猴和人T淋巴细胞表面TRBC受体和E受体的比较研究

1985年贲昆龙等首次发现人和猴T淋巴细胞能与树鼩(Tupaia belangeri)的红细胞(TRBC)形成亲和力极强的玫瑰花结，它与绵羊红细胞(SRBC)玫瑰花结(E花结)明显不同。例如，TRBC经神经氨酸酶处理之后，结花率明显下降，TRBC与T淋巴细胞所形成的玫瑰花结，经45℃保温，仍不受影响，为了进一步探讨TRBC受体和SRBC受体(CD2)，以及与其它T细胞表面分化搞原(CD)的系。我用某些抗人T细胞CD的单克隆抗体(McAb)对人和猴淋巴细胞进行玫瑰花结抑制试验，抗原调变和共调变(Antigenic modulation or co-modulation)实验，并且研究了TRBC受体在其它免疫细胞和某些人类和长臂猿细胞系的分布。结果表明，TRBC与外周血E~+-PBL形成玫瑰花结的百分率为88.8%。而E~--PBL仅为4.16%。TRBC受体存在于所有被试T细胞系(CEM, H33JHJA1, Jurkat, MLA-144, Molt-3, Molt-4, Molt-4 clone-8 和PEER)，但不存在于外周血粒细胞，B细胞，以及B细胞系的绝大多数细胞表面(Daudi, Raji和Reh)。分布于全T细胞的CD3, TCR, CD5, CD6和CD7的相McAb OKT3, T108(F1), T136(F101-15), T149(M-T604)和T152(7G5)均不能调变和共调变TRBC受体。猴和人外周血淋巴细胞与TRBC玫瑰花结的形成，不被T11.1 McAb OKT11所阻断，相反，OKT11显著地阻断猴和人外周血淋巴细胞E玫瑰花结的形成，最大抑制率分别为49.3%和77.7%。在世界各地10个实验室送交第四次国际人白细胞化化抗原讨论会待鉴定的13个CD2 McAb中，除T089 (39C1.5)因抗体量不够未作实验外，对其他12种McAb都进行试验，T081 (x/3)，T082 (GLB-T11.2/1)，T083 (GLB-T11.1/1)，T085 (RPA-2.10)，T1088 (0-275)和T092 (M-T201)对TRBC玫瑰花结和E玫瑰花结都呈现明显的阻断作用，T084 (F110.08)，T091 (AICD2.1)以及已知参数CD2 McAb-T086 (D-66 clonel)和T087 (GT-2)都不阻TRBC玫瑰花结的形成，亦不调变TRBC受体，而对E玫瑰花结则有不同程度阻断效应，并且调变E变体，使E玫瑰花结形成细胞百分率下降。T090 (6F10.3)和T198 (JOR-T2)即不阻TRBC玫瑰花结的形成，也不抑制E玫瑰花结的形成。由此可见，TRBC受体分布于全T细胞，它不同于已知的全T细胞表面分化抗原CD2 (gp50), CD3/TCR复合物，CD5, CD6和CD7。CD2分子不与TRBC玫瑰花结的形成，也不是介导E玫瑰花结的唯一分子。至少有二个或三个以上的蛋白质与E玫瑰花结和TRBC玫瑰花结的形成有关，其中有的分子为E受体和TRBC受体所共有。TRBC受体很有可能包括新的T淋巴细胞分化抗原。

