水稻种胚LOX3基因在逆境胁迫中的作用

LOX3是主要的水稻种胚脂氧合酶同工酶。为了研究水稻LOX3基因在逆境胁迫中的作用,构建了LOX3基因的反义植物表达载体,用农杆菌介导法转化水稻品种武运粳7号和Kasalath,获得了转基因植株。PCR和Southern鉴定证实基因已经导入水稻基因组中,种胚LOX3缺失鉴定和半定量RT PCR分析证实反义RNA抑制了LOX3基因的表达。对T2代转基因植株进行了水分胁迫处理及稻瘟病和白叶枯病的接种鉴定。结果显示,与非转基因对照相比,反义LOX3转基因植株对水分胁迫、稻瘟病和白叶枯病都表现敏感,说明水稻种胚LOX3基因在逆境胁迫反应中发挥一定的作用。     

Preliminary Study on Function of Calcineurin B-Like Protein Gene OsCBL8 in Rice

The homozygous T3 transgenic lines with sense OsCBL8 gene and antisense OsCBL8 gene obtained by agro-transformation were used to investigate the function of OsCBL8 in rice. Semi-quantitative RT-PCR showed that the expression of OsCBL8 extremely increased in sense transgenic lines, and decreased to some extents in antisense transgenic lines. Such up- and down-regulation of the OsCBL8 gene in these transgenic lines had little effects on main agronomic traits, but significantly decreased the number of filled grains per panicle and seed setting rate in some of transgenic lines. By evaluation of the tolerance to 150 mmol/L NaCl, 20% PEG6000 and low temperature treatments, and relevant physiological indices, 8F12, a sense transgenic line with high salt tolerance, and 8R14, an antisense transgenic line with high drought tolerance, were obtained, which suggests that the OsCBL8 gene is involved in the response of rice to abiotic stresses.     

根癌农杆菌介导的水稻转化系统的优化及转反义Wx基因植株的获得

以具不同直链淀粉含量的籼粳稻品种为受体材料,试验了适宜于根癌农杆菌介导转化的水稻愈伤组织诱导培养基及其诱导培养的时间。结果表明,一种基于MS基本培养基的商品培养基较适合作为籼粳稻幼胚愈伤组织的诱导培养基;对于籼型杂交稻亲本协青早B,转化前幼胚较适合的愈伤组织诱导培养天数为8 d左右。在此基础上,将反义蜡质（Wx）基因分别导入上述受体亲本中,获得了一批转基因水稻植株。PCR和Southern杂交分析表明反义Wx基因已经整合进了转基因水稻的基因组中。遗传分析表明外源基因在转基因水稻后代中的分离符合3∶1的理论比例。     

水稻类钙调磷酸酶B亚基蛋白基因OsCBL8功能的初步研究

 以转正义或反义OsCBL8基因的T3代株系及非转基因对照为材料, 研究了转OsCBL8基因对水稻农艺性状、内源OsCBL8基因表达和幼苗耐盐、耐旱及耐冷性的影响。结果表明, 供试的16个转基因株系的株高、单株分蘖数、单株有效穗数、穗长和百粒重等性状与对照无显著差异, 每穗实粒数和结实率则大都显著或极显著低于对照。部分转基因株系的半定量RT PCR结果表明, 转正义基因显著增强了植株中OsCBL8基因的表达, 而转反义基因则对内源OsCBL8基因的表达具有一定的抑制作用。在盐、干旱和低温等非生物胁迫条件下的生长实验发现, 1个转正义基因株系的耐盐性得到显著增强, 1个转反义基因株系的耐旱性得到显著增强。 讨论了OsCBL8基因在水稻耐非生物逆境方面可能的机理。     

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。     

应用Cre/loxP系统在转化细胞水平上高效删除转基因水稻的标记基因

 研究了Cre/loxP系统在转化细胞水平上删除转基因水稻中抗性标记基因的可行性和效率。采用农杆菌介导法将Cre/loxP标记基因剪切系统载体pNCG导入水稻细胞,用G418筛选法获得水稻抗性愈伤组织后,在组织培养不同阶段的培养基中添加25 μmol/L雌激素进行Cre基因的诱导表达和标记基因的剪切,PCR检测T0植株中标记基因nptⅡ、重组酶基因Cre和目标基因gusA的整合情况。将扩增结果为gusA（+）/nptⅡ（-）/Cre（-）的转基因植株统计为标记基因剪切成功的植株。结果表明,在抗性愈伤组织培养的预分化前、预分化和分化阶段添加雌激素诱导表达重组酶Cre均能成功切除标记基因序列,标记基因剪切成功率为6.82%~46.43%。在预分化前采用液体培养基添加雌激素诱导处理愈伤组织3 d,T0植株中标记基因的剪切效率高达173.33%,主要原因是雌激素处理提高了愈伤组织绿苗分化率。直接在分化培养基中添加雌激素诱导处理,T0植株中标记基因剪切成功率达46.43%,剪切效率（144.44%）也较高。表明雌激素诱导的Cre/loxP系统能在水稻抗性愈伤组织水平上实现对标记基因序列高效快速删除。     

OsBTF3过量表达和RNAi转基因水稻抗盐和抗低温胁迫鉴定

 为评估水稻OsBTF3基因在水稻对非生物逆境胁迫应答中的功能,利用RT Q PCR技术,对经高盐(200 mmol/L NaCl溶液)和低温(4℃)胁迫处理后水稻的OsBTF3基因表达动态进行了测定,并对前期获得的OsBTF3过量表达和RNAi转基因水稻株系进行了抗高盐和抗低温胁迫的鉴定。结果表明,高盐胁迫处理显著抑制了OsBTF3基因的表达,低温胁迫对其表达影响有一定的变化;转基因T2代过量表达株系对高盐和低温胁迫抗性显著增强,而RNAi转基因株系抗性减弱,说明OsBTF3可能在水稻对高盐和低温胁迫反应中起着重要的调控作用。     

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free (SMF) transgenic rice, two transformation methods were employed for four rice varieties (Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97). One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions (one carrying an antisense Wx gene and the other carrying a HPT gene). The other one, named as two-strain/ two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.     

