转GsCBRLK/SCMRP双价基因苜蓿耐碱性及氨基酸含量分析

从野生大豆盐碱胁迫基因表达谱中筛选并克隆到GsCBRLK基因, 与人工合成的高甲硫氨酸含量SCMRP基因构建成双价植物表达载体, 将其转入苜蓿, 获得超量表达的转基因苜蓿, 并进行耐碱性分析。结果显示, 经过100、150 mmol L^–1 NaHCO₃处理14 d后, 转基因株系生长状态良好, 而非转基因对照株系萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05), 而SOD酶活性显著高于非转基因对照(P<0.05), 说明超量表达GsCBRLK基因增强了苜蓿的耐碱能力;各转基因株系的甲硫氨酸含量均比对照植株高, 表明SCMRP基因的导入提高了苜蓿叶片甲硫氨酸的含量。     

转AtDREB2A基因苜蓿的耐碱性分析

对已获得的转基因苜蓿进行了RT-PCR检测,确定AtDREB2A基因已在转基因苜蓿中超量表达。并研究了转AtDREB2A基因苜蓿在碱胁迫(100mmol/L,pH8.5)下表型和生理生化指标的变化。结果表明,转AtDREB2A基因苜蓿的相对质膜透性及丙二醛含量显著低于野生型株系,株高、叶绿素及根系活力明显高于野生型株系,说明AtDREB2A基因的超量表达提高了转基因苜蓿的耐碱能力。     

碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析

WRKY蛋白属于锌指型转录调控因子,能够参与植物多种逆境响应。本研究利用前期野生大豆盐碱胁迫RNA-seq测序数据,从构建的碱胁迫基因调控网络中筛选并克隆到GsWRKY15基因。分析GsWRKY15在碱胁迫下野生大豆根中的表达模式,发现该基因受碱胁迫诱导显著上调表达,且在胁迫后1 h表达量最高。分析GsWRKY15基因在野生大豆各组织中的表达特异性,发现该基因在各组织中均有表达,花中表达量最高。采用根癌农杆菌侵染苜蓿子叶节方法,将GsWRKY15转化肇东苜蓿,获得39株抗性植株。通过PCR、Southern blot和RT-PCR方法分析抗性植株,获得了超量表达GsWRKY15基因的转基因株系并对其进行了耐碱性分析。在150 mmol L–1 Na HCO3处理2周后转基因苜蓿生长状态良好,而非转基因苜蓿出现萎蔫、变黄,甚至死亡;非转基因苜蓿的相对质膜透性和丙二醛含量显著高于转基因苜蓿,而叶绿素含量显著低于转基因苜蓿;同时分析碱胁迫下转基因植株中胁迫相关基因的表达模式,发现H+-Ppase、NADP-ME、KIN1、RD29A基因的表达量高于非转基因苜蓿。结果表明GsWRKY15基因的超量表达能够显著增强苜蓿的耐碱能力。     

GsZFP1基因植物表达载体构建及对苜蓿的遗传转化

从野生大豆中获得的具有耐旱、耐冷特性的锌指转录因子GsZFP1基因,构建到以CaMV35S为启动子、E12为增强子的植物表达载体pCEOM中,以bar基因为筛选标记基因,通过农杆菌介导法将构建的植物表达载体转化苜蓿品种农菁1号的子叶节,用含0.5mg/L草丁磷的筛选培养基进行筛选,获得了70株抗性植株,用PCR检测得到20株bar基因阳性植株,将获得的转基因植株进行GsZFP1基因的RT-PCR鉴定,获得3株RT-PCR阳性植株。结果表明,GsZFP1基因在苜蓿中得到表达。     

