抗草铵膦基因bar遗传转化蓖麻

在生产过程中,杂草会极大影响蓖麻的品质和产量。随着生物技术与基因编辑系统的出现,为实现蓖麻抗除草剂的定向育种带来了可能。为了提高蓖麻抗草铵膦除草剂的能力,以‘通蓖5号’大穗型蓖麻为实验材料,利用根癌农杆菌介导法将bar基因遗传转化蓖麻子叶节,研究了农杆菌菌液侵染浓度、共培养时间和Basta筛选浓度对遗传转化效率的影响。结果表明：当农杆菌菌液浓度OD₆₀₀=0.7时,侵染15 min,共培养3 d,用1 mg/L的Basta进行抗性筛选时,遗传转化效率最高。生根后移栽的转化子经PCR检测和200 mg/L的Basta除草剂叶片涂抹检测,初步证明抗草铵膦基因bar已成功整合到蓖麻基因组中,得到具有草铵膦抗性的转基因蓖麻植株。     

农杆菌介导蔗糖:蔗糖果糖基转移酶基因转化甘蔗

甘蔗是高产高蔗糖型植物的代表,把蔗糖:蔗糖果糖基转移酶基因(1-SST)转入甘蔗并使其得到有效的表达,可将甘蔗中的蔗糖转化成果聚糖.本研究以甘蔗为受体,首先接种甘蔗愈伤,培养20 d后即为胚性愈伤组织,用农杆菌菌液浸染转化.研究结果表明,出芽时PPT的最佳筛选浓度为2.0 mg/L,生根时PPT的最佳筛选浓度为5.0 mg/L.共获得42株抗性植株,对抗性植株进行PCR鉴定,其中26株呈阳性,随机选取3株阳性片段进行测序,与目的基因片段一致,初步证明1-SST基因已经转入甘蔗.     

不同抗生素对韭菜根愈伤组织诱导及再生的影响

本研究以"汉中冬韭"韭菜品种为试验材料,研究了韭菜根尖及愈伤组织对抑菌性和选择性抗生素的抗性。研究结果表明,选择性抗生素对韭菜再生的影响极显著,以PPT作为选择抗生素时,愈伤生长及不定芽分化阶段PPT适宜浓度为1mg/L,而生根阶段PPT最适浓度为0.5mg/L。当潮霉素浓度为1mg/L时,根尖愈伤组织分化率达到了8%,而浓度为2mg/L时根尖已不能分化,以1mg/LHyg为最佳浓度。用头孢霉素作为抑菌剂时,愈伤生长及不定芽分化阶段,最适浓度为400mg/L,而生根阶段适宜浓度为200mg/L;以Timentin作为抑菌剂时,最适浓度在400mg/L以内,韭菜外植体的分化没有受到影响,随着浓度的增加抑制作用越来越明显,以400mg/LTimentin为最适抑菌抗生素浓度,这为农杆菌介导韭菜遗传转化奠定基础。     

盐稻8号遗传转化体系中不同抗生素种类与浓度的抑菌效果研究

为建立粳稻遗传转化体系,筛选抗性愈伤组织,提高转化植株再生能力,选用羧苄青霉素钠（carbenicillin sodium,Car）、头孢噻肟钠（cefotaxime sodium,Cef）两种抑菌剂和一种标记选择剂潮霉素B（cygromycinB,HyB）,分别设4个抗生素浓度处理侵染转化共培养后的粳稻盐稻8号成熟胚愈伤组织。结果表明：Car的抑菌作用强于Cef,Car浓度为500mg/L时抑菌率达到100.0%,抗性愈伤组织的分化率达到42.2%;选择剂HyB浓度为30mg/L可获得86.1%较高质量的抗性愈伤组织,抗性愈伤组织的分化率达到39.5%。结论在以盐稻8号为受体材料的粳稻遗传转化体系中,抗生素的最佳搭配是500mg/L Car+30mg/L HyB。     

