侵染百合的李属坏死环斑病毒病（PNRSV）的研究

 【目的】在分子水平上鉴定百合上发生的李属坏死环斑病毒（Prunus necrotic ringspot virus,PNRSV）【方法】对待测百合种球随机抽样，在温室内种植并观察，长出叶片后，用RNA Extraction Kit (Sangon, China)从幼嫩的叶组织中提取核酸，将反转录出的cDNA用 PNRSV的特异性引物进行PCR扩增，电泳PCR产物得到了450bp的目的片段。低熔点胶回收并克隆至pGEM-T-easy载体测序后进行序列比对分析。【结果】 从百合中所扩增的450bpcDNA的核苷酸序列与27个早期已报道的PNRSV分离物的CP 基因的同源性为84.5%～99.1%。【结论】百合是PNRSV的一新寄主。     

李属坏死环斑病毒（PNRSV）RT-LAMP检测方法的建立

针对李属坏死环斑病毒（PNRSV）外壳蛋白（CP）基因保守序列,设计了1套特异性识别CP基因中6个不同区段的环介导等温扩增（LAMP）引物。通过对反应条件的优化,建立了适用于PNRSV的RT-LAMP检测技术,对优化后的方法进行特异性、灵敏度评价。结果显示,此方法能够特异性的检测出PNRSV,对马铃薯X病毒（PVX）、马铃薯Y病毒（PVY）、黄瓜花叶病毒（CMV）、蚕豆萎蔫病毒（BBWV）的检测均为阴性,灵敏度高于RT-PCR 10倍。     

Molecular Identification and Cultivar Fingerprints of Prunus persica （L.） Batsch Germplasms

[Objective] The aim was to study the molecular identification and cultivar fingerprints of Prunus persica （L.） Batsch germplasms.[Method] Sixty peach genotypes,representing China common local cultivars and European samples were screened by microsatellites （simple sequence repeats,SSRs） and Inter-Simple Sequence Repeat （ISSR） markers.[Result] 26 reproducible bands were amplified by Nine SSR primers,and 24 of which were polymorphic; 236 bands were amplified by 30 ISSR primers,and 113 of which were polymorphic.31 genotypes were discriminated with 1-3 distinct polymorphic bands generated from the primers ISSR and SSR.Seven cultivar-specific ISSR fragments and two SSR unique alleles obtained from this study were available to be converted into Sequence Characterized Amplified Region （SCAR） markers.The genetic similarity coefficient （GS） estimated from these molecular data averaged were 0.939 （ranged from 0.856 to 0.983） for ISSR and 0.646 （ranged from 0.240 to 1.000） for SSR,respectively.The combined grouping association indicated that most local Chinese peach cultivars and exotic accessions were clustered together.This could be related to the mode of introduction and maintenance of the peach cultivars involving limited foundation germplasm,exchange of cultivars between plantations,and periodic development of new recombinant cultivars following sexual reproduction.[Conclusion] The results obtained in this work would help to improve the conservation,molecular identification and management of peach germplasm in breeding.     

Preliminary Studies on Thickness of Nondestructive Rice （Oryza sativa L.） Leaf Blade

