高羊茅FaChit1基因cDNA克隆及诱导表达

应用简并RT-PCR及RACE技术,从高羊茅中克隆了1个Ⅰ类几丁质酶基因cDNA全长序列,命名为FaChit1.结果表明,该cDNA具有1个951 bp的完整编码框,编码316个氨基酸,其编码产物和其它植物的Ⅰ类几丁质酶在氨基酸序列上具有较高的同源性,包含典型的几丁质结合区、催化区以及脯氨酸、半胱氨酸富集的铰链区,但缺少定位到植物液泡所必须的C末端延伸区靶向信号.Northern杂交显示,FaChit1对真菌激发子有较强的响应,乙烯和干旱胁迫均能有效诱导FaChit1基因的表达,而对机械损伤处理的反应比较微弱,只在叶片中积累少量的mRNA.     

高羊茅FaChit1基因组DNA的克隆及其拷贝数的验定

以获得的高羊茅FaChit1基因cDNA设计引物扩增其基因组DNA,序列比对发现该基因内不存在内含子.进一步采用染色体步移方法分离FaChit1基因上游的一段935 bp启动子序列以及下游的一段470 bp 3′非翻译区序列发现,在该启动子区域内不仅含有保守的TATA盒和CAAT盒,而且包含多个潜在的与胁迫应答有关的顺式调控元件,表明该启动子可能是1个多胁迫诱导型启动子.此外,在FaChit1 3′非翻译区含有真核生物基因中高度保守的特征序列.Southern杂交结果表明,FaChit1在高羊茅基因组中有2个拷贝.     

高羊茅FaCONSTANS基因的表达及功能分析

植物由营养生长向生殖生长转变过程中光周期调控起着重要的作用.CONSTANS (CO)是光周期途径中的特有基因,为探讨高羊茅FaCONSTANS(FaCO)基因响应日照长短从而启动植物开花的机理,利用实时荧光定量qRT-PCR技术分析在长日照、短日照、持续光照、持续黑暗条件下FaCO基因的表达水平.构建过表达载体p13...     

抗除草剂高羊茅基因转化体系的建立

杂草防除是草坪建植与养护管理中的一个关键环节,对于直播草坪或新建植草坪,杂草的危害非常严重。通过抗除草剂基因工程,创造抗除草剂草坪草的新品种,这是最根本、最经济的控制杂草的方法。高羊茅(Festuca arundinacea Schreb．)是一种优良的冷季型草坪草,在国内外得到了大面积的种植,但是杂草危害非常     

高羊茅FaFT基因表达、蛋白互作及生物学功能分析

FLOWERING LOCUS T(FT)对植物开花有重要的调控作用.为探索高羊茅FaFT基因的生物学功能,从前期高羊茅转录组中克隆得到FaFT基因.qRT-PCR分析表明FaFT基因在长日照、短日照、连续光照、连续黑暗、昼夜颠倒和长日照下不同发育阶段都保持一定的昼夜节律并受光周期的调控.亚细胞定位显示FaFT基因编码...     

高羊茅组织培养再生体系及GUS基因瞬间表达研究

以成熟种子为外值体,对高羊茅纰织培养和植株再生体系进行了优化,分析了不同浓度2．4-D、6-BA和激动素对高羊茅愈伤组织诱导和愈伤组织分化成苗的影响．结果表明：9．0mg／L 2．4-L)对愈伤组织的诱导效果最佳．0．2mg／L激动素是愈伤组织分化成苗的最适浓度．二者的诱导率和分化率分别达到68．08%和45．83％。在愈伤组织继代培养基中附加1．0mg／L 2．4-D、0．5mg／L 6-BA和1．25mg／L CuSO4;有利于胚性愈伤组织的形成,可以明显促进愈伤组织分化。同时．采用基因枪法将GUS基因导入高羊茅愈伤组织中,通过组织化学染色检测到了GUS瞬间表达活性;并对影响CUS基因瞬间表达的因素进行了分析．以期为提高基因枪法遗传转化效率提供参考。     