不同FGF-2表达量的饲养层对猕猴和兔胚胎干细胞的增殖和自我更新的影响

胚胎干细胞（ESC）培养是ESC研究的基础，饲养层的选择是ESC培养的一个重要方面。本实验曾对不同的猕猴饲养层进行研究，显示能够更好的支持猕猴ESC生长的饲养层FGF-2表达量也较高。FGF-2，又名bFGF（碱性成纤维生长因子），是ESC生长所需的重要因子，但其中的分子机制现在并未完全了解。本文一方面对ESC相关研究进展进行了综述，另一方面对以下内容进行了研究：用转基因和RNA干（RNAi）扰的方法建立不同FGF-2的表达量猕猴耳部皮肤细胞（MESF）系五组：过表达FGF-2（f1），过表达的阴性对照组（f2），FGF-2 RNA干扰组（f3），RNA干扰的阴性对照组（f4）以及未作任何处理的对照组（f5），这五组MESF的FGF-2表达量相对值为f1：f2：f3：f4：f5＝4：2：1：2：2；c-fos，TGF-β1，INHBA，Gremlin1在f1中表达量上升，在f3中表达量下降；BMP4，TGF-β2在f1中表达量下降，在f3中表达量上升；表明内源FGF-2能够作用于MESF的TGF-β信号通路，引起相关基因表达量的变化。用这些细胞作为饲养层分别培养两种ESC（猕猴ESC，R366. 4和兔ESC，RFESC） ，连续培养了10代，其中在f1上培养的两种ESC增殖速度都比对照组快，并且c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达量均上升，BMP4表达下调；在f3上培养的猕猴ESC增殖较慢，BMP4表达上调，c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog，Sox2表达下调；f3上的兔ESC没有变化。表明ESC中的TGF-β信号通路也受到调节。五组猕猴和兔的ESC形成的EB均表达各胚层早期标记基因(marker)，但表达量有差异，f1上ESC形成的EB所有marker都低表达。实验结果表明饲养层中的FGF-2含量高低不仅影响自身相关基因的表达，还对ESC的增殖和维持自我更新有一定的作用。

菜花烙铁头蛇毒金属蛋白酶的研究

本论文结合生物化学与分子生物学手段对云南产菜花烙铁头蛇毒（几互刃eresrs户厂由刀打）的金属蛋白酶进行了系统深入的研究。从中分离纯化到两个新的蛇毒金属蛋白酶：二型蛇毒金属蛋白酶Jerdonit认和三型蛇毒金属蛋白酶jerdohagin。采用又卫PCR的方法得到了两个cDNA。通过蛋白质胰蛋白酶水解的内肚测序分析，其中一条为编码Jerdo址tin的，cDNA。序列分析发现，另外一条是一个含有RTS短链去整合素cDNA序列，命名为jerdos七atin，并对其进行了表达和活性鉴定。，Jerdonitin是一个表观分子量为为kDa的单链蛋白。它的c砚认全长1578bP，由金属蛋白酶、间隔区和去整合素区组成，说明它是二型蛇毒金属蛋白酶。但是与其它二型蛇毒金属蛋白酶不同的是，Jerdonitin的成熟蛋白由金属蛋白酶和去整合素结构域组成。与其它二型相比，Jerdonitin"多了两个半肤氨酸的（CysZ19andCys238）分别位于间隔区和去整合素区，Cys219可能和后面去整合素区的自由半眺氨酸残基Cys238形成一对二硫键，这对二硫键可能阻止了在后翻译加工过程中去整合素区的释放。通过Jerdonitin和其它二型蛇毒金属蛋白酶氨基酸序列比较和进化分析，结合天然蛋白结构数据，说明Jerdonitin是二型蛇毒金属蛋白酶的一个新亚型。和其它蛇毒金属蛋白酶一样，Jerdon九in是a一纤溶酶，并且它的活性都能被金属鳌合剂EDTA完全抑制，而不受丝氨酸蛋白酶抑制剂PMSF影响。像其独特的结构一样，Jerdonitin不仅具有纤维蛋白原降解的蛇毒金属蛋白酶活性，而且也有抑制ADP诱导的血小板聚集的非酶活性。Jerdohagin是一个表观分子量为96kDa单链蛋白。测定其内肤发现它有金属蛋白酶、去整合素样和富含半耽氨酸区组成，说明jerdohagig是三型蛇毒金属蛋白酶。像其它典型的蛇毒金属蛋白酶一样，jerdohagin的出血活性能完全被EDTA抑制，而不受R涯SF的影响。Jerdohagin是仪纤溶酶专一酶切人纤维蛋白原的。链。用氧化的胰岛素B链作底物，它可以水解仰r16一Leu17肤键。有趣的是，jerdohagin不激活人凝血酶原，但是它酶切人凝血酶原和凝血酶原激活后产生的激活片段F1。Jerdostotin的cDNA全长邹3bp，由信号肚、前肤和去整合素三个区组成，其与。btustotin和vieris七atin有较高的相似性（80％）。jerdostatin的序列是第一个报道的短链去整合素的cD以序列，它的产生机制和aCostatin一a有相似之处。值得注意的是，jerdostatin和obtus七atin八ipris，tatin不同的氨基酸残基（8／9）主要分布在含有整合素识别区的C末端。采用硫氧还蛋·白作为融合蛋白表达jerdostatin：，表达纯化后发现有两个j．erdostatin表达，，命名为：jerdo就。tin一1和'rjerdostotin一2。质谱分析显示它们的八个半肤氨酸都参与形成了四对二硫键，它俩可能是分别含有天然和非天然二硫键结构的多肚。但是jerdostatin一1的活性比rjerdostat还一2高两个数量级。和含有KTS的去整合素一样，重组jerdos七atin的异构体都选择性抑制整合素。lpl结合胶原IV，rjerdost就in一1的抑制活性分别是。b加stotin和viperistotill的1／1时和1／2500。氨基酸残基的组成不同尽管没有影响jerdost就in对整合素alpl的抑制选择性，但是它造成的化学环境不同可能导致抑制活性的差异。

