Isolation,cloning, and expression of five genes related to nitrogen metabolism in peach (Prunus persica L. Batsch)

An optimal dosage of nitrogen has long been known to play important roles in governing nitrogen-use efficiency and improving the yield of plants. However, there is limited information on the isolation and expression profiles of genes related to nitrogen metabolism in peach (Prunus persica L. Batsch). This study isolated five genes involved in nitrogen metabolism and evaluated the effects of a single foliar application of urea on levels of expression of these genes in the leaves of ‘Dazhenbaochiyue’ peach. Cloning resulted in the isolation of 1,767, 1,236, 1,074, 1,758, and 2,721 nt-long cDNAs with full-length open reading frames encoding 588, 411, 357, 585, and 906 amino acids representing the asparagine synthetase (AS), glutamate dehydrogenase (GDH), glutamine synthetase (GS), nitrite reductase (NiR), and nitrate reductase (NR) genes in peach, respectively. An alignment of multiple amino acid sequences revealed that the AS, GDH, GS, and NR proteins in peach shared high levels of sequence conservation or identity at the amino acid level with their homologues in Arabidopsis thaliana and grapevine (Vitis vinifera). Based on analyses of expression of the five genes in peach leaves, we demonstrated that genes related to nitrogen metabolism responded to a foliar application of urea within 2 d. This was consistent with previous reports which indicated that leaves rapidly absorbed urea-nitrogen after foliar application. Foliar application of 0.5% (w/v) urea inhibited expression of GS and NiR, but increased expression of GDH, AS, and NR compared to control leaves. The different patterns of expression of the five genes in this study suggested that their expression might reflect their various roles in nitrogen metabolism, plant N status, and responses to weather conditions such as high light intensity, temperature, or humidity, all of which affect photosynthesis. These findings provide a basis for future functional analyses of these five genes in peach.     

桃不同类型果实发育的解剖结构特性

为探讨普通桃、油桃、蟠桃和油蟠桃果实发育期表皮、亚表皮和中果皮细胞的解剖结构差异,试验以同一杂交群体内的这4种类型(各2株)为试材,通过显微和超微观察果实表皮、亚表皮和中果皮细胞发育。结果表明,4种类型果实的横径发育皆呈双S曲线,果实亚表皮细胞前期变化较小,开始膨大期为盛花后42d。普通桃、油桃和油蟠桃中果皮细胞在整个果实发育期持续膨大,类型间变化趋势基本一致,而蟠桃中果皮细胞在盛花后70～84d存在膨大延缓期。盛花后42～98d,普通桃和蟠桃亚表皮与中果皮细胞膨大倍数之比大于油桃和油蟠桃。分析认为,果实有毛/无毛基因单独影响着表皮细胞形状和亚表皮与中果皮细胞膨大倍数之比,而无毛基因与扁平基因共同影响着亚表皮淀粉粒数目,并且这种作用有累加现象。     

全基因组基因表达分析技术及其在果树上的应用

基因表达分析对于揭示基因功能、了解基因间相互关系、阐明植物生长发育过程中的生理生化调控机制起着至关重要的作用。综述了mRNA差异显示、cDNA-AFLP、基因芯片、EST数据分析、基因表达系列分析(SAGE)等全基因组基因表达分析技术的原理特点及在果树上的应用情况,并介绍了利用全基因组基因表达分析技术开展果树研究的思路。     

桃果实蒸腾强度对源叶净光合效率及相关生理反应的影响

在果核硬化期和果实最后迅速生长期,对艳丰一号桃果实浸涂10倍和100倍的液体石蜡溶液,与果实未浸涂液体石蜡的对照相比,果实蒸腾强度分别降低了57.9%和41.4%,且源叶净光合效率(Pn)和气孔导度(Gs)降低而叶表面温度(Tl)升高,并在果实迅速生长期间,果实浸涂液体石蜡处理和对照之间存在显著性差异。进一步研究表明,无论是在果核硬化期还是在果实最后迅速生长期,当Gs小于0.2mol/m2·s时,Pn和Gs呈极显著直线正相关关系,且果实浸涂液体石蜡处理的源叶的Tl随Gs减小急剧升高。源叶Pn对Tl的响应均呈极显著抛物线相关关系,但同一Tl条件下,果实浸涂液体石蜡处理的Pn低于对照。因此认为,果实蒸腾强度可能通过作用叶片的Gs调节Tl进而对源叶的Pn进行调控,气孔开张度减小、Tl升高,可能是果实蒸腾强度减弱时调控Pn重要机制之一。     

