奶牛冷冻胚胎与滋养层细胞囊泡共移植对受胎率及有关繁殖参数的影响

将奶牛冷冻胚胎与囊胚滋养层细胞囊泡(Trophoblastic vesicles,TRV)共移植,TRV来源于体外受精培养7d、体内培养7d的囊胚.胚胎移植受体牛超声波妊娠诊断共进行两次,即发情的第26～43天和38～73天.第一次妊娠诊断时试验组(与TRV共移植组)妊娠率(66.67%,18/27) 著高于对照组(45.16%,14/31)(P<0.05).实验组和对照组的分娩率分别为55.56%(15/27)41.94%(13/31),两者间无显著差异.两组间受体牛的妊娠期和后代初生重相似.     

滋养层细胞囊泡与奶牛低品质胚胎共移植对受胎率的影响

将奶牛低品质胚胎与囊胚滋养层细胞囊泡（trophoblastic vesicles,TRV）共移植,TRV来源于体外受精培养 7 d、体内培养7 d囊胚的囊胚滋养层细胞。胚胎移植受体牛超声波妊娠诊断共进行2次,即发情的第26～43天和第38～73天。第1次妊娠诊断时试验组（与TRV共移植组）妊娠率（66.7%,16/24）显著高于对照组（34.5%,10/29）（P＜0.05）。在第1次和第2次妊娠诊断期间试验组有3头牛的胚胎死亡。试验组和对照组的分娩率分别为41.7%（10/24）和27.6%（8/29）,两者间无显著差异。两组间受体牛的妊娠期和后代出生重相似。     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

Influence of post harvest temperature treatments,storage period and harvest date on development of spraing caused by tobacco rattle virus and potato mop-top virus

Summary The frequency of PMTV-spraing and TRV-spraing increased during storage in several cultivars. The only exception was cv. Saturna, where a decrease was observed in 1992. A spraing inducing treatment of one week at 18 °C followed by one week at 8 °C immediately after an early harvest increased the frequency and provided a rough estimate of the development of spraing in untreated tubers during storage. Under most circumstances wound healing at 25 °C compared with 15 °C resulted in a lower frequency of spraing during the storage period. The date of harvest had an important influence on development of spraing during storage and on the effect of post harvest temperature treatments. Therefore, physiological processes that induce or erase spraing are strongly related to tuber maturity. The mechanisms controlling development of symptoms seemingly are identical for the two spraing inducing vira.     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

VIGS实验技术体系在月季中的应用及优化

旨在月季中建立高效、快速的VIGS实验技术体系,以期能够改进VIGS技术体系在月季等木本植物表达效率低等缺陷。采用RT-PCR方法进行月季RhPDS 基因片段的克隆,在以pTRV2 为原载体,采用双酶切的方法构建了pTRV2-RhPDS 重组载体。经双酶切及测序验证后,通过电击法转化农杆菌GV3101,并分别采用高压喷枪法对月季植株进行侵染、采用真空渗透法对扦插苗以及组培苗进行侵染。采用qRT-PCR方法分析RhPDS 基因的表达。结果显示：构建的重组载体pTRV2-RhPDS,经双酶切验证,显示条带单一清晰,符合预期目标。在侵染方法和材料上,采用真空渗透法侵染组培苗是最为快速、高效的方式。该试验获得了较为高效、快速VIGS 技术体系,提高了VIGS 技术体系在月季上表达效率。     

不同分割液对奶牛不同阶段胚胎分割效果的影响

本试验探讨了3种不同分割液对奶牛桑葚胚和囊胚分割效果的影响。借助显微操作仪，将发育至第6～8天的体内常规生产的桑葚胚和囊胚进行分割，体外培养半胚，观察其发育情况，选择形态恢复好的半胚与一个囊胚滋养层细胞囊泡（trophoblastic vesicles，TRV）共移植。结果显示，在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割桑葚胚，其分割成功率显著高于PBS（P<0.05），分别为89.13%、86.73%和69.67%，而半胚的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；在PBS+0.2 mol/L蔗糖与PBS+5%PVP中分割囊胚， 其分割成功率显著高于PBS（P<0.05），分别为94.52%、92.52%和70.52%，而半胚培养的囊胚发育率及移植妊娠率三者均无显著差异（P>0.05）；说明在PBS中分别添加0.2 mol/L的蔗糖和5%的PVP有利于提高奶牛桑葚胚和囊胚的分割成功率。     

Potato Transformed with a 57-kDa Readthrough Portion of the Tobacco Rattle Virus Replicase Gene Displays Reduced Tuber Symptoms when Challenged by Viruliferous Nematodes

Summary A 57-kDa readthrough portion of the Tobacco rattle virus (TRV) replicase gene was isolated in two replicates and sequenced. Sequence comparisons showed the isolated sequences to have a homology of 93–94% compared to other TRV isolates at the DNA level and code for proteins with 98–99% similarity to other corresponding TRV sequences at the amino acid level. The isolated sequences were cloned into binary vector constructs, which were transformed to the potato cultivar Matilda. Ten obtained transgenic potato lines were challenged with TRV mediated by the natural nematode vector in greenhouse trials. In a first trial all transgenic lines demonstrated fewer symptoms compared to an untransformed Matilda control. However, the infection pressure in this first trial was in general very low and to get more reliable results a second greenhouse trial with the two most promising lines was performed the following season. In this trial both transgenic lines had significantly fewer symptoms compared to the untransformed control. The symptom reduction for the best line was 78% compared to the control. Thus, introduction into potato of a gene encoding the 57-kDa TRV replicase portion is a possible way to improve resistance to TRV in potatoes.     

Molecular cloning and characterization of a novel gene encoding an EF-hand calcium-binding protein related to fruit seedlessness of grapevine

EF-hand calcium-binding protein (CBP) gene plays a critical role during embryonic development in animals, but it remains unclear whether this gene possesses similar function in plants. In this study, a novel CBP gene was isolated from Vitis vinifera cv. Thompson Seedless and designated as VvCBP1. Using a combination of sequence structural investigation, expression pattern analysis of different embryo development in grapevine, and silencing assay of VvCBP1 homolog in tomato, the functional role of VvCBP gene in embryo development of seedless grapevine was studied. Structural investigations showed that VvCBP1 is 907 bp in length, and contains two EF-hand motifs. Expression pattern of this gene is different in embryo between seedless and seeded grapevine species. Moreover, the silencing of homology gene led to the seeds number significantly decreased in tomato. These data demonstrate clearly that VvCBP1 plays an important role in embryo development of seedless grapevine.     

Effects of Tuber-borne M-type Strain of Tobacco Rattle Virus on yield and Quality Attributes of Potato Tubers of the Cultivar Wilja

It was previously thought that Tobacco Rattle Virus (TRV) was self-eliminating from seed potato stocks and that the principal effects of the virus were the spraing symptoms (arcs or lines of corky brown tissue) formed in the tuber flesh. Recent work has clearly demonstrated that the virus can become fully and systemically established in some potato cultivars, with few, if any, tuber flesh symptoms. The studies reported here demonstrate that, in at least one such cultivar, an M-type strain of the virus can have a considerable effect on the growth and quality of the plant and its produce. When infected material was compared with healthy material, overall yield and yield components were severely affected by TRV, as were quality traits such as dry matter, after-cooking blackening and chemical components such as sugars, glycoalkaloids and chlorogenic acid. The results are discussed in terms of plant response to virus infection and plant protection mechanisms.