转抗菌肽B基因和bar基因籼稻植株的再生

 运用基因枪法将含有bar基因和cecropin B基因的质粒pCB1导入籼稻品种特青的幼胚细胞,筛选后得到3株转基因植株。PCR检测和Southern杂交结果表明,外源基因已整合到水稻基因组中。转化当代植株表现出对Basta很强的抗性,同时也增强了对水稻白叶枯病的抗性。     

转基因水稻经花药培养获得纯系的研究

 利用基于苯乙酸的一步成苗法,获得了来源于多个品种转基因水稻当代植株的花培苗上百株。对其中两个含bar基因插入的转基因粳稻当代植株京引119-B3和京引119-B4的花培植株及其后代进行了详细的研究分析。bar基因在所获得的单倍体和二倍体花培植株中均可正常表达出对除草剂Basta的抗性。PCR、Southern分析进一步证明导入的bar基因在绝大多数花培植株中没有发生变化。即使对粳稻京引119-B4这一有多位点插入的转基因植株,花药培养也可达到使外源基因一次纯合、后代不再分离的目的。结果表明对转基因植株进行花药培养可用于快速纯合外源基因以获得纯系。并就这一技术体系的技术要点进行了讨论。     

基因枪转化的bar基因表达盒在水稻中的重组结构

应用Southern杂交和引物组合PCR技术研究了基因枪转化的bar基因表达盒（包括启动子、编码区和终止子）在水稻（Oryza sativa L.）中的重组结构。bar基因表达盒的多个拷贝通过重组连接形成1～3个转基因串联子,彼此邻近整合在受体基因组内一个较大的染色体区域,不同转基因串联子之间由水稻基因组DNA间隔,形成转基因簇。bar基因表达盒形成转基因串联子时,存在头接头、头接尾、尾接尾3种连接方式,通常伴随着bar基因片段的截短。     

花粉管通道介导的抗除草剂基因(bar)对大豆的遗传转化

采用花粉管通道介导法将含有bar基因的pPTN140质粒DNA导入8个黑龙江省大豆主栽品种（系）,导入1384朵花,共获得1164粒种子,经2次Basta除草剂筛选后共获得3个品种的除草剂抗性植株8株.对其进行PCR以及PCR-Southern杂交检测均为阳性结果,Southern杂交结果显示,8个除草剂抗性后代植株整合数目不同,其中2株整合单拷贝基因,另外6株分别整合3～8个拷贝的bar基因.对8个抗性植株的后代进行了除草剂抗性分析,D1-D3代均出现除草剂抗性植株,表明bar基因可以在转基因植株后代中遗传,抗性遗传分析结果表明不同株系间以及株系内除草剂抗性缺乏规律性,分离比率不符合孟德尔遗传规律.说明大豆花粉管通道法转化基因存在多拷贝共抑制现象.目前获得除草剂抗性稳定的D3代株系82个.试验说明利用花粉管通道技术进行大豆转化是可行的.     

CbDREB1A基因表达载体构建及转化水稻光温敏核不育系的研究

为了增强荠菜DREB1A基因在转基因水稻中的表达,构建了由Ubi启动子驱动的植物表达载体pUΩCbDREB3300.通过基因枪转化法将CbDREB1A基因导入水稻光温敏核不育系4008S,获得5个抗干旱胁迫的再生植株.运用PCR及Southern杂交技术对抗性再生植株进行鉴定,结果显示CbDREB1A基因已整合到水稻基因组中.干旱胁迫后转基因水稻植株脯氨酸含量显著高于对照,显示耐旱性增强.     

两种不同的水稻标记基因删除技术的比较

Cre/loxP位点特异性重组系统是一种利用cre重组酶的瞬时表达来调控位于两个loxP位点之间DNA序列的删除系统。以反义LOX-3基因作为目的基因,以bar为选择标记基因,应用热激启动子驱动的Cre/loxP系统构建标记基因诱导删除型载体,得到转基因植株后,对比两种不同的热激方法中选择标记基因bar的删除效率,发现以生长到四叶期的转基因水稻植株为热激材料,标记基因删除效率较高,达到83.3％,因此,确定此方案为水稻标记基因删除的最佳方案。     

转WYMV-Nib8基因抗黄花叶病小麦的鉴定及优良株系的选育

小麦黄花叶病是我国冬麦区亟待解决的重要病害之一。为了给应用病毒复制酶基因介导的转基因抗病小麦新品种（系）选育提供依据，对利用基因枪法将WYMV-Nib8基因和bar基因共转化扬麦158获得的15个T2代株系进行了黄花叶病抗性鉴定，筛选出4个高抗病株系和1个剔除bar基因的高抗病株系；连续多年抗病性鉴定结果表明，通过转基因技术获得的5个株系不但抗病性优良而且可以稳定遗传。以转基因材料为亲本之一，通过常规杂交、回交获得1个兼抗小麦黄花叶病和白粉病且农艺性状优良的新品系BR1014。     