表达bar基因的抗除草剂转基因甘薯的获得

用农杆菌介导法将bar基因导入甘薯主栽品种徐薯18的胚性悬浮细胞中,获得了抗除草剂的转基因植株.农杆菌菌株EHA105携带的双元载体pCAMBIA3300上含有bar基因.来自于徐薯18胚性悬浮细胞的直径为0.7～1.3 min的280个胚性细胞团用于遗传转化.共培养3 d后,首先在含有2 mg/L 2,4-D、100 mg/L Carb的液体MS培养基中培养1周,然后将胚性细胞团转移到添加2 mg/L 2,4-D、100 mg/L Carb 和0.3 mg/L PPT的固体MS培养基上进行选择培养.选择8周后,将获得的37个PPT抗性愈伤组织转移到添加1 mg/L ABA、100mg/L carb和0.3 mg/L PPT的固体MS培养基上,其中的34个愈伤组织诱导得到体细胞胚并发芽形成小植株,共获得了164株拟转基因植株.PCR分析表明,其中的123株为转基因植株.Southern blot和Northern blot分析表明,bar基因稳定整合到转基因植株的基因组中并正确表达.除草剂喷洒试验结果表明,转基因植株具有高度除草剂抗性.     

用根癌农杆菌介导法转化大豆萌动子叶节细胞

利用根癌农杆菌转化萌动大豆成熟子叶节获得了高频率的转基因大豆。从萌发12～16 h的大豆种子切取半片种子作为目标组织,对其子叶节组织进行伤处理后,接种于含有pGB载体的LBA4404农杆菌溶液。pGB载体含有除草剂（phosphinothricin, PPT）抗性基因bar和绿色荧光蛋白基因sgfp。将接种的外植体分别在含有3或5 mg/L PPT的芽诱导培养基、3 mg/L PPT的芽伸长培养基及2～4 mg/L PPT的根诱导培养基上进行了筛选。利用萌发5 d的外植体进行PPT浓度筛选的结果表明,芽诱导、芽伸长及根诱导阶段的PPT浓度分别为5、3及3 mg/L时,转化效率达到最大值,为5.3%。PCR和Southern杂交结果证实了外源基因稳定地整合在大豆基因组中。Northern杂交和GFP分析结果表明被整合的外源基因在大豆细胞中得到了稳定的表达。成熟植株对体外喷洒100 mg/L PPT溶液产生抗性。转化效率达8.9%。     

水稻植株再生与农杆菌介导的遗传转化研究

本研究以不同籼稻品种的成熟胚为材料, 研究了不同培养基对愈伤组织诱导、分化和植株再生的影响,并研究了利用农杆菌介导bar基因和ipt基因的遗传转化技术.结果表明,采用NBD (N6salts B5Vitamin casamino acids 500 mg/L proline 500 mg/L glutamine 300 mg/L mannitol 36.4 g/L sucrose 30 g/L 2,4-D 2 mg/L 6-BA 0.2 mg/L phytagel 2.5 g/L,pH 5.8) 为诱导培养基,MSD(MS casamino acids 500 mg/L proline 500 mg/L glutamine 300 mg/L mannitol 36.4 g/L sucrose 30 g/L 2,4-D 2 mg/L 6-BA 0.2 mg/L phytagel 2.5 g/L,pH 5.8)与NBD培养基交替继代培养,籼稻愈伤组织诱导率高、质量较好;采用MSR3c(MS casamino acids 500 mg/L glutamine 300 mg/L proline 500 mg/L mannitol 36.4 g/L maltose 30 g/L TDZ 0.2 mg/L phytagel 5 g/L pH 5.8)为分化培养基,愈伤组织分化率均在90%以上.本研究利用农杆菌介导,将Act启动子驱动的抗除草剂基因(bar)和异戊烯基转移酶基因(ipt)构建的融合基因表达载体对水稻进行遗传转化.初步确定了愈伤组织筛选及分化培养基附加除草剂(glufosinate ammoniun)4 mg/L、头孢霉素300 mg/L,愈伤组织预培养2-3天,共培养基中加入乙酰丁香酮浓度为39 mg/L,共培养2-3天.通过上述培养条件,已获得移栽成活的水稻转基因植株.     