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety (Digu) by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 (pZH01-tp309) and three different expression constructs, pCB-Pi-d25.3kb (pCB5.3kb), pCB-Pi-d26.3kb (pCB6.3kb) and pZH01-Pi-d22.72kb (pZH01-2.72kb) of Pi-d2, driven by Pi-d2 gene's own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.     

几个转bar基因水稻外源基因的遗传学行为研究

利用春江03等4个品种的转基因株及后代稳定系进行遗传研究表明,bar基因作为一个显性单位已整合在水稻核基因组中,转基因株稳定系TR3、TR4、TR5、TR6对除草剂Basta的抗性均受1对显性基因控制,且4个转基因株稳定系间bar基因互不等位,不同位点的bar基因之间存在基因互作,表现为基因重叠作用。Southern杂交分析结果表明,TR5、TR6的bar基因整合位点互不相同,但可通过杂交育种重组到同一个水稻核基因组中。     

Development and Substance Accumulation of Caryopsis in Transgenic Rice with Antisense Wx Gene

China is one of the leading nations in production as well as consumption of rice. Almost more than half of Chinese people live on rice. But most of the widely grown rice is of low quality, especially with high amylose content [1]. The higher level of amyl…     

两份转Fer基因富铁水稻特异种质应用价值的初步评价

在获得了105份转豌豆铁蛋白基因（Fer）水稻转化体的基础上,通过连续7代自交纯化,并结合GUS标记辅助选择,得到了82份独立转基因纯系。经稻米矿质元素检测,发掘到2份富铁转基因种质（Fer34和Fer65）,其精米全铁含量为野生型亲本秀水11的4.82倍和3.46倍。进一步鉴定表明,所筛选到的2份富铁转基因种质的外源基因（Fer）遗传简单,增加精米粉铁含量明显,对植株重要农艺性状、其他矿质营养、精米外观和食用品质等均无明显的负作用,是较为难得的富铁转基因优良新种质。此外,还就该2份富铁种质的实用价值及其应用前景进行了讨论。     

不同转化方法培育无抗性选择标记转基因水稻效率的比较

 为研究不同的转化方法培育无抗性选择标记转基因水稻的效果,以4个籼粳稻品种为受体材料,比较了用农杆菌介导的双菌双载体和双T DNA单载体两种共转化技术的共转化频率及随后获得的无抗性选择标记基因转化子的效率。结果表明,在双菌双载体介导的转化中,4个水稻品种的平均共转化频率为10.1%,其中55.6%的共转化植株自交后代中可筛选出无抗性选择标记的转基因水稻植株。在单载体双 T DNA介导的共转化中,平均共转化频率为45.0%,从其中600%的共转化植株自交后代中可获得无抗性选择标记的转基因植株,即双T DNA单载体法的去选择标记的效率（270%）要较双菌双载体法（5.6%）高。     

1-磷酸甘露醇脱氢酶基因转化水稻的研究

摘要：PCR和Southern blotting检测表明,来自大肠杆菌的[i]mtlD[/i]基因已通过农杆菌介导整合进水稻基因组。[i]mtlD[/i]基因在T1代出现分离, T2代出现纯系。在0.75% NaCl胁迫下,7个转基因T3代株系都能检测到mtlD酶活性,与对照相比细胞膜的相对电导率和大分子渗漏值明显降低。部分转基因株系能在 1.0% NaCl浓度下正常生长,而对照在0.5% NaCl浓度下已不能存活。通过有性杂交途径实现了[i]mtlD[/i]和[i]gutD[/i]两个基因的聚合,部分杂交后代株系能在1.25%NaCl胁迫下正常生长结实。     

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。     

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 (RACK1) is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference (RNAi), were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower, and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants. It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.     

转PAL基因水稻抗稻瘟病性和过氧化物酶活性的研究

 用PAL(苯丙氨酸解氨酶）基因正义和反义转化水稻，获得了70株转基因植株。选择正义转化植株(1s)和反义转化植株(4a)进行稻瘟病菌([i]Magnaporthe grisea[/i])接种，针对病原物侵染，1s的PAL活性上升更快，幅度更大。观察水稻叶片超微结构发现，1s的细胞具有更强的抵抗病原菌入侵的能力，其过氧化物酶活性也比对照和4a要高。     

转反义Wx基因水稻颖果的发育及物质积累

以转反义Wx基因粳稻和籼稻品系为材料,研究其颖果的发育和物质积累。结果表明,直链淀粉含量降低后的转基因水稻品系籽粒的粒重会有所下降,而且直链淀粉含量下降越多,粒重的下降幅度也越大。单个颖果胚乳细胞的数目也有不同程度的下降,但在籽粒发育早期（花后6 d前）,转基因水稻的胚乳细胞增殖速率明显高于其亲本。直链淀粉降低后的转基因品系籽粒可溶性糖含量在发育初期（花后9 d前）低于其同时期的亲本,而花后9 d后则明显高于其亲本。转基因水稻籽粒中的总淀粉含量有不同程度的下降,而支链淀粉的含量却相对增加,从而改变了籽粒中淀粉的组成,但对籽粒中蛋白质的积累没有显著影响。