茶树CsWRKYIIcs转录因子的克隆及功能分析

【目的】以‘陕茶1号’为材料,克隆CsWRKYIIcs转录因子并分析序列特征,了解其在不同组织和非生物胁迫下的表达模式以及转录活性,为深入研究茶树在逆境胁迫中的功能提供理论依据。【方法】根据茶树基因组数据库注释的WRKY序列设计特异性引物,采用RT-PCR技术从‘陕茶1号’中扩增CsWRKYIIcs的cDNA序列,用生物信息工具分析序列的特点,实时荧光定量PCR（qRT-PCR）研究基因的表达模式,用酵母试验验证其转录活性。【结果】获得9条CsWRKYIIcs的cDNA序列,开放阅读框长度分别为561、960、936、978、897、912、720、1 008和969 bp,分别编码186、319、311、325、298、303、239、335、322个氨基酸。除了CsWRKYIIc7缺少锌指序列外,其他CsWRKYIIcs均含有1个WRKY保守结构域和典型的C₂H₂型锌指结构。不同物种中的WRKYIIcs具有相似的保守基序,而茶树CsWRKYIIcs和拟南芥、葡萄等双子叶植物的WRKYIIcs氨基酸序列相似性更高。另外,CsWRKYIIcs启动子区域均预测到多个与非生物胁迫相关的顺式作用元件,意味着其可能参与非生物胁迫响应。qRT-PCR结果表明,9个CsWRKYIIcs在根和花中的表达量高于茎和叶,具有一定的特异性。同时,在干旱、ABA、高温和高盐胁迫下被不同程度的诱导表达,其中CsWRKYIIc1和CsWRKYIIc7的表达量变化显著,与推定的顺式作用元件结果相符。酵母试验表明9个茶树CsWRKYIIcs均具有转录激活活性。【结论】克隆获得9个茶树CsWRKYIIcs转录因子,它们参与了茶树对ABA、干旱、高温和高盐胁迫的响应,也可能发挥转录激活因子的调控作用。CsWRKYIIc1和CsWRKYIIc7可作为候选基因深入研究茶树的抗逆功能。     

Effect of apple cultivars and storage periods on the virulence of Neofabraea spp.

Bull’s eye rot is a typical quiescent postharvest apple disease in major fruit-growing areas. The susceptibility of different apple cultivars to Neofabraea spp. (N. vagabunda and N. malicorticis) was assessed, with Granny Smith showing the most resistance and Cripps Pink the most susceptibility. To assess the factors involved in conidial germination, Neofabraea spp. were grown on crude protein extracts (CPEs) collected from apple fruits at different storage periods. Fungal germ tube growth rate and pathogenic enzyme (cellulase and xylanase) activity were assessed. Results showed that CPEs collected after 2 and 4 months of storage progressively stimulated conidial germination and germ tube elongation, while a lesser effect was observed from CPEs after 1 month of storage. Xylanase proved to be the main degrading enzyme secreted by all the isolates, while cellulase was produced only by N. vagabunda isolates. Overall, the isolate ID02 was the most virulent, based on more rapid germ tube elongation and greater activity of the lytic enzymes.     

甜瓜质地差异及相关细胞壁酶活性变化

对两种不同质地类型甜瓜果实发育过程中果肉质地的变化及相关酶活性进行研究。结果表明,花后35~40 d为软、脆两种甜瓜质地形成的关键时期。在该时期,软肉型甜瓜‘NSL’细胞面积与间隙增大,果肉细胞排列疏松,细胞面积为脆肉型甜瓜的115.35%,软肉型甜瓜‘NSL’与脆肉型甜瓜‘XZM’质构参数差异显著,脆肉型甜瓜果实4种细胞壁酶活性总体低于软肉型甜瓜。花后35 d,软肉型甜瓜‘NSL’细胞壁扩展酶基因(CmEXP3、CmEXP5、CmEXP9)相对表达量为最大值,显著高于脆肉型甜瓜‘XZM’。     

外源褪黑素对干旱胁迫下大豆鼓粒期生理和产量的影响

干旱胁迫降低大豆产量,探究提高大豆耐旱能力和降低产量损失的机制对大豆生产具有重要意义。施褪黑素能缓解干旱胁迫对植株生长的抑制和氧化损伤。本试验于2017—2018年研究叶面喷施褪黑素对干旱胁迫下大豆鼓粒期叶片光合、抗逆、碳氮代谢和产量的影响表明,外源褪黑素提高干旱胁迫下大豆叶片抗氧化酶活性,抑制活性氧的产生和细胞膜损伤,缓解干旱胁迫对光合能力的抑制,提高碳氮同化能力,最终缓解干旱胁迫造成的产量损失。与干旱胁迫相比,褪黑素处理下单株荚数、单株粒数和百粒重两年平均提高了2.9%、0.8%和17.2%,产量(单株粒重)平均提高了14.7%。     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

黄连中盐酸小檗碱的提取纯化及抑菌活性研究

为了探究盐酸小檗碱在植物病原真菌方面的抑制作用,以黄连根粉为材料,采用酸水法提取和乙醇重结晶获得盐酸小檗碱纯品,采用生长速率法研究了盐酸小檗碱对常见植物病原菌的抑制活性。结果表明,盐酸小檗碱的提取率为3.3%,经高效液相色谱法测得其含量为93.56%;抑菌活性试验表明,盐酸小檗碱在100μg/mL时对玉米大斑病菌的抑制率达99.9%,EC_(50)为8.20μg/mL,表现出比药剂对照噁霉灵和百菌清更高的抑制活性;进一步的试验表明,与噁霉灵及百菌清相比,盐酸小檗碱对玉米大斑病菌的抑制作用具有持效期长的特点。     