单边掺杂InAlAs／InGaAs单量子阱中二维电子气的磁输运特性

研究了双子带占据的In0.52Al0.48As／In0.53Ga0.47As单量子阱中磁电阻的Shubnikov-de Haas（SdH）振荡效应和霍耳效应，获得了不同子带电子的浓度、迁移率、有效质量和能级位置．低磁感应强度（B〈1．5T）下由迁移率谱和多载流子拟合相结合的方法得到的各子带电子浓度与通过SdH振荡得到的结果一致．在d^2ρ／dB^2-1／B的快速傅里叶变换谱中，观察到除了通常强烈依赖温度的对应于各子带的频率f1和f2以及f1的倍频（2f1）外，还观察到对温度不敏感的频率f1-f2．这是由于量子阱中不同子带的电子具有相近的有效质量，两个子带之间发生了强烈的磁致子带间散射．

镉、铅污染土壤的植物-微生物联合修复研究

植物－微生物联合修复是利用微生物作为植物修复重金属土壤的一种强化手段，在弥补单纯植物及微生物修复技术不足的同时，利用植物和微生物的共存体系提高植物修复效率。本研究考察了向日葵（Helianthus annuus L.），芥菜（Brassica juncea L.），紫花苜蓿（Medicago sativa L.）和蓖麻（Ricinus communis L.）对Cd、Pb的富集特征，并筛选出向日葵作为富集植物；探讨了向日葵根系分泌物在重金属胁迫下的变化；通过重金属耐受菌株与向日葵的配伍对Cd、Pb污染土壤进行联合修复，结果如下： 液体培养实验结果表明，四种植物对Cd、Pb富集能力明显不同，其中向日葵对两种重金属的提取效果较好。四种植物对重金属的富集量随着浓度的增加而增加，而富集系数随重金属浓度的增加而减小，转移系数同重金属浓度及地上部/地下部生物量比值呈现一定的相关性；Cd、Pb复合处理中，一种重金属的存在会在不同程度上影响植物对另一种重金属的吸收；此外，不同植物及重金属处理中根际区域的酸碱度及氧化还原电位呈现负相关性。 砂培实验结果表明，向日葵对重金属的富集规律基本同液体培养实验相似。富集系数与重金属浓度和培养时间呈现线性相关关系。复合处理中，当Cd和Pb在适当浓度比例时，向日葵可以增加对某一重金属的吸收效率。向日葵的根系分泌物组成因重金属的存在而明显减少，根系分泌物中的草酸、酒石酸、苹果酸、柠檬酸、乙酸及丁二酸含量随着不同浓度Cd、Pb而发生不规则变化。 在以根系分泌物作为唯一营养来源筛选重金属耐受菌株实验中，细菌和真菌对Cd及Pb的耐性及吸附效率不同。总体上看，微生物生物量随重金属浓度升高而降低，而重金属吸附量随浓度升高而增加，重金属复合毒性也减少了微生物对单一重金属的吸收；另外，培养基中酸碱度因微生物种类及重金属浓度而有所差异。 将筛选出的优势微生物与向日葵配伍处理Cd、Pb污染土壤的实验中，由于Cd、Pb污染模式及菌株种类的不同，微生物对向日葵吸收重金属的强化效果呈现很大差异，其中真菌对植物吸收重金属的强化能力较细菌强；同时，根系分泌物中6种有机酸的含量在不同处理中变化较大；在处理单一Cd或Pb污染的时候选用菌株B1、F1或混合菌B1+B2、F1+F2、B2+F1、B1+F2与向日葵配伍修复的效果较好；混合菌F1+F2、B1+F2、B2+F1的添加能较好的强化Cd、Pb复合污染中向日葵吸收重金属的能力。

重金属对矮小拟丽突线虫的毒性效应研究

为了探讨重金属对矮小拟丽突线虫（Acrobeloides nanus）的毒性作用，本文采用室内培养的方法，研究了不同浓度Cu、Zn、Pb、Cd对矮小拟丽突线虫死亡率、生长发育和繁殖的影响，并对线虫世代间的毒性效应进行了比较。结果表明：1）Cu、Zn、Pb、Cd对矮小拟丽突线虫24 h的半致死浓度LC50值分别为 0.80、71.20、12.94 和 1.42 mg • L-1，48 h 的半致死浓度LC50值分别为 0.71、35.08、2.65 和 0.32 mg • L-1。不同重金属离子对矮小拟丽突线虫产生的毒性具有一定的差异，24 h四种重金属离子对矮小拟丽突线虫的毒性大小依次为Cu > Cd > Pb > Zn，48 h毒性大小依次为Cd > Cu > Pb > Zn；2）与对照组相比，各暴露组当代（P0）和后代（F1）线虫平均体长均显著降低（P<0.05, P<0.01）；随着暴露浓度的增加，当代（P0）和后代（F1）线虫平均体长呈逐渐降低的趋势。生物量比体长更能敏感地反应重金属对矮小拟丽突线虫生长发育的影响，可作为重金属污染的敏感检测指标；3）供试线虫的产卵数随着Cu、Zn、Pb、Cd暴露浓度的增加而降低；其中Cu对线虫产卵数的72h-EC50、EC20和EC10值分别为1.35、0.49 和 0.2mg • L-1，Zn的72h-EC20和EC10值分别为330.29 和 163.9 mg • L-1，Pb的72h-EC20、EC10分别为17.41、4.36 mg•L-1，而Cd对线虫产卵数的72h-EC50、EC20和EC10分别为4.47、0.91 和 0.53 mg • L-1。研究结果表明，重金属Cu、Zn、Pb、Cd暴露可显著抑制矮小拟丽突线虫的生长发育和繁殖；线虫体长、产卵数表现出明显的重金属浓度依赖性。