Bar基因的亚麻花粉管通道法转化

[研究目的]为鉴定T1代转基因亚麻植株中抗除草剂基因(bar)整合是否成功。[方法]通过花粉管通道法将bar基因导入转化植株,以叶片涂抹法对T1代转化植株进行田间草丁膦最低致死浓度筛选;以最低致死浓度草丁膦进行田间喷施后筛选耐受草丁膦药液的抗性植株,并用PCR检测bar基因阳性的抗性植株数目。[结果]草丁膦对T1代转化植株的最低致死浓度为100 mg/L。得到耐受草丁膦药液的抗性植株44株,PCR检测结果表明,8株为PCR阳性植株,平均转化率为18.2%。[结论]花粉管通道法将bar基因成功转入亚麻基因组。     

双丙氨磷在橡胶树遗传转化中的应用

本研究以GV3101::pMP90RK为工程菌株,bar基因作为筛选标记基因,研究了双丙氨磷在橡胶树根癌农杆菌介导法转基因体系中对转基因愈伤组织的筛选效果,并利用该体系将橡胶树转录因子HbWRKY5转入橡胶树无性系热研8-79中。实验结果表明,3mg/L双丙氨磷可作为橡胶树转基因体系中抗性愈伤组织筛选的最佳浓度。本研究中共获得了3个抗性愈伤组织系,抗性愈伤经过体细胞胚发生共获得234个体细胞胚,经植株再生培养后共获得22株再生植株。对再生植株进行的PCR鉴定结果表明,所获得的再生植株均为转基因植株。因此,bar基因结合双丙氨磷的筛选体系可以作为橡胶树转化体系中的有效筛选体系。     

农杆菌介导胡萝卜转化体系的诱导培养基优化

高频再生系统是外源基因成功转化的先决条件,但再生系统不等同于转化系统。为提高农杆菌共培养后胡萝卜的再生频率,以日本新黑田五寸参和林丰改良五寸参2个品种胡萝卜下胚轴为外植体,利用农杆菌介导法将人源白细胞介素-2基因(IL-2)导入其中,探讨了共培养后诱导培养基中激素浓度和筛选剂对再生的影响。结果显示：不同基因型胡萝卜的分化对激素浓度的反应不同,林丰改良五寸参在3种培养基上的胚性愈伤率差异不显著;日本新黑田五寸参胚性愈伤率和每个外植体分化的抗性株数随激素浓度的提高而呈上升趋势,2,4-D和6-BA浓度为1.0mg/L时胚性愈伤率和每个外植体分化的抗性株数最高,分别为87.50%和59株;日本新黑田五寸参以bar基因为标记基因、PPT作筛选剂时,分化较好。因此,遗传转化过程中,为提高共培养后胡萝卜胚状体的分化率,对共培养后的诱导培养基进行再优化是必要的。     

Increased regeneration from NaCl-tolerant callus in rice

Summary NaCl-tolerant calli were selected from two Japonica and two Indica rice (Oryza sativa L.) cultivars on basal media containing 6,000, 9,000, 12,000 or 15,000 ppm NaCl. Frequency of callus formation decreased with the increase of NaCl in the medium, especially in Indica. About half of the calli of Japonica cultivars selected on NaCl-ammended media survived 20,000 ppm NaCl but none of the Indica callus survived. In Japonica, more plants were regenerated from calli selected on all concentrations of NaCl media than from NaCl-free medium. Concentration of Cl^- in callus increased dramatically with increased NaCl content but peroxidase activity decreased.     