转水稻OsSIK1基因玉米植株的获得及抗旱性分析

类受体激酶基因Os SIK1具有通过激活抗氧化系统,增强水稻对于干旱和盐胁迫抗性的作用。为了丰富可利用的作物抗旱基因,获得具有较高抗旱水平的玉米新种质,通过超声波辅助花粉介导法,将水稻类受体激酶基因Os SIK1导入玉米自交系郑58中,并对转化株进行卡那霉素筛选及T1、T2、T3的PCR及Southern Blotting杂交等分子检测,获得转化植株并在T3获得转基因纯合株系。对T3转基因玉米和非转基因玉米对照以16.1%的PEG模拟水分胁迫进行抗旱性分析。结果表明,与对照相比,在水分胁迫处理下,转基因玉米株系叶片相对含水量提高了7.4%~19.8%,叶绿素含量提高了11.3%~106.9%,SOD活性上升45.8%~93.4%,而转基因玉米叶片的相对电导率下降了35.4%~58.1%,MDA含量下降了25.7%~50.4%,说明转Os SIK1基因玉米植株抗旱性得到提高,其中,5个转化株系与对照在抗旱性方面有显著差异,且生长状况明显优于对照。综上所述,研究最终获得5个转Os SIK1基因玉米株系,并证明导入水稻Os SIK1基因可以提高玉米植株的抗旱性。     

转GhMAPK基因烟草植株的获得及其耐盐性分析

促丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)是普遍存在于真核生物中的一类保守的丝氨酸/苏氨酸类蛋白激酶,在植物生长发育和多种生物、非生物胁迫信号转导过程中起着关键作用.GhMAPK是首个在棉花(Gossypium hirsutum)中克隆的MAPK基因,为了进一步研究其功能,本研究利用农杆菌介导法将GhMAPK转入本氏烟草,Northern杂交和Western杂交分析证实,在转基因植株中GhMAPK能够高效表达.烟草耐盐性分析显示,在高盐胁迫下,转基因植株种子萌发率高于野生型烟草,苗期转基因株系能正常生长;进一步试验证明,转基因植株中的抗氧化酶(POD、SOD)活性也有明显提高.这些结果表明,GhMAPK过表达的转基因植株可以通过有效清除活性氧来提高植物对盐胁迫的抗性,GhMAPK可能参与了植物高盐胁迫信号传递过程.     

转BADH基因玉米植株的获得及其耐盐性分析

采用超声波辅助花粉介导植物转基因方法, 将甜菜碱醛脱氢酶(BADH)基因导入玉米自交系郑58, 获得了耐盐性强的转基因玉米植株。经卡那霉素抗性初筛、PCR扩增、Southern blot杂交分析, 证明BADH基因已导入转化植株并整合到其基因组中。用不同浓度的NaCl溶液对T₂代转基因玉米植株与对照进行盐胁迫处理, 结果表明, 转BADH基因玉米植株表现出一定的抗逆性, 生长状况明显优于对照; 根据非转化苗对NaCl的反应以及生长状况, 确定250 mmol L^-1 NaCl溶液为玉米幼苗耐盐性筛选的适宜浓度; 依据此临界浓度下形态指标和生理生化指标的测定结果, 与对照相比, 转基因植株的株高提高10.94%~25.7%, 鲜重增加8.62%~18.2%, 干重增加9%~18.18%, 相对电导率降低37.21%~58.14%, 叶绿素含量增加15.89%~90.65%, 超氧化物歧化酶(SOD)活性提高64.92%~148.29%, 丙二醛(MDA)含量减少26.97%~48.05%。综上所述, 转入甜菜碱醛脱氢酶(BADH)基因提高了玉米的耐盐性。这是首例将BADH基因导入优良玉米自交系郑58的报道。超声波辅助花粉介导法是一种经济、高效、实用和无基因型依赖性的植物基因转化方法。     

转低温胁迫转录因子BcICE1基因水稻提高抗寒性

低温冷害是制约黑龙江省水稻安全生产最主要的气象灾害之一,严重影响其产量和品质.为提高寒地水稻抗低温冷害能力,本研究将从大白菜分离的低温胁迫转录因子BcICE1基因(GenBank登录号为HQ902162)构建到植物表达载体pCAMBLA1301上,利用农杆菌介导法导入粳稻品种龙粳24中.PCR、Southern杂交和R...     