木霉的种间融合重组及生物学特性研究

本文利用原生质体融合技术，以四株营养缺陷型突变株（康宁木霉UV2－1、UV2－15、绿色木霉UV2－11和另一木霉菌株UV2－2）为亲本进行种间融合重组。研究了原生质体形成和再生的最适条件及高产的重组菌株筛选的方法。筛选到了一株优良的融合株F1－18，它固体发酵稻草粉产生的滤纸酶活（FPA）、Cx酶活、Cl酶活和β－葡萄糖苷酶活分别为1148、5705、6042和1036 U/g基质，分别是其双亲－康宁木霉F224和绿色木霉F264－的1.86、1.38、1.95、1.52倍和9.10、6.78、4.79、0.85倍。对F1－18的生物学特性进行了研究。经菌落形态观察、分生孢子大小及其DNA含量测定，认定它为单倍性重组体。经非特异性酯酶和过氧化物酶同工酶酶谱分析及纤维素酶SDS－PAGE电泳图谱分析表明，它既有与双亲共有的谱带，也有它独有的谱带，说明在融合－分离过程中，发生了基因重组和基因突变，产生了新的蛋白组分。与生产用菌SN9706相比，F1－18固体发稻草粉产生的滤纸酶活提高了39％，β－葡萄糖苷酶活提高了86％。在固体发酵玉米秸粉时也获得较高的纤维素酶产量，具有广泛的应用前景和推广价值。F1－18的最适产酶条件为：培养基起始PH值为5.5 ～ 6.0，培养基中加2.5 ～ 3倍于原料的水，28 ～ 30 ℃发酵4天，培养基中添加0.35%脲和0.35% KNO3，最高酶产量可达滤纸酶活1347 U/g基质。

