番木瓜抗病突变体阻碍环斑病毒体内运转

应用RT-PCR一步法检测了PRSVYs株系在感病番木瓜及其抗病突变体植株体内的运转动态,结果表明在感病植株中,接种后48hr接种叶的未接种部位可检出病毒,第4天部分接种叶柄可检出病毒,第6天植株各部位均能检出病毒;而在抗病植株中,接种后可以而且仅能在接种部位检出病毒;因而认为抗病突变体能够阻碍病毒从接种部位运出及（或）向未接种部位运入。     

兼抗抗白粉病和黄矮病小麦育种材料的创造与鉴定

白粉病和黄矮病是小麦生产上的重要病害,近几年来这两种病害经常在我国一些小麦产区同时发生。为解决该问题,本研究通过杂交、回交方法将抗黄矮病的Bdv2基因（源自于YW642）和抗白粉病的Pm21基因（源自于CB037）聚合在一起,育成了兼抗黄矮病和白粉病的小麦新材料。通过田间抗病性鉴定与分子标记辅助选择相结合,得到聚合了Bdv2基因和Pm21基因的BC1代小麦22株,F2代小麦51株。农艺性状调查显示,这些含Pm21和Bdv2基因的双抗白粉病和黄矮病小麦新材料的农艺性状优于感病植株和原先的亲本,可以在小麦白粉病和黄矮病兼性抗病育种中作为优异种质资源加以利用。     

番木瓜抗病突变体阻碍环斑病毒体内运转

应用RT－PCR一步法检测了PRSV Ys株系在感病番木瓜及其抗病突变体植株体内的运转动态,结果表明：在感病植株中,接种后48hr接种叶的未接种部位可检出病毒,第4天部分接种叶柄可检出病毒,第6天植株各部位均能检出病毒;而在抗病植株中,接种后可以而且仅能在接种部位检出病毒;因而认为抗病突变体能够阻碍病毒从接种部位运出及（或）向未接种部位运入。     

抗黄矮病小麦新品系YW443的分子细胞遗传学鉴定

以小麦－中间偃麦草二体附加系Ｌ１衍生抗病系ＰＰ９－１为抗源,与小麦推广品种陕７８５９．丰抗８号杂交并自交,在Ｆ６代中选到农艺性状优良的高抗黄矮病小麦新品系ＹＷ４４３。对ＹＷ４４３及其亲本进行抗病性鉴定。结果表明：ＹＷ４４３高抗大麦黄矮病毒ＧＰＶ、ＧＡＶ株系。利用基因组原位杂交,ＲＦＬＰ分析和ＲＡＰＤ分析,研究诉遗传构成及其抗病基因染色体归属。结果表明：ＹＷ４４３（２ｎ＝４３）的遗传构成了４０条（２     

转病毒来源发夹RNA小麦表现对大麦黄矮病毒的抗性

将大麦黄矮病毒GPV株系的复制酶基因片段和CP基因片段构建成可在植物细胞内表达含有双链复制酶RNA(茎)和反义CP RNA(环)的复合发夹RNA结构, 希望能够诱发植物体针对病毒的RNA干扰作用, 从而达到抗病毒目的。利用基因枪法将该结构导入小麦幼胚愈伤组织细胞后, 通过在幼苗再生阶段进行以叶片为模板的快速PCR来加速阳性植株的筛选过程, 最终共获得基因组整合有外源基因的小麦再生植株21株。对再生植株接种不同剂量的病毒, 其中9株对BYDV-GPV有低度抗性, 表现在低接毒量时无症状, 接毒量提高时发病且严重; 6株具中度抗性, 表现在低接毒量时无症状, 接毒量提高时局部有不严重症状; 6株具高度抗性, 两种情况下均无症状。抗性实验结果表明, hpRNA介导对BYDV的抗性可能受到BYDV含量的影响, 具有剂量效应的特点。     