空间飞行水稻pi-hit-1基因启动子功能分析

pi-hit-1基因是本实验室通过空间诱变找到的一个水稻新基因。为了对pi-hit-1基因启动子结构和功能进行研究,首先使用植物启动子分析数据库(PlantProm DB-TSSP,TFSEARCH,PLACE及PlantCARE)对该基因转录调控区序列进行预测分析,结果显示该基因上游调控区存在多个顺式元件,主要集中在翻译起始位点前300bp的区域,转录起始位点位于翻译起始位点前100bp,在转录起始位点前132bp存在TATA box元件。凝胶电泳迁移率实验(EMSA)发现翻译起始位点上游约300bp存在转录因子特异结合位点,为该基因的核心启动子,这与预测结果一致。采用系统生物学的方法研究水稻新基因pi-hit-1启动子结构,发现了该基因的核心启动子元件,为研究空间环境如何影响基因的转录调控提供了重要依据。     

水稻OsNRT1-d启动子的分离和功能分析

采用PCR技术,从水稻基因组中分离到OsNRT1-d读码框上游2 019 bp序列.序列分析表明:在起始密码ATG上游-189 bp和-127 bp处分别存在CAAT-box和TATA-box,具有典型的启动子结构.推测的转录起始位点CAC位于起始密码ATG上游-93 bp处.将OsNRT1-d启动子5′-端系列缺失后,分别与GUS报告基因融合,获得的NRT2019∷GUS、NRT1196∷GUS 和NRT719∷GUS转基因载体.农杆菌介导转化水稻,获得的转基因水稻均能启动下游GUS报告基因在水稻的根、叶、花颖和种子中表达;将转基因水稻幼苗放在滤纸上紧急干旱处理和用15% PEG6000进行模拟干旱处理,GUS基因的表达量明显升高,且干旱应答元件在-719 bp～-1 bp的范围内;GUS活性分析表明:OsNRT1-d启动子对ABA、NaCl、(NH4)2SO4、KNO3和Gln等信号没有应答反应.     

高羊茅春化基因FaVRN1亚细胞定位与差异表达分析

高羊茅在生长季出现生殖枝,抑制新枝形成,不利于草坪质量及其持久性生长.研究春化基因的分子特征,探索抑制生殖生长的分子育种新途径,对坪用型高羊茅品种改良具有重要意义.本研究在克隆高羊茅春化基因FaVRN1的基础上,构建高羊茅春化基因FaVRN1与绿色荧光蛋白基因GFP融合的植物表达载体p-FaVRN1-hGFP,利用基因枪转化法转入洋葱表皮细胞,荧光显微镜检测融合基因的瞬时表达,并运用实时荧光定量PCR分析春化基因FaVRN1在春化与非春化条件下的表达差异.研究结果表明,FaVRN1基因编码的蛋白产物位于细胞核,符合它作为转录因子特性;春化条件下,FaVRN1基因的表达随处理时间延长逐渐增加.非春化条件下,FaVRN1基因的表达随处理时间延长而降低.FaVRN1基因在春化条件下的表达水平远高于非春化条件,FaVRN1基因的表达受春化条件正调控.     

高羊茅甘油醛-3-磷酸脱氢酶基因FaGAPDH的克隆与分析

以高羊茅茎基部与叶基部的mRNA为模板,引用基于多年生黑麦草GAPDH基因序列设计的引物,进行RT-PCR分析,同时结合3'RACE和5'RACE方法从高羊茅中扩增出两个GAPDH基因,命名为Fa-GAPDH1(GQ480772)与FaGAPDH2(GQ480773)。两序列cDNA全长分别为1281bp和1348bp,具有完整的开放阅读框(ORF,FaGAPDH1:74～1087bp;FaGAPDH2:106～1119bp),编码蛋白都为337个氨基酸。保守结构域功能分析表明两基因编码的蛋白质都具有典型的Rossman-NAD连接折叠和C-末端结构域。FaGAPDH1和FaGAPDH2蛋白与禾本科植物的该基因蛋白产物比较发现氨基酸序列的同源性都在90%以上,说明两基因具有高度的保守性。     

Genetic variability of mineral concentrations in Festuca arundinacea Schreb.