小麦—偏凸山羊草6Mv/6B代换系的研究

小麦条锈病（Puccinia striiformis f. sp. tritici）是世界性小麦病害，可导致受害小麦减产30%以上，甚至绝收。小麦条锈病在我国西南、华北麦区危害严重，四川麦区是小麦条锈病发病最重的地区之一，每年因条锈病流行造成小麦产量损失巨大。利用抗条锈病品种是控制该病害最安全、经济的有效途径，因此挖掘利用抗病新基因，开展抗病遗传基础研究是当前育种工作中面临的重要任务。 偏凸山羊草（Aegilops ventricosa，DDMvMv，2n=28）是一年生草本植物，起源于地中海西部沿岸地区，具有对小麦白粉病、锈病等高抗或免疫、耐盐、抗寒、蛋白质含量高等优良性状，是小麦遗传育种很好的种质资源。本研究以高抗条锈病的小麦—偏凸山羊草6Mv/6B代换系（Moisson 6Mv/6B）为材料，对其含有的带条锈病抗性基因的偏凸山羊草6Mv染色体在四川小麦背景中的传递情况、与小麦—簇毛麦双端体附加系所具有的白粉病抗性的聚合以及对Moisson 6Mv/6B进行电离辐射诱变筛选抗条锈病的小麦—偏凸山羊草易位系三个方面进行了研究。取得的主要研究结果如下： 1. Moisson 6Mv/6B与高感条锈病的四川地区普通小麦品种绵阳26、绵阳93-124和SW3243的杂种F1与其普通小麦亲本分别作为父、母本回交，通过对其BC1和F2的结实率、根尖细胞有丝分裂中期染色体的观察以及对条锈病抗性的鉴定，发现含6Mv染色体的F1植株作母本时的回交结实率（83.10%）普遍高于含6Mv染色体的F1植株作父本（48.61%），结实率与普通小麦基因型密切相关（χ2=34.15>>χ20.05=5.99（df=2））；6Mv染色体在三种四川小麦中通过雌、雄配子传递的传递方式与其传递率间没有显著相关性，其传递率与普通小麦基因型呈显著相关性（χ2=6.42>χ20.05=5.99（df=2））。 2. Moisson 6Mv/6B与高抗白粉病的小麦—簇毛麦双端体附加系Pana（2n=42+2t）正反杂交，希望在聚合两者抗性的同时观察不同受体背景下的抗性反应。对Moisson 6Mv/6B和Pana正反杂交的结实率、杂交后代的农艺性状进行观察，并对杂交后代进行基因组荧光原位杂交（GISH）分析及条锈病和白粉病的抗性鉴定。结果表明Moisson 6Mv/6B作母本时杂交结实率（80.56%）高于Pana作母本时（58.33%），结实率与杂交方式间紧密相关（χ2=4.96>χ20.05=3.84（df=1））；Moisson 6Mv/6B和Pana杂交后代株高比最高亲本高约10cm，成熟期也较两亲本提前两个星期左右；正反杂交后代中具有偏凸山羊草6Mv染色体的植株具有条锈病抗性，具有簇毛麦端体的植株具有白粉病抗性，同时筛选到4株含有偏凸山羊草和簇毛麦遗传物质并对条锈病和白粉病兼抗的材料，证明来自偏凸山羊草6Mv染色体的条锈病抗性与来自簇毛麦端体的白粉病抗性已经聚合在一起，且没有产生相互抑制的作用，暗示通过这两个抗性基因的聚合是完全能获得兼抗条锈病和白粉病的小麦新种质。 3. 对Moisson 6Mv/6B在减数分裂时期的成株进行总剂量为6Gy、辐射频率为120rad/min的60Co-γ射线辐射，对辐射植株自交后代进行农艺性状及根尖细胞有丝分裂中期染色体形态观察和条锈病抗性鉴定。结果为辐射植株自交结实率为2.22%，根尖细胞有丝分裂中期的染色体存在明显碎片，辐射自交后代植株对条锈病具有成株期抗性。 小麦—偏凸山羊草6Mv/6B代换系对条锈病抗性稳定，是培育条锈病抗性品种的良好供体。本研究证明在四川小麦背景中要利用该品种抗性，在结实数满足需要时，可将其作父本，亦可作母本，但关键是要选择好一个优良的受体基因型；同时其条锈病抗性与来自簇毛麦的白粉病抗性没有相互抑制作用，可将两者抗性有效聚合用于小麦育种中。 Wheat stripe rust (Puccinia striiformis f. sp. Tritici) is a worldwide disease of wheat, and could lead to victims of 30 percent or even total destruction of wheat production. Wheat stripe rust harms badly in China's southwest and North China. Sichuan province is one of the regions damaged by wheat stripe rust heavily. The use of resistant varieties is the most secure and economical way to control the wheat stripe rust. Therefore, it is essential to identify new disease-resistant genes and genetically research of disease resistance. Aegilops ventricosa (DDMvMv, 2n = 28) is an annual herbaceous plant, originating in the coastal areas of the western Mediterranean, with good characters such as resistance of wheat powdery, rust, salt, cold and high protein content. It is a good germplasm resource. In this study, the wheat- Aegilops ventricosa 6Mv/6B substitution line Moisson 6Mv/6B (highly resistant to the wheat stripe rust) was used to study on the transmission of chromosome 6Mv of Aegilops ventricosa in different genetic background of Sichuan wheat varieties, hybridization with wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew) and screening of wheat- Aegilops ventricosa translocation line by exposuring Moisson 6Mv/6B under ionizing radiation. The main results are as following: 1. Moisson 6Mv/6B was crossed with Sichuan wheat varieties mianyang26, mianyang93-124 and SW3243 (highly susceptible to stripe rust), respectively. Their F1 hybrids were further backcrossed as male and female to corresponding wheat varieties. The seed-setting rate, chromosomes confirmation