大麦黄矮病毒的冰核活性与作物霜冻的关系

对大麦黄矮病毒(Barley Yellow Dwarf Virus,BYDV)的冰核活性,以及它的侵染与作物霜冻的关系进行了研究.利用人工摸拟霜箱,测试了接种BYDV的小麦等作物植株的霜冻温度,并用ELISA法检测了供试植株的病毒含量.结果表明,接种样品与对照相比,感病品种的霜冻温度升高1—2℃以上,抗病品种的霜冻温度变化不大.离体叶片测定结果表明,“中7902”的叶片中,病毒含量与霜冻温度成正相关,说明BYDV可以起到异源冰核(heterogeneous ice nuclei)的作用,它的侵染能影响作物的抗霜冻能力.用“Vali小液滴冻结法”检测提纯的BYDV,证明BYDV具冰核活性,从而首次发现病毒也能起到生物冰核的作用.     

大麦黄矮病毒的冰核活性与作物霜冻的关

对大麦黄矮病毒(Barley Yellow Dwarf Virus,BYDV)的冰核活性,以及它的侵染与作物霜冻的关系进行了研究.利用人工摸拟霜箱,测试了接种BYDV的小麦等作物植株的霜冻温度,并用ELISA法检测了供试植株的病毒含量.结果表明,接种样品与对照相比,感病品种的霜冻温度升高1—2℃以上,抗病品种的霜冻温度变化不大.离体叶片测定结果表明,“中7902”的叶片中,病毒含量与霜冻温度成正相关,说明BYDV可以起到异源冰核(heterogeneous ice nuclei)的作用,它的侵染能影响作物的抗霜冻能力.用“Vali小液滴冻结法”检测提纯的BYDV,证明BYDV具冰核活性,从而首次发现病毒也能起到生物冰核的作用.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

用分子标记定位源于中间偃麦草的小麦抗黄矮病基因

利用生物素标记的中间偃麦草基因组总DNA作为探针 ,对以L1为抗源的抗黄矮病小麦新品系H960 642的有丝分裂中期染色体进行原位杂交 ,结果表明 :H960 642是纯合的小麦 中间偃麦草易位系 ,携有抗黄矮病基因的中间偃麦草染色体片段易位到小麦染色体端部 .采用小麦第 7部分同源群上的 8个RFLP探针进行Southern分析 ,结果表明 :H960 642的小麦 7D染色体长臂末端片段被中间偃麦草染色体 7X长臂末端片段所取代 ,即该染色体为T7DS·7DL -7XL ,易位断点位于Xpsr680和Xpsr965之间 ,距着丝点的遗传距离约 90～ 99cM .筛选出了与 7X上抗黄矮病基因紧密连锁的RFLP标记psr680和psr687.将该抗黄矮病基因定位于 7XL端部、RFLP遗传图Xpsr680与Xpsr687位点附近 .Ep 1同工酶分析结果佐证了RFLP分析结果 .     

谷子、大麦、高粱抗黄矮病候选基因的生物信息学比较分析

黄矮病是禾本类作物的主要病害之一,该病主要由大麦黄矮病毒侵染所致,侵染该病毒后植株生理活动紊乱,叶片黄化至红化,结实率低,严重影响农作物的产量。大麦黄矮病毒最早发现于大麦作物中,对大麦抗黄矮病的研究较早,但至今仍未从大麦克隆到黄矮病抗病基因。利用病毒诱导基因沉默系统和转基因技术,TIRB基因被验证具有抗黄矮病功能,是小麦抗黄矮病的关键基因。我们通过用小麦TIRB基因序列,在谷子、高粱和大麦基因库进行BLAST比对,发掘了谷子、高粱和大麦抗黄矮病候选基因,并运用生物信息学软件,分析、比较了这些基因所表达蛋白的理化性质,蛋白的亲疏水性及亚细胞定位,信号肽预测及三级结构等。不同物种中的这些蛋白相似的性质暗示着它们可能执行相近的抗病机制。总之,三种候选基因的发现,对于通过生物信息手段发掘作物抗病基因具有一定的指导意义。     

Research progress in BYDV resistance genes derived from wheat and its wild relatives