应用双单倍体克服籼粳交的不亲和性初探

通过对籼粳交Ｆ１体细胞愈伤组织的秋水仙素处理和四倍体花药培养，建成了水稻四倍体诱导及双单倍体分解系统。应用这一系统获得了４４个籼粳交组合的双单倍体及其自交４后代。对这些双单倍体及其后代的遗传分析表明，籼粳交育性可能受寡基因控制，双单倍体途径通过改变正常的世代交替过程，能部分克服籼粳交的不育性及倾籼或倾粳的偏态分离，并具有比Ｆ２更丰富的遗传多样性。在双单倍体一代群体中，育性正常的植株约占４０％，这是     

Competition of Barnyardgrass with Rice Varieties

A field study was conducted to examine the competition of barnyardgrass with various rice varieties and its effect on propanil effectiveness. Six rice varieties, Ispaniki A', Strymonas and Roxani (Japonica type) and Rea, Skybonnet and Blue belle (Indica type), were grown in a silty loam soil, in 1994 and 1995. The varieties had different 1000 seed weights, which varied from 24 to 40 g. The herbicide propanil was applied at the four-leaf stage of barnyardgrass on half the plot area. Japonica type varieties emerged earlier and had lower numbers of barnyardgrass plants compared to Indica varieties. The time of seedling emergence was significantly and negatively correlated with the seed weight and positively with the number of barnyardgrass plants. The delayed establishment of Indica type varieties favoured rapid barnyardgrass growth at the early stages and resulted in lower effectiveness of propanil, compared to Japonica type varieties. Thus, in controlling barnyardgrass in rice fields it is necessary to take into account the variety grown in order to reduce production costs and the pollution risk to the environment.     

旱作对籼粳稻产量及相关性状的影响

研究了旱作对籼、粳亚种间产量及其相关性状的影响,并用逐步回归分析法建立亚种间水稻旱作的产量回归模型。研究表明,旱作在生育期、分蘖数、有效穗、穗长、株高、总粒数、结实率、千粒重等性状上对籼型品种的影响较粳型品种的影响大。在旱作条件下,粳型产量总体表现比籼型品种高。     

Morphological Classification of Traditional Philippine Upland Rice Cultivars in Upland Nurseries Using Cluster Analysis Methods for Recommendation, Breeding and Selection Purposes

Using eight morphological parameters with six cluster analysis methods, a set of 21 traditional and four improved Philippine upland rice cultivars (cvs) could be separated into five groups: (1) improved semi-dwarf plant type, (2) improved or 'moderately modified' (short) Indica plant type, (3) traditional tall Indica plant type, (4) traditional tall Japonica plant type and (5) traditional short Japonica plant type. The results emphasized the importance of the separation into Indica and Japonica plant types, but the cultivars could be classified more accurately using growth characteristics such as tillering ability, followed by plant height and leaf area parameters. The clusters containing Indica cvs consistently showed a higher tiller number per plant, a higher growth stage (DC classification) and narrower leaves than clusters with Japonica cvs. The parameters culm length, plant height, leaf area, leaf length and width were represented in subgroups with both long and short expression of the respective characteristic. Use of the existing variability of morphological traits within the group of traditional upland rice cvs should be beneficial in recommendation, breeding, selection and development of upland rice cvs for the different rice ecosystems as well as for the upland rice/rice blast pathosystem.     

栽培稻的基因型差异程度和分类

本文利用来自一个籼/粳交(圭630/02428)的双单倍体(DH)系和由多种类型的籼、粳品种(系)组成的对照组,从分子水平上研究亚洲栽培稻的基因型差异。结果表明,栽培稻中存在两种主要的差异类型,即质的差异和量的差异。前者主要存在于籼、粳分化程度比较高的对照组中,85%以上的有关位点已经发生了籼或粳的分化趋向;后者主要存     

Indica-Japonica differentiation of paddy rice and its relationship with heterosis