in the mitotic metaphase of root tip cells, and resistance to stripe rust of the subsequent BC1 and F2 plants were investigated. The average seed-setting rate of backcross via 6Mv as female donor (83.10%) was higher than that of backcross via 6Mv as male donor (48.61%), suggesting that the seed-setting rate was associated with the wheat genotypes（χ2=34.15>>χ20.05=5.99（df=2））. In all analyzed populations, transmission frequencies of chromosome 6Mv were not correlated with the ways of 6Mv through male or through female. However, transmission frequencies of chromosome 6Mv were significantly correlated with Sichuan wheat genotypes（χ2=6.42>χ20.05=5.99（df=2））. 2. To aggregating the resistances to stripe rust and powdery mildew, as well as research on the resistance reactions in different genetic background, Moisson 6Mv/6B was reciprocally hybrided with the wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew). The seed-setting rate, agronomic characters, genomic in situ hybridization (GISH) of hybrid progenies，and resistances to stripe rust and powdery mildew were investigated. The results showed that the seed-setting rate of hybridization via Moisson 6Mv/6B as female donor (80.56%) was significant higher than that via Pana as female donor (58.33%). The seed-setting rate was associated with the hybrid methods (χ2 = 4.96> χ20.05 = 3.84 (df = 1)). The plant height of hybrid progenies was about 10 cm higher than Pana, the parent with maximum height. And the maturity of hybrid progenies was about two weeks earlier than that of the parents. In the hybrid progenies, the plants with the 6Mv chromosome have the resistance to stripe rust and the plants with the telosome from Haynaldia villosa have the resistance to powdery mildew. It was found that four plants with both the 6Mv chromosome and the telosome from Haynaldia villosa were resistant to stripe rust and powdery mildew. It indicated that the resistance to stripe rust and powdery mildew aggregated, and no mutual inhibition was found. It implied that the aggregation of the two resistance genes was able to provide the new wheat germplasm with the resistances to stripe rust and powdery mildew. 3. Moisson 6Mv/6B was irradiated with 60Co-γ rays of 6Gy (120rad/min) during meiosis. The agronomic characters and chromosomes confirmation in the mitotic metaphase of root tip cells，as well as resistance to stripe rust were investigated. The seed-setting rate of irradiated plants was only 2.22%. The chromosomes in mitotic metaphase had clear fragments. The resistance to stripe rust of progeny of irradiated plants was the adult-plant resistance. The wheat- Aegilops ventricosa 6Mv/6B substitution line is a good stripe rust resistance donor for its stabile resistance. Our study demonstrated that the key for use the resistance is to choose a good receptor. There is no difference between Moisson 6Mv/6B be the female and be the male if the seed number meets the requirement. At the same time, the stripe rust resistance of Moisson 6Mv/6B did not have the mutual inhibition with the powdery mildew resistance from Haynaldia villosa. It is able to aggregate the two resistances for wheat breeding.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

