、20.02%、32.21%、22.77%,而转基因玉米叶片的电导率、MDA含量比非转基因玉米下降14.38%、29.41%。研究表明,通过导入SAMS基因,可以提高玉米的抗旱性。     

根癌农杆菌介导的转aroAM12基因棉花植株的草甘膦抗性

以中棉35无菌苗下胚轴为外植体,采用农杆菌介导法将含有通过基因优化技术获得的草甘膦抗性突变基因aroAM12导入棉花中.以aroAM12为选择标记,利用草甘膦直接筛选获得65棵再生植株.PCR和Southerablot分析表明,经过草甘膦筛选出的To代植株均整合有aroAM12基因.Western blot分析表明整合进的aroAM12基因得到了有效表达,且不同植株之间的表达不尽相同.大棚喷洒的实验结果表明To代转化植株具有很高的草甘膦抗性.对T1代棉花的草甘膦抗性遗传分析表明,aroAM12基因以孟德尔方式遗传.     

转新型双抗虫基因棉花的遗传分析

首次用含合成的BtCrylAc活性杀虫蛋白嵌合基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体,通过土壤根癌杆菌介导转化棉花品种冀合321,获得一批抗虫的转化再生棉花植株。利用叶片涂抹卡那霉素、叶片离体养虫和PCR扩增等检测方法对6个不同转双抗虫基因株系的抗虫性进行遗传分析。结果显示,转化株系自交的T1抗虫性状遗传较为复杂;农杆菌介导获得的转基因抗虫棉在早期世代不易选到纯合系,但是随着对抗虫性状进行单向选择,到T4和T5就能获得抗虫纯合系。利用抗虫性稳定的转化后代材料和转化受体进行田间杂交,发现F2抗虫性分离完全符合一对或两对显性基因的分离规律,并证明了DR248和DR193两个材料为外源基因双拷贝插入。转化株系的Southern杂交也证明了上述结果。     

转双抗虫基因杂种741毛白杨的研究

用部分改造后的苏云金芽孢杆菌 (Bt)杀虫蛋白基因和慈菇蛋白酶抑制剂 (API)基因A构建了植物表达载体。然后通过根癌土壤杆菌 (Agrobacteriumtumefaciens (SmithetTownsend)Conn .)介导将此表达载体上的双抗虫基因转入杂种 741毛白杨 [PopulusalbaL .× (P .davidianaDode P .simoniiCarr.)×P .tomentosaCarr.]获得了一批抗卡那霉素的转化再生植株。用杨扇舟蛾 (Closteraanachoreta (Fabricius) )进行虫试的结果表明有 3株抗虫杨树 ,其中有1株杨树的叶片可使试虫在 6天内的死亡率达 90 %以上 ,而且存活幼虫的生长发育受到了明显的抑制。PCR检测及基因组DNASouthern杂交分析的结果都表明Bt杀虫蛋白基因和API基因已整合到以上 3株抗虫杨树的基因组中 ,而且表现为单拷贝整合。用Bt毒蛋白抗血清进行滤膜免疫反应及ELISA检测结果表明 3株转基因杨树都有Bt杀虫蛋白的表达 ,表达量约占叶总可溶性蛋白的 0 .0 15 %。这是国内外首次报道用双抗虫基因获得的抗虫 741毛白杨植株。     

农杆菌转化获得转B．t．基因水稻及其生物学鉴定

以水稻主栽品种"秀水11"和"春江11"预培养4d未成熟胚为转化受体,经农杆茵LBA4404／pGBI4A2B(含B．t．基因)感染后,筛选出抗性愈伤组织并获得转化植株。其中抗性愈伤组织产生频率达44％～70％,转化植株产生频率达27％～64％。转基因植物总DNA经PCR和Southernblot分子杂交试验表明,T-DNA上的外源基因已整合到水稻的基因组里。对转B．t．基因水稻植株进行了两种水稻主要害虫纵卷叶螟和二化螟的饲虫试验。与对照相比,7d后纵卷叶螟幼虫死亡率达31％～49％,同时纵卷叶螟对转基因水稻叶片的危害程度大大减轻。二化螟在咬食转B．t．基因水稻植株7d后的校正死亡率达15％～71％,30d后多数转基因水稻仍能正常抽穗,表明转基因水稻对上述害虫具有一定抗性。     

Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5

This report describes the disarming of Agrobacterium tumefaciens Chry5, a strain highly tumorigenic on soybean. Disarming was achieved by removing an approximately 16.5-kb segment of the 285-kb Ti plasmid pTiChry5, including approximately 4 kb of the oncogenic T-DNA and an extended region right of the T-DNA, and replacing it with a gene for carbenicillin resistance, through homologous recombination. The deletion was confirmed with Southern analysis, and the loss of tumorigenicity was verified in tobacco and tomato plant stem inoculation assays. The deletion mutant, named KYRT1, successfully transferred the β-glucuronidase (GUS) gene into tobacco leaf tissue, producing GUS-expressing callus which could be regenerated into viable plants. In a comparative study, the transformation efficiency of A. tumefaciens KYRT1, GV3850, and EHA105 was assayed by inoculating cotyledonary node explants. The results of this study revealed that, in a binary vector system, KYRT1 is equally or more effective than EHA105 or GV3850 at delivering DNA into soybean. Received: 30 January 1997 / Revision received: 10 June 1997 / Accepted: 5 July 1997