The Indica‐Japonica differentiation of three photoperiod‐sensitive and/or thermosensitive genetic male‐sterile rice (PGMS or TGMS, respectively) lines and 47 male parental lines from seven ecotypes were studied for their restriction fragment length polymorphism marker data to determine which ecotype crosses with the three PGMS and/or TGMS lines could lead to higher yield potential, and to estimate the relationship between the Indica‐Japonica differentiation of parents and heterosis in grain yield and its components. The results indicated that hybrids derived from ‘N422s’ and the early‐middle ripening Indica varieties from southern China, and hybrids between ‘Pei'ai64s’ and three Japonica ecotypes, including North‐eastern Japonica varieties, restoring lines of Japonica hybrid rice and north China Japonica varieties, showed the highest grain yields. There was less variation of yield among the F1s between ‘108s’ and the seven ecotypes than among the other F₁s. Highly significant positive correlations between heterosis of the F₁ yield and genetic distance of the parents were detected, although the correlation between F₁ yield performance and genetic distance did not reach a significant level. Considerable variation of correlation between heterosis and genetic distance was also detected in the Indica × Indica crosses and Indica × Japonica crosses. There was much higher correlation (r = 0.63) between the F₁ yield performance and the genetic distance of parents in the Indica × Indica crosses than in the others. It is proposed that a genetic distance of 0.4‐0.8 between the two parents of hybrid rice might be appropriate not only for F₁ performance, but also for heterosis.     

Analysis of Vietnamese rice germplasm provides an insight into Japonica rice differentiation

Genetic differentiation among landraces of rice in northern Vietnam that lie in the centre of genetic diversity of Asian cultivated rice was analysed using DNA markers, and morphological and physiological traits to obtain an insight into the genetic differentiation of Asian rice. Principle coordinate analysis, based on nuclear RFLP data, divided Vietnamese rice varieties into three groups, designated A, B and C. Chloroplast DNA variation discriminated group A, which corresponded to the Indica rice varieties, from both groups B and C, which corresponded to the Japonica rice varieties. Physiological characteristics for Indica‐Japonica and tropical‐temperate Japonica discrimination suggested groups A and B consisted of typical Indica and Japonica varieties, respectively, while the characteristics of group C were distinct from those of the other two groups. This suggests that the genetic differences between groups B and C represent a new aspect of genetic variation that has not been previously reported in Japonica rice. Groups C and B corresponded with upland and lowland rice, respectively. Eight marker loci were associated with upland‐lowland differentiation in Japonica rice. Highlighted chromosomal regions might have been selected during the history of cultivation under different cultural conditions, and thus contain genes related to adaptation.     

水稻种子耐干性机理的研究

以籼稻C38、粳稻京21、杂交稻秋优3为材料,进行种子超干储藏特性的研究,进而探讨种子耐干性的机理。通过超干水稻种子生活力和活力的比较,发现籼稻C38耐干性较强。超干有利于保持种子抗氧化酶系统的完整性,因此超干能提高种子的耐储性。对超干种子可溶性糖含量与组分进行分析,结果表明随着种子含水量的降低,蔗糖含量增高,多糖和葡萄糖含量降低,籼稻C38的蔗糖含量明显低于粳稻京21和杂交稻秋优3。用热重仪测定的种子含水量与烘干法得到的数据相吻合,用差示热量扫描仪测定束缚水/自由水的比例和玻璃化转变温度Tg,发现籼稻C38的Tg高于粳稻京21和杂交稻秋优3,籼稻C38束缚水/自由水比例大于粳稻京21和杂交稻秋优3,而束缚水/自由水的比例与耐干性成正相关。由此表明,耐干性在不同的水稻品种间有明显的差异,籼稻C38的耐干性较强。     

生态条件对籼粳稻杂交F2代亚种特性分化与经济性状的影响

以籼粳稻杂交F2代群体(七山占/秋光)为供试材料,2010年分别在广东省与辽宁省同年种植,研究籼粳亚种属性和经济性状的分化规律以及两者之间的相互关系.结果表明,广东和辽宁2个生态地区群体,亚种特性呈相似正态分布,未出现偏分现象,说明在F2群体中遗传重组对群体的作用呈主导性.经济性状发生较显著的分化,穗数、千粒重、1次枝梗数及粒数辽宁地区显著高于广东,而产量、穗粒数及结实率呈现相应生态条件的偏向性,籼型及偏籼型在广东地区较高,粳型及偏粳型在辽宁地区较高,说明在籼粳交F2代中不同的籼粳类型存在较为显著的生态适应性,并且该结果在相关性分析也得了验证.此外,对进一步研究籼粳分化机制的相关问题进行了讨论.