Barley yellow dwarf virus （BYDV） may cause a serious disease affecting wheat worldwide. True resistance to BYDV is not naturally found in wheat. BYDV resistance genes are found in more than 10 wild relative species belonging to the genera of Thinopyrum, Agropyron, Elymus, Leymus, Roegneria, and Psathyrostachy. Through wide crosses combining with cell culture, use ofph mutants, or irradiation, 3 BYDV resistance genes in Th. intermedium, including Bdv2, Bdv3 and Bdv4, were introgressed into common wheat background. Various wheat-Th, intermedium addition and substitution, translocation lines with BYDV-resistance were developed and characterized, such as 7D-TAi#1 （bearing Bdv2）, 7B-7Ai#1, 7D-7E （beating Bdv3）, and 2D-2Ai-2 （bearing Bdv4） translocations. Three wheat varieties with BYDV resistance from Th. intermedium were developed and released in Australia and China, respectively. In addition, wheat-Agropyron cristatum translocation lines, wheat-Ag, pulcherrimum addition and substitution lines, and a wheat-Leymus multicaulis addition line （line24） with different resistance genes were developed. Cytological analysis, morphological markers, biochemical markers, and molecular markers associated with the alien chromatin carrying BYDV resistance genes were identified and applied to determine the presence of alien, chromosomes or segments, size of alien chromosome segments, and compositions of the alien chromosomes. Furthermore, some resistance-related genes, such as RGA, P450, HSP70, protein kinases, centrin, and transducin, were identified, which expressed specifically in the resistance translocation lines with Bdv2. These studies lay the foundations for developing resistant wheat cultivars and unraveling the resistance mechanism against BYDV.     

Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat.

Wheatgrasses (species of Agropyron complex) have previously been reported to be resistant to barley yellow dwarf virus (BYDV). To introgress this resistance into wheat, Triticum aestivum x Thinopyrum (Agropyron) intermedium hybrids were advanced through a backcrossing program and reaction to BYDV, as determined by enzyme-linked immunosorbent assay (ELISA), is reported for the first time in backcross populations of wide hybrids between wheat and wheatgrasses. ELISA values revealed highly resistant to highly susceptible segregants in backcrosses. BYDV resistance was expressed in some backcross derivatives. Continued selection, based on cytology and ELISA in each generation, eliminated most of the unwanted wheatgrass chromosomes and produced self-fertile BYDV resistant wheat lines. The BYDV resistant lines with 2n = 42 had normal chromosome pairing similar to wheat, and their F1 hybrids with wheat had two univalents. DNA analyses showed that the source of alien chromatin in these BYDV resistant wheat lines is distinguishable from that in other Th. intermedium derived BYDV resistant wheat lines. Chromosome pairing and restriction fragment length polymorphism analyses indicated that the 42 chromosome resistant Purdue wheat lines are substitution lines in which chromosome 7D was replaced by a chromosome from Th. intermedium that was carrying gene(s) for BYDV resistance.     

Identification and characterization of wheat-wheatgrass translocation lines and localization of barley yellow dwarf virus resistance.

Stable introgression of agronomically important traits into crop plants through wide crossing often requires the generation and identification of translocation lines. However, the low efficiency of identifying lines containing translocations is a significant limitation in utilizing valuable alien chromatin-derived traits. Selection of putative wheatgrass-wheat translocation lines based on segregation ratios of progeny from gamma-irradiated seed using a standard phenotypic analysis resulted in a low 4% success rate of identifying barley yellow dwarf virus (BYDV) resistant and susceptible translocation lines. However, 58% of the susceptible progeny of this irradiated seed contained a Thinopyrum intermedium chromosome-specific repetitive sequence, which indicated that gamma-irradiation-induced translocations occurred at high rate. Restriction fragment length polymorphism (RFLP) analysis of susceptible lines containing alien chromatin, their resistant sister lines and other resistant lines showed that more than one third of the progeny of gamma-irradiated double monosomic seeds contained wheatgrass-wheat translocations. Genomic in situ hybridization (GISH) analysis of selected lines confirmed that these were wheatgrass-wheat translocation lines. This approach of initially identifying BYDV susceptible deletion lines using an alien chromosome-specific repetitive sequence followed by RFLP analysis of their resistant sister lines efficiently identified resistant translocation lines and localized the BYDV resistance to the distal end of the introgressed Th. intermedium chromosome.     