桃叶片再生不定芽的研究

以桃栽培品种曙光、金童5号和甜桃王试管苗叶片为外植体,研究了基本培养基、植物生长调节剂种类及质量浓度组合、基因型和试管苗继代次数等因素对叶片再生的影响。结果表明,适宜暗培养的培养基为LP+1.5mg·L-1TDZ+0.15mg·L-1NAA;光培养基为LP+0.5mg·L-1TDZ+0.3mg·L-1KT+0.3mg·L-1NAA和LP+0.6mg·L-1TDZ+0.3mg·L-1KT+0.4mg·L-12,4-D;金童5号和曙光具有较强的再生能力,再生率分别为21.8%和14.5%;曙光和金童5号试管苗继代第1～11次叶片的再生能力与继代前3次愈伤组织相比形成率较高;曙光再生苗在培养基1/2MS+0.5mg·L-1NAA上生根率为100%,平均生根条数为7.9。     

喷施磷酸二氢钾对桃叶片和果实性状的影响

以不同成熟期的桃品种豫甜、豫香、秋甜、秋蜜红为试材,研究了喷施磷酸二氢钾对其叶片叶绿素含量、干鲜质量比及果实的可溶性固形物含量、可溶性糖含量、可滴定酸含量、维生素C含量等品质特性的影响。结果表明,喷施磷酸二氢钾能提高叶片中叶绿素含量、干鲜质量比和果实可溶性固形物含量、可溶性糖含量;对果实的可滴定酸含量、维生素C含量影响不明显;叶片喷施不同浓度和次数的磷酸二氢钾均可推迟桃果实成熟期;喷施清水和对照处理间叶片性状和果实品质差异不明显;喷施磷酸二氢钾浓度和次数以500mg/L3次效果最显著,以300mg/L3次最经济有效。     

Prognosis and correction of iron chlorosis in peach trees and relationship between iron concentration and Brown Rot

This study investigates the possible correlations between bark, floral, and leaf iron (Fe) concentrations and SPAD measures to be used as early methods for prognosis of iron chlorosis in peach trees. The results showed that there were significant correlations between bark, floral and leaf Fe concentrations and SPAD measurements. This study shows for the first time the possibility of using bark analysis as an early predictive method of iron chlorosis in peach trees.Differences in mineral composition of leaves of peach trees, in relation to rootstocks were also found.This study also investigated the distribution of mineral elements in different parts of peach leaves. Tissue analysis of different leaf parts showed that the peripheral and petiole of leaves had the highest P, Ca, Mg, Zn, Fe, B and Cu concentrations. In contrast, the highest K concentration was found in the internal parts of leaves. High Mn concentration was found in the laminar of leaves, but was lower in the petioles.In other experiments, the effect of different sources of Fe application on the leaf Fe concentrations and development of Brown Rot in the year of application and one and 2 years later was also examined. No application increased significantly the leaf Fe concentrations in the year of application and 1 year later. Leaf Fe concentrations were significant higher in trees treated with FeSO₄·7H₂O + S 2 years after application.The possible effect of flesh Fe concentration to susceptibility of peach (cv. Sun Crest) to Monilinia laxa was also evaluated. The results showed no correlation between flesh Fe concentration and susceptibility of peach to M. laxa. Besides, no statistical difference was found in the susceptibility of peach to M. laxa collected from the cultivar Sun Crest grafted on different rootstocks.     

采后玉露桃果实冷害发生与ROP基因的表达调控

以软溶质玉露桃果实为材料,于8℃、5℃、0℃和LTC(8℃锻炼3d再转到0℃贮藏)下贮藏30d后转至货架(20℃)2d,对各个处理果实的腐烂和冷害程度、果实品质变化规律以及ROP基因的表达模式进行了比较,以探讨ROP基因与低温胁迫的关系。结果表明,经5℃和0℃贮藏30d的玉露桃果实在货架期间褐变加重,经5℃处理的果实褐变最重,经0℃处理果实表现后熟障碍;经8℃贮藏30d以及后续2d货架期的果实不出现冷害症状,但果实腐烂严重;LTC处理果实经冷藏后在货架期后熟正常,且果实冷害和腐烂均被明显抑制。各处理桃果实在采后低温贮藏过程中总可溶性糖和总有机酸质量分数均趋下降,其中5℃处理果实总可溶性糖质量分数下降幅度最大。结合应用EST检索和RT-PCR克隆扩增,分离出桃ROP基因(PpROP)。实时定量PCR结果显示,PpROP在5℃下表达迅速增强,第5天达到高峰,其丰度明显高于其他处理,LTC处理使PpROP表达维持较低水平。     

新疆桃树上苹果锈果类病毒(ASSVd)的检测与全序列分析

在以往研究苹果和梨ASSVd的基础上,利用RT-PCR技术对新疆的桃树进行了检测,检测过程中首次发现桃树上有ASSVd类病毒的存在,利用RT-PCR技术分别获得了其全长序列,通过回收克隆测序,发现桃树苹果锈果类病毒基因组长为331 nt,将克隆测序结果在GenBank中登录,获得了桃树上的10条ASSVd的序列登录号为:EU031477～EU031486。为快速鉴定桃树苹果锈果类病毒奠定了良好基础。     