Summary Nine randomly chosen clones of tall fescue (Festuca arundinacea Schreb.) were mated in all possible combinations to determine the nature of genetic variation for Mg, Ca, K, and P concentrations in a broad genetic base population. General combining ability mean squares were significant for most variables, whereas specific combining ability mean squares were not significant in most instances indicating that additive genetic variance was more important. Genotype x year interactions were significant for most variables, suggesting that selection should be evaluated over many environments. Broad-sense heritability estimates based on parental and progeny variance components were generally high for P, K, Ca, and Mg but low for the ratio K/(Ca + Mg). Narrow-sense heritabilities for these minerals were close to the broad sense values since the additive genetic variance was the largest component of the total genetic variation. Correlations between mineral concentrations and herbage dry matter yield were low. It was concluded that adequate genetic variation exists to improve mineral concentration without altering herbage dry matter yields.Journal Series no. 5886     

盐胁迫对高羊茅(Festuca arundinacea)幼苗生长和离子分布的影响

盐胁迫环境抑制植物的生长，影响植物组织的离子分布，不同的盐分组成对植物的抑制伤害存在差异，为了研究上海市临港新城滨海盐渍土的生态恢复和重建，模拟该地区的盐分组成，进行了高羊茅（Festuca arundinacea）幼苗的盐胁迫试验。高羊茅种子在非盐胁迫条件下萌发，出苗5d后，进行了不同浓度NaCl：0、50、100、150、200、300、400mmol／L处理，15d后测定生长情况、组织含水量和Na^＋、K^＋、Ca^2＋、Mg^2＋等离子含量。研究结果表明：盐分对高羊茅幼苗的抑制作用随NaCl浓度增加而加剧，低盐胁迫环境下，幼苗地上部分和根系的鲜重、干重和含水量都与对照没有显著性差异，但是高盐环境严重影响了高羊茅幼苗的生长，而且对地上部分的抑制作用大于根部；盐胁迫影响植物组织的离子分布，Na^＋浓度持续增加，Ca^2＋和K^＋浓度下降，Mg^2＋含量的影响不大；各组织中K／Na、Ca／Na和Mg／Na随盐胁迫增加而下降。     

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea)

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.     

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric -glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.     

Genome mapping of polyploid tall fescue (Festuca arundinacea Schreb.) with RFLP markers

Genetic mapping using molecular markers such as restriction fragment length polymorphisms (RFLPs) has become a powerful tool for plant geneticists and breeders. Like many economically important polyploid plant species, detailed genetic studies of hexaploid tall fescue (Festuca arundinacea Schreb.) are complicated, and no genetic map has been established. We report here the first tall fescue genetic map. This map was generated from an F₂ population of HD28-56 by Kentucky-31 and contains 108 RFLP markers. Although the two parental plants were heterozygous, the perennial and tillering growth habit, high degree of RFLP, and disomic inheritance of tall fescue enabled us to identify the segregating homologous alleles. The map covers 1274 cM on 19 linkage groups with an average of 5 loci per linkage group (LG) and 17.9 cM between loci. Mapping the homoeologous loci detected by the same probe allowed us to identify five homoeologous groups within which the gene orders were found to be generally conserved among homoeologous chromosomes. An exception was homoeologous group 5, in which only 2 of the 3 homoeologous chromosomes were identified. Using 12 genome-specific probes, we were able to assign several linkage groups to one of the three genomes (PG₁G₂) in tall fescue. All the loci detected by the 11 probes specific to the G₁ and/or G₂ genomes, with one exception, identified loci located on 4 chromosomes of two homoeologous groups (LG2a, LG2c, LG3a, and LG3c). A P-genome-specific probe was used to map a locus on LG5c. Comparative genome mapping with maize probes indicated that homoeologous group 3 and 2 chromosomes in tall fescue corresponded to maize chromosome 1. Difficulties and advantages of applying RFLP technology in polyploids with high levels of heterozygosity are discussed.Journal Series No. 12, 190     