转抗除草剂基因小麦植株的筛选方法研究

为了降低小麦转bar基因时产生较高比例的假阳性植株,采用测定T0代转基因植株叶片释放NH4 浓度和对T1代植株喷施Basta除草剂等辅助筛选方法,建立了简便有效的转bar基因小麦筛选体系。研究结果表明,2叶期和5叶期叶片释放的NH4 浓度与bar基因PCR检测结果呈极显著相关,相关系数分别为0.86和0.75。对391株T0代再生植株进行叶片释放NH4 浓度测定,筛选到34株转基因植株,其中27株为bar基因的PCR检测所证实。用100 mg/L Basta除草剂对381株T0代植株的后代(3 800余株)进行喷施筛选,根据后代抗性分离筛选到13株T0代阳性植株,与PCR检测结果吻合度达到100%。这两种方法可用于以bar基因为选择标记基因的转基因植株的筛选。     

转cry 1Iem基因大豆的培育及抗虫性检测

以大豆子叶节为外植体,应用农杆菌介导法,将抗虫(cry1Iem)基因转化大豆.筛选标记为bar基因.经Glufasinate筛选,获得大量抗性植株.对转基因T_0、T_1、T_2代植株进行PCR检测,初步证明cry1Iem基因已经整合到大豆基因组中.对T2代PCR阳性植株幼嫩豆荚,采用圆盘分隔法接人初孵幼虫,进行初步的抗虫性检测,得到1株具有明显抗虫效果和7株抗虫效果较好的转基因植株.     

抗除草剂转基因水稻稳定系TR4的获得及其遗传研究

 用Bar基因通过基因枪法轰击京引119未成熟胚,得到抗除草剂Basta的转基因植株,经加代和选择得到抗除草剂的稳定系TR4,其基本农艺性状与京引119相仿。用TR4与4个粳稻品种杂交进行遗传分析表明,TR4的抗除草剂特性由一对显性基因控制,符合孟德尔遗传规律。该外源基因可通过有性杂交转育,在杂交稻和常规直播稻的选育上有较大的利用价值。     

Allelic Analysis for bar Gene from Different Rice Transformation Events

Abstract

Mechanism(s) of gene transformation and integration in rice (Oryza sauva L.) is/are not currently well understood. This research was conducted to determine whether a transgene is inserted into the rice genome specifically or randomly. Seven homozygous transgenic Taipei (T) 309 and Nipponbare plants with the bar transgene from different rice transformation events were crossed. The segregation of F2 and F3 populations from a total of 21 crosses was studied in a greenhouse and field to determine if the genes were allelic or non-allelic. Five genomic locations appeared to be involved among the seven transgenic plants. An additional 20 homozygous transgenic T309 plants, with the bar transgene from different transformation events, were crossed reciprocally with the previous seven plants. One hundred and fifteen crosses made during 1999 and 2000 were analyzed for allelism. In some combinations, the genes were allelic, but most of them were non-allelic, with two or more pairs of genes being expressed. Twenty loci among the 27 transgenic plants were involved and some plants had several inserted genes expressed. Genes in nine out of 27 transformed plants were allelic. We concluded that the functional foreign (bar) gene was restrictively/preferentially inserted into the rice genome in some cases and was not completely randomly inserted and expressed in the rice genome. If the mechanism(s) for preferential insertion were identified, rice researchers could possibly control insertion sites of transgenes to optimize gene expression.     

农杆菌介导的转ICE1基因提高水稻的耐寒性

利用农杆菌介导的转基因技术,成功地将通过RT PCR克隆的拟南芥ICE1基因导入垦鉴稻10号中,经PCR和Southern检测确认目的基因已整合到水稻基因组中。潮霉素抗性测定结果表明,与未转基因水稻相比,T1代表现出对潮霉素较高的抗性和孟德尔式的单位点遗传。抗寒能力检测结果表明,在同等低温胁迫条件下T1代转基因株系的死亡率明显低于未转基因对照。脯氨酸含量增幅明显高于未转基因对照。上述结果表明,转ICE1水稻提高了抗寒能力。     

转基因与常规杂交相结合改良水稻耐盐性

通过农杆菌介导法和基因枪法将CMO、BADH、mtlD、gutD和SAMDC基因以单价或双价的形式导入水稻常规品种中,再结合常规杂交育种,选育5价强耐盐性的转基因水稻植株。1~5价9类组合的水稻品系,经PCR分子检测,在转基因后代中多价目的基因聚合,遗传稳定,且分子检测与田间耐盐性的表现一致。转基因植株在盐碱地中能正常生长,拓展了水稻常规品种耐盐性。并已获得耐0.5%~1.0%NaCl的T秀水11——品3、品6和品7等9份优良株系或中间材料。     