High expression level of TAL1 gene in newly diagnosed acute myeloid leukemia

AIM: To investigate the characteristic of T-cell acute lymphocytic leukemia 1 (TAL1) gene expression in acute myeloid leukemia (AML) cell lines and in primary AML cells from de novo AML patients with different subtypes. METHODS: Real-time PCR was used to determine the expression of TAL1 mRNA in acute leukemia cell lines (Jurkat, CCRF-CEM, HL-60 and NB4 cell lines) and peripheral blood mononuclear cells from 47 newly diagnosed AML patients. Twelve healthy individuals were served as healthy control group. RESULTS: A significantly increased level in TAL1 mRNA was found in AML cell lines (HL-60 and NB4), T-cell acute lymphacytic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and primary AML cells compared with the healthy controls. Over-expression of TAL1 was found in all detected AML subtypes, the highest level of TAL-1 mRNA was found in AML-M1 and AML-M5 subtype (P<0.05). CONCLUSION: High expression of TAL1 in AML might influence the differentiation and proliferation of myeloid cells, further investigation needs to confirm whether it would be as a biomarker for pathogenesis of AML.     

一种植物表达载体的构建及其在金柑上的瞬时表达

以植物表达载体pCambia1301的带内含子的GUS(iGUS)基因替换植物表达载体pBI121中的GUS基因,构建了以pBI121为基础载体并含有iGUS的植物表达载体pLZ13。农杆菌染色表明,pCamiba1301和pLZ13两载体中的iGUS基因均不会导致GUS染色反应。以金柑不同组织为材料,通过农杆菌介导开展瞬时表达研究,结果表明,pLZ13和pCambia1301两载体的iGUS在CaMV35S启动子调控下的表达没有差异,证明构建的pLZ13是一种同时适于瞬时表达和转基因研究的植物表达载体。     

Drying half of the root-zone from mid fruit growth to maturity in ‘Hass’ avocado (Persea americana Mill.) trees for one season reduced fruit production in two years

We tested the effect of extended drying of half the root system on fruit yield and fruit Ca concentration, an indirect measure of fruit quality, in avocado (Persea americana Mill. cv Hass). In a field experiment on a sandy soil, withholding irrigation and plastic sheeting was used to dry the root-zone beneath the whole canopy (DD) or half the canopy (WD), compared with well-watered trees (WW). The irrigation water contained added nutrients and was slightly saline. Yield, shoot growth, leaf conductance, leaf and fruit water status and mineral concentrations of leaves and fruit were studied. The responses of treated trees were assessed in the following season during which normal irrigation practices were restored. With respect to yield, the WD treatment behaved the same as the DD treatment. It reduced yield by more than half and proportionately more than the reduction in water supply thus reducing irrigation efficiency. Re-watering did not restore yield of WD or DD-trees in the next season. The WD and DD treatments had no effect on the concentration of Ca in the fruit mesocarp and so are unlikely to affect fruit quality. The main impact of reduced water supply on the trees was fruit abscission and this was linked to dry soil around the roots rather than the water status of the leaves or fruits. We conclude that extended drying of half of the root-zone in one season reduced irrigation efficiency for two seasons by promoting the abscission of developing fruit to the same extent as occurred when the whole root system was exposed to extended drying.     

Sequence analysis and prokaryotic expression system construction of PIA genes isolated from Neisseria gonorrhoeae

AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N.gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N.gonorrhoeae were amplified by using high fidelity PCR.The target amplification fragments were sequenced after T-A cloning.Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed.A prokaryotic expression system of PIA gene was constructed.Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA.rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962),the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%,respectively,which indicated that all the isolates were belonging to serovars IA6.Output of rPIA was as high as 50.1% of the total bacterial proteins.The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N.gonorrhoeae isolates and sequences of the encoding gene are relatively conserved.The constructed prokaryotic expression system is able to express rPIA with high efficiency,which may lay a foundation for further development of serological detection kit and vaccine of N.gonorrhoeae.     

梨矮化砧木中矮1号GA20-氧化酶基因克隆与表达分析

中矮1号是中国农业科学院果树研究所选育的梨优良矮化砧木,为研究其矮化机理,利用RT-PCR结合RACE方法克隆了梨矮化砧木中矮1号的1个GA20-氧化酶基因,并对该基因及其氨基酸序列以及该基因在不同生长类型梨品种中不同时期新梢叶片中的表达进行了分析。结果表明,该基因全长1 179 bp,推导编码392个氨基酸,其编码的...     

甜瓜果实酸性转化酶基因反义表达载体的构建(英文)

应用RNA反义技术抑制甜瓜果实发育过程中酸性转化酶活性,促进蔗糖积累,从而为培育优质品种提供了可行的新方法。将已克隆到pMD18-T载体上的甜瓜酸性转化酶基因用BamHⅠ和HincⅡ双酶切,得到该基因编码区1038bp的cDNA片段,将其定向插入到植物表达载体pROK2的BamHⅠ/SmaⅠ克隆位点,构建了甜瓜酸性转化酶cDNA反义表达载体(Anti-MAI1)。采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。利用叶盘法转化烟草,经PCR和PCR-Southern杂交检测,证明此基因已整合入烟草的核基因组中。