转BADH基因苜蓿T-DNA侧翼序列分析及转化事件特异性分析

为了从分子水平上鉴别不同的转基因株系,以转BADH基因苜蓿的T₀代基因组DNA为模版,采用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列,获得了B127株系的左翼序列和右翼序列,以及B125、B138、B295和B196株系的左翼序列。侧翼序列特征分析表明,有的T-DNA边界序列被删除,有的边界序列被保留,并填充了一段未知来源的核苷酸序列。根据侧翼序列中插入载体序列和紧邻插入序列的基因组序列特征,分别设计PCR扩增的上、下游引物,并对获得的42个转BADH株系分别进行左、右翼序列的扩增,结果表明,转基因植株B106、B125、B138、B157、B158、B289、B295、B305和B127具有相同的扩增条带,B203、B220、B223和B196具有相同的扩增条带,说明这些株系可能仅来源于2个转化事件。本研究建立的事件特异性检测方法可以准确地将不同的转化株系区别开来。     

转Cry1Ab-t基因玉米YA108的获得及其抗虫性鉴定

为了培育优良的抗虫玉米自交系,通过农杆菌介导的方法,将含有自主改造合成的抗虫基因Cry1Ab-t的植物表达载体pCAMBIA3300m导入玉米杂交种HiII基因组中,通过双丙氨磷筛选、PCR检测获得阳性转基因植株,采用RT-PCR、试纸条、ELISA方法检测外源基因的表达情况,并进行抗虫性鉴定.结果表明,用双丙氨磷筛选...     

超量表达苹果酸脱氢酶基因提高苜蓿对铝毒的耐受性

在酸性土壤中,铝毒是许多作物正常生产的主要限制因子。虽可通过施用生石灰来改良土壤酸碱度,但只能改善土壤表层,在实际应用中受到很大限制。紫花苜蓿作为一种非常重要的牧草、饲料,在酸性土壤条件下苜蓿生长缓慢甚至死亡。我国南方水热资源丰富,但土壤多偏酸性,限制了苜蓿南移。有机酸能螯合铝离子,减轻铝对植物根系的危害。苹果酸是游离铝离子的有效螯合剂,而苹果酸脱氢酶(Malate Dehydrogenase,MDH)催化草酰乙酸形成苹果酸。本研究在苜蓿中超量表达苹果酸脱氢酶基因,通过抗生素筛选、组织化学染色和PCR扩增等技术鉴定出转化植株,并对转化株系进行了耐铝胁迫试验,比较分析了转基因株系与非转基因株系的的相对伸长量,转基因株系根相对伸长量比对照高出3．6％～22．5％。说明超量表达neMDH基因可提高了转基因苜蓿对铝毒的耐受性。     

甘蓝型油菜BnADH3基因的克隆及转BnADH3拟南芥的耐淹性

利用RT-PCR技术从甘蓝型油菜耐淹品系WR-4中克隆获得Bn ADH3基因,其完整开放阅读框为1137 bp。该基因编码379个氨基酸,与甘蓝Bo ADH3基因和拟南芥At ADH3基因高度同源,同源性分别达到96%和91%。利用定量RT-PCR检测Bn ADH3基因在油菜耐淹系WR-4和不耐淹系WR-24中的表达表明,在淹水处理下该基因表达受到一定的诱导,淹水处理6 h后表达开始上调,说明Bn ADH3基因在油菜耐淹机制中发挥作用;将其转化到模式生物拟南芥中,采用幼苗淹水3 d后去水处理,测定表明转基因株系叶片和根系中乙醇脱氢酶活性均高于野生型;生长4周和6周的拟南芥植株淹水3 d后的表型显示,Bn ADH3的表达可增强拟南芥对淹水胁迫的耐性,处理的T2代转基因幼苗大部分恢复,但野生型幼苗枯死;调查表明,淹水5 d后野生型植株的存活率为26.7%,转基因株系ADH33和ADH44的存活率分别为80.0%和66.7%。     

