褐飞虱取食水稻幼苗cDNA文库的构建及筛选

以褐飞虱取食 3 2 h的水稻幼苗为材料构建了 c DNA文库 ,初始文库含 3 .2× 1 0 6 个克隆 ,重组率为 86%,取 1 .0× 1 0 6个克隆子扩增一次 ,收集到 80 m L滴度为 1 .2× 1 0 9pfu/ m L的扩增文库 .随机挑取 2 0个克隆以 T7和 M1 3 reverse为引物进行 PCR扩增 ,以鉴定插入片段的大小 ,结果发现多数片段的长度在 1～ 4.0Kb左右 ,少数片段在 4Kb以上 ,个别片段在 6Kb左右 .以在受褐飞虱取食的水稻幼苗中特异表达的 ESTBp Hi0 0 8为探针 ,筛选 c DNA文库 ,得到该基因的 c DNA全长     

水稻BpHi008A基因对褐飞虱取食应答及其编码产物在大肠杆菌中的高效表达

对抗虫相关蛋白BpH i008A进行了生物信息学分析,发现BpH i008A含有一个PKC磷酸化位点、一个CK2磷酸化位点、三个N-myristoylation site位点;另外,BpH i008A蛋白含有一跨膜螺旋,和位于细胞内的N-末端及伸向细胞外的C-末端.Northern分析证明褐飞虱的取食是维持BpHi008A基因的表达的必要条件.以pET28 a为载体构建了非融合表达的重组质粒,并且实现了BpHi008A基因在大肠杆菌菌株BL21(DE3)中的高效表达.     

一个水稻抗纹枯病突变体的遗传分析及其基因的初步定位

高水平抗纹枯病突变体和高感纹枯病品种蜀恢881杂交构建分离群体,经F2分离世代的遗传分析,抗、感单株比例符合3 1(χc2=0.563,χ12,0.05=3.84),初步确定该突变体对纹枯病的抗性由一对显性主效基因所控制,命名为Rsb-2(t)。利用已合成的530对微卫星引物,对抗纹枯病突变体和蜀恢881进行多态性引物筛选,用多态性引物对上述F2分离群体的全部感病单株和部分抗病单株的DNA进行PCR分析,借助MAPERMAKER/EXP3.0软件,对其微卫星标记实验数据进行连锁分析,将Rsb-2(t)定位于第3染色体的p臂,发现RM218、RM251、RM4321和RM5748与Rsb-2(t)连锁,它们均位于着丝粒端,连锁距离分别为32.1 cM,41.1 cM,42.4 cM和49.7 cM。研究结果为进一步对该基因的精细定位奠定了基础。     

一个新的水稻矮秆突变体sd-sl的遗传与基因定位研究

新的矮秆基因的发掘、研究和利用对水稻育种和植物生长发育机制研究有重要的作用.用60Coγ射线辐照粳稻9522,获得一个能稳定遗传的突变体.该突变体表型为株高较野生型矮,叶片短而微卷.将该突变体与籼稻广陆矮杂交,F2代呈3∶1分离,说明该突变体受隐性单基因控制.通过InDel分子标记对F2代分离群体进行遗传定位,将该基因定位于第6染色体InDel标记OS604附近.随后又发展了多对有多态性的InDel分子标记,将该基因座位精细定位在InDel标记XL6-6和XL6-1之间,AP003490和AP005619上,两个引物之间的物理距离为118 kb.本研究为该克隆基因及其作用机理的探究奠定了基础.     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

一个水稻白化致死突变体abl25鉴定及其基因定位

经Co60辐照的粳稻嘉花1号得到一个新的致死白化突变体albino lethal 25(abl25),该突变体从发芽至4叶期表现为白化苗,之后逐渐死亡.与野生型嘉花1号相比,abl25突变体的叶绿素含量和类胡萝卜素的含量大大降低,叶绿体结构不正常,说明其叶绿体发育受到严重阻碍,导致植物死亡.遗传分析表明:该突变体受一对隐性核基因(abl25)控制,进一步利用abl25与广占63S杂交的F2分离群体,将该突变体基因(abl25)定位于第2染色体上SSR标记RM424与Indel分子标记ID7330之间,随后利用新开发的分子标记和扩大群体将其定位在Indel分子标记ID9111和ID9261之间的150 kb内,发现abl25是一个新的水稻苗期白化致死基因.     

Effects of herbivore-induced rice volatiles on the host selection behavior of brown planthopper, Nilaparvata lugens

It has been suggested that herbivore would react to volatiles produced by herbivore infested plant due to potential change, either positive or negative, in the acceptability of the host plant. This hypothesis was tested for the brown planthopper (BPH) in the laboratory. Sixteen components of the headspace volatiles from rice seedlings with different treatments were collected with SPME and Tenax-TA trap and analyzed with GC and GC-MS. Significant differences in volatile emissions were observed for rice plants with different treatments. Undamaged control plants, mechanically damaged plants and the plants infested by BPH for 1 or 2 d emitted much lower amounts of volatiles compared to the plants infested by BPH for 3 or 5 d. The plants infested by BPH for 3 or 5 d emitted several volatiles that were not detected in undamaged control plants, mechanically damaged plants or the plants infested by BPH for 1 or 2 d. Spodoptera litura infested plants released much higher amounts of volatiles than those in all other treatments, and the contents of several green leaf volatiles, methyl salicylate and terpenoids increased dramatically. In dual-choice flight tunnel experiments, adult BPH females showed no significant preference between the untreated healthy plants and mechanically damaged plants or the plants infested by BPH adult females. However, rice plants damaged by S. litura had a clearly repellent effects on BPH adult females compared to healthy undamaged plants, mechanically damaged plants or the plants infested by BPH.     