Frankia与链霉菌融合子特性的研究

将Frankia菌株CcOl与金色链霉菌GL原生质体融合,得到3株融合子,均具有GL生长快的特性与CcOl的结瘤固氮能力.固体培养时,3株融合子呈现出与GL不同的颜色;且均具有Frankia菌的顶囊形态,以及链霉菌的链状孢子丝结构.与两亲本相同,3株融合子均对大肠杆菌有抗性,其中F4与GL的抗菌谱基本相同.在传10代之后,它们仍具有结瘤与固氮能力.血清学分析表明,F1与F6兼具两亲本的特异抗原,而F4仅具有GL的特异抗原,融合子F1、F6较F4在遗传上更为稳定.

卤代烷烃脱卤酶基因在拟南芥中反式失活系统的建立

以细菌Xanthobacterautotrophicus卤代烷烃脱卤酶基因为遗传负选择标记 ,建立了该基因在拟南芥中反式失活的实验系统 .在卤代烷烃脱卤酶转基因拟南芥中 ,有 1株表现为转基因失活 ,离体核run off转录实验表明为基因转录后沉默 (这里特指沉默位点 ) .用这一转基因沉默植株与同源转基因高效表达植株 (这里特指同源转基因位点 )杂交 ,结果 96 %的F1代植株表现为同源转基因反式失活 .将F1代植株自交 ,使部分沉默位点与反式失活的同源转基因位点分离 ,结果 2 0 0株子代中有 42株表现DhlA活性 ,15 8株无DhlA活性 ,即dhlA沉默植株与表达植株之比为 3 .76∶1,表明沉默位点是以孟德尔显性因子方式使同源转基因位点反式失活的 .