Two varieties, Yuexinzhan and Guangchao 3, were used to study leaf thickness in rice in this experiment. The thickness of the leaf blade was measured by the nondestructive leaf thickness instrument, which was modified from the thickness instrument for steel objects （John Bull, England）. The contacting area between the leaf and the probe of the instrument was 0.5 cm^2. There was no significant difference between the thickness of steel materials measured by the nondestructive rice leaf thickness instrument and the micrometer. The correlation between the thickness of the rice leaf blade measured by the nondestructive rice leaf thickness instrument and the specific leaf weight （SLW） was significant （P 〈 0.05 or P〈 0.01）. The results also showed that the rice leaf thickness was uneven and asymmetric. The thickness and SLW of flag leaf tended to increase from the base to the tip of the leaf blade. The middle part of the second and third top leaf was the thickest, but no significant difference in thickness between the basal part and the fore part was found. Drawing a line on the main vein in the top three leaves, the left part was thinner than the right part. The thickness of the lower leaves （6/0-9/0） on the main culm tended to increase with increasing positions of the leaves in the early and middle stages, but the tendency was not the same for the higher leaves （10/0 upwards）, although the higher leaves （10/0 upward） were thicker than the lower leaves （9/0 or downward）. Furthermore, different CO2 concentrations （550±30, 460 ± 30 μmol mol^-1） in the growth boxes had no effect on the thickness of rice leaf blades. It can be concluded that the measurement of rice leaf thickness using the nondestructive rice leaf thickness instrument is simple, precise, and nondestructive.     

Studies on some characteristics of nitrate reductase from sugar beet （Beta vulgaris L.）leaves

Some characteristics of nitrate reductase from sugar beet leaves shown in this paper were as follows:The nitrate reductase from sugar beet leaves required NADH as an electron donor.Accordingly,the nitrate reductase was classified as NADH-dependent(E.C.1.6.61).The Km value of the nitrate reductase for NADH and NO3^- were 0.86m mol and 0.18μ mol respectively.The optimum pH in reaction mixture solution for nitrate reduction activity was 7.5.The effect of variable concentrations of inorganic phosphorus in the reaction buffer on nitrate reductase activity was investigated.When the inorganic phosphorus concentration was below 35m mol,the nitrate reductase activity was increased with increase of inorganic phosphorus concentration.Conversely,when the inorganic phosphorus concentration was over 35m mol,the nitrate reductase activity was inhibited.The nitrate reductase activity assayed in vitro was 3.2 and 5.6times of that assayed in vivo under the condition of exogenous and endogenous ground substance respectively.     

李属坏死环斑病毒怀柔分离物(PNRSV-HR)外壳蛋白基因片断的克隆与序列分析

应用RT-PCR方法检测了采自北京市怀柔区的樱桃叶片,将此PCR扩增产物连接到pMD18-T载体上,通过转化大肠杆菌DH5α感受态细胞,克隆了含有目的片段的重组质粒,并进行序列测定和分析。序列分析结果表明,认为该分离物外壳蛋白基因片断与李属坏死环斑病毒的NRSiz8株系的同源性最高,为98%,与德国分离物Ring17的同源性达99%;通过进化关系分析,认为该分离物属于引起温和症状的group,认为该分离物的第5,62,81和126位氨基酸也与CH9血清型引起温和症状的保守的替换相同。     

安徽省水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的 分子检测和序列分析

近年来,水稻黑条矮缩病毒(RBSDV)和南方水稻黑条矮缩病毒(SRBSDV)在安徽各稻区危害日益严重,生产上仅凭症状难以区分2种病毒。本研究建立的RT-PCR方法可以实现一次性检测和鉴定RBSDV和SRBSDV 2种病毒,根据RBSDV和SRBSDV S9保守序列设计3个引物,不仅可以从单独感染RBSDV或SRBSDV的水稻样本中分别扩增出1条大小不同的特异性条带,而且还可以从感染RBSDV和SRBSDV的混合水稻样本中一次性扩增出2条大小不同的特异性条带。从安徽省庐江县、郎溪县、怀宁县和宣州市4个地区水稻田采集表现明显矮缩病症状的水稻样本,RT-PCR检测结果表明,庐江和郎溪水稻样本受到RBSDV侵染,怀宁和宣州水稻样本受到SRBSDV侵染。选取庐江县、郎溪县、怀宁县和宣州市各1个水稻样本,利用RT-PCR扩增出特异性条带,再分别克隆和测序。序列分析表明,庐江、郎溪2个样本的核苷酸序列与RBSDV-Shandong和RBSDV-Zhjr S9部分片段序列相似性高达99.3%～99.8%,且与RBSDV的亲缘关系最近,说明庐江和郎溪样本感染的病毒是RBSDV的2个分离物;怀宁、宣州2个样本的核苷酸序列与SRBSDV-Shangdong和RBSDV-2 S9部分片段序列相似性高达99.5%,且与SRBSDV亲缘关系最近,说明怀宁和宣州样本感染的病毒是SRBSDV的2个分离物。     