转GNA+SBTi双价基因抗虫水稻的遗传分析及抗虫性研究

研究了转GNA SBTi双价基因籼稻的遗传规律及对褐飞虱和稻纵卷叶螟的抗虫实验。分子检测结果表明：外源基因在R2代株系中发生了分离，部分株系分离符合3：1或15：1的盂德尔遗传规律，但也有分离比为1：1及其它不正常分离的株系存在。对这些株系的特别追踪发现这几种分离式样在R3代中出现交叉，对R2和R3代转基因植株进行抗虫实验，结果表明分别有83.3%和76.9%的株系对褐飞虱的抗性较原种对照均有了显的提高，78.6%的R2代株系对稻纵卷叶螟的抗性比原种对照的抗性强，且大部分R3代株系继续保持良好的抗稻纵卷叶螟的性质，获得了9个分子检测阳性率高且对褐飞虱和稻纵卷叶螟抗性增强的水稻株系。     

水稻叶色突变研究进展

水稻叶色突变体表型变异明显,易于观察、利用和鉴别。叶色相关基因在水稻叶绿体形成与发育、叶绿素代谢等过程中,具有极其重要的作用。目前,在水稻所有的染色体中都发现了控制叶色相关的基因。文章从叶绿素合成、降解、水稻叶色突变表型以及叶色变化的分子机制等方面阐述了水稻叶色突变形成的机制。     

Wolbachia在灰飞虱体内的分布

灰飞虱（Laodelphax striatellus)能传播水稻条纹叶枯病,是我国主要的水稻害虫,有些灰飞虱体内含有细胞内共生菌－Wolbachia。Wolbachia是一种细胞质遗传的细胞内共生菌,这类细菌改变了宿主的生殖行为,引起细胞质不相容（CI）,雌性化及孤雌生殖等现象,导致带有Wolbachia的宿主具有生殖优势。采用PCR和Western杂交的方法分析Wolbachia的灰飞虱体内不同组织的分布状况,发现灰飞虱体内Wolbachia除存在于生殖组织外,还广泛分布在头、胸、腹、唾液腺、消化道等非生殖组织中。这种广泛的分布状态说明Wolbachia是在昆虫体内引入、表达与传播外源基因的良好媒介。     

活跃传毒介体灰飞虱(Laodelphax striatellus)品系的杂交与选育

对水稻条纹叶枯病毒传播介体进行连续4个世代的杂交和严格的人工选育，获得活跃传毒的介体品系，其传毒率从F0群体的5.31%上升到25%，该介体品系经2年时间无人工选择压力的连续饲养，在间断选育的第5代，其传毒率明显下降，仅有14.75%，可见人工选择压对介体传毒力这一性状的稳定起着重要的作用，但对介体获毒没有影响，单头昆虫的DIBA技术使测定标本用量可以少至1/30头灰飞虱，带毒虫率从F0群体的12.09%上升到F4代的32.6%。     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

一个新的水稻幼苗黄叶突变体的遗传分析及其基因的初步定位

在60Coγ射线辐照的水稻突变体库中,发现了一个以粳稻品种日本晴为遗传背景的幼苗叶色黄化突变体syl11(seedling yellow leaf 11).与野生型相比,突变体幼苗第二和第三叶表现黄色,在其完全展开之前叶片自其顶端开始转绿,长到四叶期其叶色恢复正常;并且该突变体syl11幼苗黄色叶片光合色素含量明显下降.遗传分析表明,该突变体的遗传性状由1对隐性核基因控制.本研究以培矮64S/syl11的F2代突变型植株作为定位群体,应用微卫星(SSR)分子标记以及新发展的InDel分子标记,将基因syl11定位在水稻第11号染色体长臂上的RM26652和处于着丝粒附近的ID11974分子标记之间,其遗传距离分别为0.5 cM和0.7 cM.     

一个水稻苗期白化致死突变体asl6的鉴定及其基因定位

经60Coγ诱变处理粳稻\"嘉花1号\"得到一个稳定遗传苗期白化致死突变体asl6(albino seedling lethality 6).与野生型(WT)相比,该突变体从发芽出苗起一直表现白化,四叶期逐渐死亡,叶合色素含量几乎没有且没有完整的叶绿体结构.通过qRT-PCR分析发现,与叶绿体发育、叶绿素合成及光合作用相关的基因表达量明显下调.对利用asl6突变体与\"培矮64S\"杂交获得的F2代分离群体进行遗传分析,发现该突变表型受单个隐性核基因控制.利用图位克隆技术将该asl6基因定位于第2号染色体的InDel分子标记ID31982与SSR分子标记MM5712之间约293 kb的区域内.目前,该范围内没有叶色相关基因的报道,可能为一新的调控水稻叶绿体发育的基因.     

一个新水稻低温敏感叶色突变体tcm11的鉴定及基因定位

对粳稻\"嘉花1号\"经~(60)Coγ诱变处理获得的稳定遗传低温敏感叶色突变体tcm11(thermo-sensitive chloroplast mutant 11)进行了表型鉴定与遗传分析.在20℃条件下,该突变体三叶期之前幼苗均表现为黄色,光合色素含量明显下降,叶绿体发育不完整,从第4叶开始逐渐转为浅黄绿色直至最后死亡.而在32℃条件下,其表型与野生型相比没有明显差异,具有低温敏感属性.通过对培矮64S与tcm11杂交的F_2代分离群体进行遗传分析,发现该低温敏感突变体性状是受单个隐性核基因(tcm11)控制,利用图位克隆技术对tcm11进行定位,将其定位在第11号染色体的InDel分子标记ID13252与SSR分子标记MM1361之间一个约1 566 kb的区域内.这也为后续的研究奠定了基础.