9个高羊茅品种初期生长对镉胁迫的响应

用Cd²⁺浓度0（对照）、25、50、75 mg/L分别处理9个高羊茅品种种子,测定供试品种种子发芽势、发芽率、幼苗苗高、根长、鲜重、叶绿素含量、丙二醛含量、质膜透性等8项指标,比较9个品种耐镉性。结果表明:与对照相比,在不同浓度Cd²⁺胁迫下,家园的发芽势和发芽率以及麦哲伦鲜重呈先略上升后下降趋势,其余品种的均呈下降趋势;9个高羊茅品种幼苗根长、苗高、叶绿素含量均呈下降趋势;9个高羊茅品种丙二醛含量和质膜透性均呈上升趋势。对测定的8项指标进行综合评价结果显示:在Cd²⁺25 mg/L胁迫下,9个品种耐Cd²⁺性由强到弱的顺序为热浪>翠碧A>里园2号>缤狗>麦哲伦>家园>宇宙星>回报>爱瑞3号;在Cd²⁺50 mg/L胁迫下,耐Cd²⁺性的顺序为热浪>翠碧A>缤狗>麦哲伦>里园2号>家园>宇宙星>爱瑞3号>回报;在Cd²⁺ 75 mg/L胁迫下,耐Cd²⁺性的顺序为热浪>缤狗>里园2号>翠碧A>家园>麦哲伦>爱瑞3号>宇宙星>回报。所以,在治理Cd²⁺污染土壤时,要根据污染严重程度,合理选用高羊茅品种。     

高羊茅和其他羊茅植株再生与遗传转化研究进展

高羊茅、紫羊茅和草地羊茅均为很重要的多年生冷季型牧草与草坪草,生物技术在其品种改良中具有很大的应用潜力.30年来,三种羊茅的组织培养、胚性培养物的长期保存以及遗传转化等研究取得了较大进展,已建立起多种植株再生体系和遗传转化技术,但作为单子叶植物,这些草种的组织培养和转基因遗传改良也还存在一些问题.本文就以上几方面的内容进行了综述.     

城市垃圾堆肥对高羊茅生长及土壤性质的影响

采用盆栽实验方法,比较了在每盆施用130 g垃圾堆肥(T1)或1.5 g化肥(T2)以及130 g垃圾堆肥与1.5 g 化肥混合施用(T3)条件下高羊茅(Festuca arundinacea L)生长及土壤性质的变化.结果表明:T1和T3处理组的土壤容重显著低于对照(不施肥)和T2处理组,土壤中的阳离子代换量及有机质、碱解氮、速效磷和速效钾含量显著高于对照及T2处理组,且T3处理组的增加幅度更大;T1和T3处理组土壤中Pb、As、Cu、Cr和Cd含量均高于对照和T2处理组,且T1处理组土壤中Pb、As、Cu和Cd含量均显著高于仍处理组,但均小于土壤环境质量一级标准,不会造成土壤环境污染.T1和T3处理组高羊茅种子萌发率均明显低于T2处理组和对照,但随萌发时间的延长逐渐提高,表明垃圾堆肥对高羊茅种子萌发的抑制作用是暂时性的.施肥处理对高羊茅生长均有明显的促进作用;T1、T2和T3处理组高羊茅的株高、地上部分和根于质量、叶片N含量和叶绿素含量总体上均显著高于对照;其中T1处理组在栽培前期高羊茅的株高、地上部分干质量和叶片N含量低于T2处理组,但在后期显著高于T2处理组;而T3处理组高羊茅的各项指标总体上均最高.结果显示:使用垃圾堆肥能明显改善土壤性质、增加土壤肥力;垃圾堆肥具有缓释效应,能改善高羊茅的生长状况、提高草坪质量,与化肥混合施用效果更佳.     

淋洗后生活垃圾堆肥建植高羊茅草坪生理生态响应

以不同淋洗方式处理后的生活垃圾堆肥为基质进行草坪植物培植,研究了高羊茅的生理生态响应。结果表明,淋洗处理堆肥中高羊茅的发芽率及株高等生长指标均优于未淋洗的原堆肥对照组,其中水肥淋洗比例为1∶1的处理中,高羊茅的最大发芽率比对照提高89.6%。通过对超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）3种保护酶的活性及脯氨酸和丙二醛（MDA）含量等指标的测定可以看出,经淋洗处理后的堆肥基质对草坪植物的胁迫程度均小于原堆肥,更有利于草坪植物的正常生长。综合高羊茅生长指标及生理指标来考虑,堆肥处理的最佳淋洗比例为水∶堆肥的质量比为2∶1。