基于半马尔科夫条件随机场的命名体识别及其关系抽取研究

随着互联网和电子化办公的发展，出现了大量的文本资源。信息抽取技术可以帮助人们快速获取大规模文本中的有用信息。命名体识别与关系抽取是信息抽取的两个基本任务。本文在调研当前命名体识别和实体关系抽取中采用的主要方法的基础上，分别给出了解决方案。论文开展的主要工作有：（1）从模型选择和特征选择两个方面总结了命名体识别及实体关系抽取的国内外研究现状，重点介绍用于命名体识别的统计学习方法以及用于实体关系抽取的基于核的方法。（2）针对当前命名体识别中命名体片段边界的确定问题，研究了如何将 Semi-Markov CRFs 模型应用于中文命名体识别。这种模型只要求段间遵循马尔科夫规则，而段内的文本之间则可以被灵活的赋予各种规则。将这种模型用于中文命名体识别任务时，我们可以更有效更自由的设计出各种有利于识别出命名体片段边界的特征。实验表明，加入段相关的特征后，命名体识别的性能提高了 4-5 个百分点。（3）实体关系抽取的任务是判别两个实体之间的语义关系。之前的研究已经表明，待判别关系的两个实体间的语法树结构对于确定二者的关系类别是非常有用的，而相对成熟的基于平面特征的关系抽取方法在充分提取语法树结构特征方面的能力有限，因此，本文研究了基于核的中文实体关系抽取方法。针对中文特点，我们探讨了卷积（Convolution）核中使用不同的语法树对中文实体关系抽取性能的影响，构造了几种基于卷积核的复合核，改进了最短路依赖核。因为核方法开始被用于英文关系抽取时，F1 值也只有40%左右，而我们只使用作用在语法树上的卷积核时，中文关系抽取的F1 值达到了35%，可见核方法对中文关系抽取也是有效的。

灵长类的减数分裂细胞遗传学研究

I.四种猕猴属(Macaca)动物精母细胞联会复合体的比较研究 本工作采用去污剂微铺展－－硝酸银染色技术比较研究了熊猴、平顶猴、藏酋猴、恒河猴及其亚种毛耳猴的精母细胞联会复合体（SC）核型和SC的结构、形态及其在减数分裂过程的行为。结果表明：1.这几种动物的SC核型及SC的发育过程基本一致。粗线期SC的相对长度和臂比与体细胞染色体的相对长度和臂比具有较好的吻合性。SC的形成开始于线期，成熟于粗线期，解体于双线期。2.在减数分裂前期，性染色体轴表现强烈的嗜银性，配对明显落后于常染色体。根据性染色体的形态和行为，可分为五种类型。此外，本文还对XY染色体的同源性和侧轴加粗等现象进行了讨论。II.食蟹猴（M.fascicularis）和熊猴（M.assamensis）杂种（F1）的细胞遗传学研究 本工作采用染色体显带、组织学观察以及低渗铺张－－硝酸银染色等方法较为详细地研究了食蟹猴和熊猴种间杂种（F1）体细胞染色体的G带、C带、Ag-NOR、精母细胞联会复合体的结构、形态和行为以及精子发生。结果表明，①杂种亲本的染色体级具有高度的同源性；②杂种的精子发生过程完全正常；③食蟹猴和熊猴的种间生殖屏障可能主要是生态隔离。此外，本文还对食蟹猴和熊猴染色体高度同源的原因、二者的分类地位以及杂种细胞中Ag-NOR的多态性等问题进行了分析和讨论。