应用RAPD标记对百合杂交种真实性鉴定的研究

应用RAPD标记对百合杂交种真实性进行了鉴定。研究表明:采用RAPD分子标记对百合杂交种进行早期鉴定是一种快速有效的方法。在本试验中杂种既具有相加性带型、单亲本带型,又具有融合双亲的特异带,并出现了新的带型。这样可以通过多个引物的应用,对杂交种进行早期鉴定。     

马哈利樱桃砧木组培快繁技术研究

以马哈利樱桃砧木的嫩梢茎尖为外植体进行离体培养,筛选出的起始培养基为MS+6-BA 0．2- 0．5 mg／L;最佳增殖培养基为MS+6-BA 2．0 mg／L+IAA 0．5 mg／L+GA3 0．5 mg／L,此时增殖倍数达7．06,平均 苗高2．79 cm;最佳生根培养基为1／2 MS+IAA 2．0 mg／L或1／2 MS+IBA 0．6 mg／L,暗培养7 d后移至温室或 大棚,在自然光下生根炼苗,生根率达85％以上,单株平均根数3-6条,根系白嫩,组培苗生长整齐健壮;以营养土 为基质,用50 mg／L生根粉泥浆溶液处理生根组培苗的移栽成活率达87．0％以上。该技术体系适宜工厂化生产的 要求。     

RT-PCR方法检测烟草环斑病毒的研究

 根据烟草环斑病毒（TRSV）外壳蛋白基因非编序列设计的引物P1,P2,用感病及健康组织总RNA为模板,进行cDNA 合成和PCR试验,感病组织中扩增出了600 bp的目的片段,而健康组织中无此扩增带。摸索各项实验条件,建立了RT-PCR检测烟草环斑病毒（TRSV）的方法。     

硝酸银对中国李原生质体分离和培养的影响

以种胚愈伤组织为试材,研究了硝酸银对中国李原生质体分离和培养效果的影响。结果表明,在酶解液中加入4～10mg/L质量浓度的硝酸银,可提高原生质体的活力,其中以8mg/L的效果最好,原生质体的活力比对照提高21.1%,但其对原生质体产量无明显作用。在培养基中加入4～8mg/L质量浓度的硝酸银,原生质体的分裂频率和植板率分别比对照增加19.85%～38.24%和9.23%～36.53%。硝酸银对愈伤组织不定芽的再生能力也具有明显的促进作用。     

白芷花叶病的毒原鉴定

在陕西杨陵地区,从严重受害呈现花叶症状的白芷植株上分离到一种病毒。该病毒通过磨擦接种,能侵染10科25种以上植物;TDP为65℃,DEP为10~(-3),L则为4天;桃蚜及蚕豆蚜不能传播,但能通过种子和汁液进行传播;可与烟草环斑病毒(TRSV)抗血清产生明显的沉淀反应;该病毒粒子呈圆球形,大小30nm。此病毒被鉴定为TRSV。     

日光温室油桃光合特性的观察研究

观察结果表明,日光温室内油桃的净光合速率以晴天10:00和14:00时较高,12:00时稍低,表面为“双峰”曲线。而阴天时表现为“单峰”曲线,12:00时净光合速率最高。晴天时,日光温室内油桃的净光合速率在12:00时降低的主要原因是由日光温室内空气中CO2浓度不足引起的,适宜日光温室内主要环境因子CO2 的浓度不得低于310．1μL·L 1．适宜的温度为21．0-32．5℃,空气相对湿度不得低于45．3％。