转hrf1基因水稻对稻瘟病多小种非专化的稳定抗性

以表达水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）编码harpin广谱抗性激发子的hrf1基因的转基因系为材料,在温室和田间病圃鉴定它们对稻瘟病菌的抗性,分析转基因水稻抗稻瘟病的作用机制。研究结果显示,转hrf1基因水稻对稻瘟病菌ZC3、ZD1和ZG1小种表现高抗,对ZB13表现中抗,表明hrf1基因在水稻中表达可以产生非小种专化抗性。在稻瘟病圃中对水稻稻瘟病抗性鉴定结果显示,转hrf1基因水稻在T1、T3、T5和T7代对稻瘟病菌都表现很好的抗性,表明转hrf1基因水稻对稻瘟病菌的抗性能稳定遗传。 转hrf1基因抗病水稻中防卫反应基因OsPR1a、OsPR1b、PAL和Chia4a以及正向调控水杨酸介导信号传导的NPR1基因的表达显著增强。转基因抗病水稻中硅含量显著提高。Harpin编码基因在水稻中表达,可能通过激发水稻中防卫反应基因的表达,提高水稻中硅含量等,从而使水稻产生对稻瘟病菌的广谱抗性。     

四个云南水稻品种对白背飞虱的抗性遗传分析

 分析了鬼衣谷、便谷、大齐谷备和大花谷四个籼型云南地方品种的单显性抗白背飞虱基因与与Wbph3、Wbph5间，以及四个云南地方品种抗性基因间的等位性关系。结果表明，鬼衣谷等四个品种所携带的单显性抗虫基因均与Wbph3和Wbph5间为非等位关系；鬼衣谷、便谷、大齐谷和大花谷各所携带的单显性抗虫基因间互为等位关系。在鬼衣谷等四个云南地方品种中发现了一新的抗白背飞虱抗性基因，暂将这一新的抗性基因定名为Wbph6(t)。     

Allelic Effects on Eating and Cooking Quality of SSⅡa and PUL Genes Under Wxmp Background in Rice

【Objective】Our aim is to analyze the genetic effects of soluble starch synthase gene SSⅡa and debranching enzyme gene PUL on eating and cooking quality under the same main gene background of Wxmp, so as to lay a theoretical basis for rice quality improvement. 【Method】In this study, a semi-glutinous rice line Ning 0145 and japonica rice variety Wuyunjing 21 were crossed to obtain F2 population and F3 lines. There was polymorphism in soluble starch synthase gene SSⅡa and debranching enzyme gene PUL but no polymorphism in other starch synthase related genes between the two parents. With molecular markers, some F2 plants and F3 lines containing Wxmp gene were selected and divided into four genotypes, SSⅡanPULn, SSⅡanPULw, SSⅡawPULnand SSⅡawPULw(n and w indicated that the genes were contributed by Ning 0145 and Wuyunjing 21, respectively). The allelic effects of SSⅡa and PUL genes on eating and cooking quality under the same Wxmp gene background was investigated by analyzing the eating and cooking quality and its differences among different genotypes. 【Result】There were significant differences for eating and cooking quality among genotypes of different parental origins. The allelic gene SSⅡaw and PULw from Wuyunjing 21 increased amylose content by 0.29%–1.00% and 0.62%–1.18% respectively, and the effect of PUL was greater than that of SSⅡa. There was interaction between SSⅡa and PUL genes. The SSⅡaw and PULw genes also decreased gel consistency and breakdown viscosity, increased hot paste viscosity, cool paste viscosity, setback viscosity and consistency viscosity, but had little effect on gelatinization temperature, peak viscosity and peak time. 【Conclusion】The genetic effects of SSⅡa and PUL genes on cooking and eating quality of rice under the background of Wxmp gene were clarified. The results lay a theoretical basis for improving rice quality by molecular marker-assisted selection of SSⅡa and PUL genes.     

转bar基因水稻除草剂抗性遗传研究及其应用

试验用３个恢复系－测６４,明恢６３和特青作母本分别与３个抗除草剂转ｂａｒ基因水稻品种－Ｂｅｎｇａｌ－Ｈｕ１０,Ｃｙｐｒｅｓｓ ＰＢ－６和Ｇｕｌｆｍｏｎｔ杂交,对其后代（Ｆ１,Ｆ２和ＢＣ１）进行遗传分析表明,除草剂的抗性是受一对显性核基因控制,通过杂交可将这种基因转育到恢复系,配出抗除草剂杂交组合,应用这项技术可放宽对不育系完全雄性不育的严格要求,有利于优良不育系的培育及高产、优质,多抗新组合的选配