Screening for Barley Yellow Dwarf Luteovirus Resistance in Barley on the Basis of Virus Movement

The movement of barley yellow dwarf luteovirus (BYDV) was evaluated in susceptible and resistant barley and bread wheat genotypes. After leaf inoculation, the virus infected the root system and the growing point of susceptible earlier than resistant, barley genotypes. No difference in virus movement occurred in resistant and susceptible wheat genotypes. It was possible to reliably differentiate susceptible from resistant genotypes when root extracts of 41 barley genotypes were tested by DAS-ELISA 3 or 4 days after inoculation at the oneleaf stage. When barley plants inoculated at the two- or three-leaf stage were assayed by tissue-blot ELISA on nitrocellulose membrane, virus was detected in the phloem vessels of the growing points of the susceptible, but not of the resistant genotype, 4–6 days after inoculation. Our results thus suggest that screening for BYDV resistance in barley could be done quickly and cheaply especially when assays are made by the tissue-blot test.     

Molecular characterization of the genomic region harboring the BYDV-resistance geneBdv2 in wheat

Barley yellow dwarf virus (BYDV) can cause significant losses of wheat worldwide. The long arm segment ofThinopyrum intermedium chromosome 7Ai#1 carrying the BYDV resistance geneBdv2 was translocated to the distal region of the long arm of wheat chromosome 7D in translocation line Yw642. In this study, 40 wheat EST sequences located in the distal region of 7DL were explored to identify specific PCR markers for theBdv2 region on the basis of the homoeologous relationship between wheat chromosome 7D and Th.intermedium chromosome 7Ai# 1. Our results revealed 8 novel EST-PCR markers specific to theBdv2 region, including 5 EST-STS markers of BE404744, BE498985, BE591497, BG606695 and BQ161842, and 3 EST-SSCP markers of BE404953, BG312663 and BE498985. These EST-PCR markers could distinguishBdv2 from another BYDV-resistance gene located onTh.intermedium chromosome 2Ai-2. These specific bands for theBdv2 region were further cloned and sequenced. The sequencing analysis indicated that the specific sequences for theBdv2 region were highly homologous with the original wheat EST sequences that were used to design primers, and encode respectively a protein kinase, P450, centrin, transducin, and a hypothetical protein. This study created a starting point for eventual cloning of theBdv2 gene and understanding the defense mechanism.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Effect of BYDV Infection on the Cell Surface Glycoproteins in Common Wheat,Wheatgrass and Octoploid Wheat-Wheatgrass

Changes in the cell surface glycoproteins in common wheat 3B-2, Agropyron intermedium and octoploid wheat-wheatgrass Zhong 5 after the inoculation with barley yellow dwarf virus (BYDV) were sdudied using electron microscopy and ruthenium red staining. The results indicated that, after the inoculation with BYDV, different changes in cell surface glycoproreins were observed in the plant species with different levels of resistance. In A. intermediurn which is immune to BYDV, inoculation with BYDV did not cause significant change in cell surface glycoprotein layer. In cotoploid wheat-wheatgrass Zhong 5 which is highly resistent to BYDV, BYDV infection caused significant thickening in most cell surface glycoprotein layer. In common wheat 3B-2 which is susceptible to BYDV, BYDV infection did not cause thickening in cell surface glycoprotein layer, but in most cells, glycoproteins on the cell surface were partially peeled off or disappeared completely. Therefore, it is suggested that the glycoproteins on cell surface play certain roles in BYDV resistance. The phenomenon of the thickening of cell surface glycoprotein layer caused by BYDV infection was possibly a resistant reaction to the virus.