粳稻超优1号背景回交导入系的耐热性筛选与评价

全球气候变暖不可避免地影响水稻的生产。本研究以粳稻品种超优1号为轮回亲本与9个来自不同国家的供体品种杂交培育的BC₂F₄回交导入群体为材料, 通过开花期高温胁迫, 共初筛到124个耐高温单株; 以粳稻为供体的回交导入群体出现耐热个体的频率高于籼稻供体的导入群体, 表明粳稻资源中同样存在耐热有利基因。经对初筛后代的耐热性重复鉴定和在正常条件下的性状评价, 发现有80个株系的结实率显著高于轮回亲本, 耐热性选择效率为64.5%, 这些耐热导入系的产量及其相关性状在高温胁迫和正常条件下均出现广幅分离, 从中鉴定出耐热性和产量性状均显著好于轮回亲本的8个优良导入系。在3个耐热导入系的聚合F₂群体中, 筛选出耐热个体的平均结实率在80%以上, 极显著高于轮回亲本和最高聚合亲本, 从中获得106株耐热性极显著好于聚合亲本的单株, 显示出较理想的耐热性聚合效果。通过对耐热导入系和轮回亲本在正常和高温胁迫条件下的产量以及相关性状的表型比较分析, 有助于提高对水稻耐热性状筛选中的供体选择、选择效率和筛选方法等一些重要问题的认识。本研究获得的高产耐热聚合系, 将为水稻耐热有利基因发掘和耐热性标记辅助选择聚合育种提供宝贵材料。     

The potential of selection and breeding for improved salt tolerance in lucerne (Medicago sativa L.)

Summary Four cultivars of Medicago sativa L. were subjected to selection for improved salt tolerance using a salinized solution culture technique at 250 mM NaCl. Selections were made after two weeks growth, based upon seedling shoot length differences. High (shoot lengths 14–31 mm) and low (7–12 mm) selection lines were established. Unselected shoot lengths ranged from 0–11 mm. Selection intensities ranged from 0.17% to 0.22% for the high selection lines, and from 0.30% to 1.05% for the low selection lines.Eleven selected plants from cv. CUF 101, and 10 from Local Syria were grown on and polycrossed. A sample of progeny seed from each was grown at 8 NaCl concentrations in solution cultures. Selected line seedlings produced shoots at 225, 250, 260, and 275 mM NaCl, whereas unselected material failed to grow above 225 mM NaCl. A second cycle of selection at 280 mM NaCl with these two cultivars achieved selection intensities of 0.21% and 0.45% for high and low selection lines, respectively. Selected lines grew and produced shoots at 300 mM NaCl.Seedlings derived from 1st and 2nd selection cycles and unselected control material were grown for 6 weeks at 0, 150, 175, and 200 mM NaCl in a sand culture experiment. The high selection line produced significantly greater shoot fresh weight, dry weight, and % live shoot weight than the unselected control material. Clearly selection as practised here can isolate individual seedlings having enhanced genetically based tolerance to NaCl, which is manifest in mature plants derived from polycrossing those selected individuals.     

盐胁迫对苜蓿种子发芽性状的影响及其耐盐性评价

为了阐明盐胁迫条件下紫花苜蓿种子的发芽性状表现和种子萌发期耐盐性能强弱,同时为改良盐碱化土壤提供更加耐盐碱的植物品种,提高盐碱化土地资源利用率,本试验以5个苜蓿(Medicago sativa)品种为材料,采用不同浓度NaCl处理(0、0.4%、0.8%、1.2%),研究了不同盐浓度对苜蓿种子发芽率、发芽势、苗高、根长的影响。结果表明,盐胁迫对苜蓿种子发芽性状均有较大影响,随着NaCl浓度的提高,苜蓿种子发芽率和发芽势降低,苗长和根长明显缩短,而且,高质量浓度盐胁迫对苜蓿种子发芽性状的影响尤为显著。不同苜蓿品种在不同盐浓度间的差异显著;品种间各指标的表现不尽相同,发芽势与发芽率指标在品种间的差异显著,苗高和幼根长指标在品种间的差异不显著。综合单项指标耐盐系数和品种综合耐盐指数来看,5个苜蓿品种的耐盐性能强弱顺序为‘Magnum salt’>‘中苜1号’>‘维多利亚’>‘巨人201T’>‘皇冠’。     

An improved breeding strategy for autotetraploid alfalfa (Medicago sativa L.)

Alfalfa cultivar development will be enhanced by breeding strategies whichutilize the full potential of autotetraploid population genetic structures. Thisstudy evaluates the effectiveness of an allelic selection scheme, which wasdeveloped to overcome limitations of inbreeding depression and to exploitgeneral and specific combining ability effects in autotetraploid populations.Allelic selection entails the minimization of non-additive genetic effects byselecting among full-sib families (F1) which are at uniform levels ofheterozygosity. Such F1 lines are developed by crossing individuals fromtwo unrelated random mating populations. Selected F1 lines wereintercrossed to form an improved population. Eight random matingpopulations of alfalfa were developed to study the effectiveness of allelicselection. Selection for increased dry matter yield resulted in alfalfapopulations with 38 percent greater yield than the parent populations. Twocycles of intercrossing, among selected F1 lines, did not dissipate the gainfrom selection. This result has important implications for synthetic cultivardevelopment in which a major limitation is the decline in productivity withadvancing generations of seed increase. A positively correlated response toselection for dry matter yield was observed for plant height and stemdiameter. The results of this research indicate that continued testing of theallelic selection scheme is warranted and could have a significant impact onthe breeding of autotetraploid alfalfa, particularly for synthetic cultivardevelopment.     

Germplasm Variability and Environmental Effects on Stem Cellulose and Lignin Concentrations in Alfalfa*

Stem cellulose and lignin concentrations are major determining factors of alfalfa (Medicago sativa ssp. sativa L.) forage quality. Only limited information is available on the genetic variability and the influence of environmental effects on these two stem-quality traits. Our objectives were to: evaluate the variability for stem cellulose and lignin concentrations in modern alfalfa germplasms across several harvests; observe the environmental stability of these two quality traits in 32 alfalfa clones selected high or low for either stem cellulose or lignin concentration; and examine the relationships between these two stem-quality traits and leaf and stem crude protein (CP). Fifty alfalfa entries (cultivars and experimental populations) were established May 1993, and sampled for stem acid-detergent lignin (ADL), stem acid-detergent cellulose (ADC), and stem CP on Sep 1993, June and Aug 1994. Clones were vegetatively propagated from individual plants selected for extremes in stem ADL and ADC and transplanted into blocks at two locations in May 1994 and sampled for quality analysis in September 1994. For all samples, leaves were hand-separated from stems and stem ADL, ADC, CP and leaf CP concentration were determined using near infrared reflectance spectroscopy. Entry differences for stem ADL and ADC were detected only at the June 1994 harvest date in the cultivar study. Spearman's ranked correlations over years in the clonal study demonstrated greater environmental stability for stem ADC (r = 0.70, P ≥ 0.01) than for stem ADL (r = 0.54, P # 0.05). Environmental effects had an impact on both traits, but stem ADC showed greater potential for improving forage quality. Simple correlations showed that decreasing stem ADL or ADC would have minimal effect on leaf CP and may increase stem CP.     

转TiERF1-RC7双价基因小麦的鉴定及其全蚀病抗性

为探讨TiERF1和RC7基因对小麦抗全蚀病的防御反应,本研究对转TiERF1-RC7双价基因小麦进行了分子检测以及全蚀病抗性的室内和田间鉴定。结果表明,转入的TiERF1和RC7基因在转基因小麦中可以遗传和转录;与受体扬麦18相比,5个转TiERF1-RC7小麦株系在整个生育期抗病性显著提高,苗期的全蚀病严重度在10%以下,成熟期的白穗率在13%以下,而扬麦18的严重度为62.98%,白穗率为26.09%。电子显微镜观察结果表明抗病转基因小麦根表的全蚀病原菌菌丝数量及生长势明显低于感病材料。上述结果说明,转入的TiERF1和RC7基因抑制了全蚀病原菌的侵染及在转基因小麦中繁殖,进而提高了转基因小麦对全蚀病的抗性。