秸秆复合有机酿热物对大棚土壤环境的影响

为解决黑龙江省早春大棚土壤温度低及设施土壤环境恶化问题,以‘千禧’番茄为材料,研究秸秆+牛粪(NGH)、秸秆+马粪(MGH)、秸秆+羊粪(YGH)、单一秸秆(JG),以未进行反应堆技术为对照(CK),对早春大棚‘千禧’番茄土壤温度、土壤理化性状、酶活性及微生物群落结构的影响。结果表明,与对照相比,各处理降低了土壤pH,提高了土壤温度、土壤EC、碱解氮、速效钾、有机质含量及土壤蔗糖酶、脲酶、磷酸酶、过氧化氢酶活性。经过秸秆生物反应堆处理的20 cm土层温度有明显的提升,其中秸秆+羊粪处理在反应启动后提升土壤温度效果最好;秸秆+羊粪(YGH)处理提高了土壤速效磷含量,是对照的2倍;秸秆+羊粪(YGH)处理土壤中4种酶活性均处于最高水平,分别比对照提高了91.9%、220%、18.5%、7.3%。各处理微生物多样性指数均显著高于对照,变化规律一致为秸秆+羊粪(YGH)>秸秆+牛粪(NGH)>秸秆+马粪(MGH)>秸秆(JG)>对照(CK);秸秆+羊粪(YGH)及秸秆+牛粪(NGH)处理对糖类、氨基酸类、酯类、醇类、胺类、酸类6类碳源的利用能力最强,显著高于其他处理和对照。应用主成分分析和聚类分析方法将大棚番茄土壤分为4个等级,秸秆+羊粪(YGH)处理为第一级,秸秆+牛粪(NGH)处理为第二级,秸秆+马粪(MGH)及单一秸秆(JG)处理为第三级,对照(CK)为第四级。应用秸秆复合有机酿热物生物反应堆促进了土壤环境的改善,以秸秆+羊粪(YGH)作为复合有机酿热物效果最佳。     

黄瓜叶酸合成关键基因克隆与分析

【目的】分析黄瓜基因组中与叶酸合成代谢相关的基因数量、定位以及表达特征,对关键酶基因进行生物信息学分析与克隆,旨在为黄瓜叶酸合成调控研究奠定基础。【方法】根据已报道的拟南芥叶酸合成相关基因,利用黄瓜基因组数据库中9930_V3版本进行BLAST比对。利用MapChart绘制黄瓜染色体物理图谱并对基因定位。利用qRT-PCR分析这些基因在黄瓜果实发育不同时期和不同材料中的表达量。通过MEGA、WebLOGO、ExPASy等工具对关键酶基因进行生物信息学分析。通过PCR扩增对关键酶基因进行克隆,并测序分析基因的序列差异。【结果】同源比对获得19个黄瓜叶酸代谢相关基因,这些基因不均匀分布在黄瓜7条染色体上,且以Chr.4和Chr.5上分布最多。通过对其中11个调控叶酸合成的基因在测序黄瓜9930果实发育不同时期以及果实叶酸含量高低差异显著的2份材料的表达量分析,发现CsFPGS、CsHPPK/CsDHPS和CsDHNA 3个基因与果实叶酸含量变化趋势完全一致;CsADCS、CsADCL、CsDHNA、CsHPPK/CsDHFS、CsFPGS、CsDHFS等基因的表达量在2份材料中具有显著差异。通过对2个调控叶酸合成限速步骤的关键酶基因CsGCHI和CsADCS的蛋白序列及蛋白结构域分析,发现各物种中CsGCHI的同源基因均具有2个GTP_cyclohydroI结构域;CsADCS的同源基因均具有2个GATase结构域、1个Anth_synt_I_N结构域和1个Chorismate_bind结构域。它们在不同物种中高度保守,进化树分析亲缘关系近的物种聚类到一起。分别扩增黄瓜果实低叶酸含量自交系65G和高叶酸含量自交系02245中CsGCHI和CsADCS的同源基因,序列分析表明CsaV3_1G041250全长为3 012 bp,CDS序列长度为1 413 bp,3个SNP位点的突变导致了氨基酸序列的变异;CsaV3_7G026240全长为3 047 bp,CDS长度1 407 bp,序列无变异;CsaV3_5G036360全长7 941 bp,CDS序列长度为2 706 bp,序列无变异。【结论】鉴定出19个不均匀分布在7条染色体上的黄瓜叶酸代谢相关基因,基因CsFPGS、CsHPPK/CsDHPS 、CsDHNA和CsADCS是影响黄瓜果实叶酸含量变化、导致叶酸含量高低显著差异的关键基因,调控叶酸合成限速步骤的关键酶基因GCHI及ADCS功能相对保守,CsGCHI在65G、02245中有3个SNP位点的突变导致了氨基酸序列的差异。     

Straw mulching with fertilizer nitrogen: An approach for improving crop yield,soil nutrients and enzyme activities

Field experiments were conducted to study soil properties, soil enzymes activities, water use efficiency (WUE) and crop productivity after six years of soya bean straw mulching in the semi‐arid conditions of China. The experiment included four treatments: CK (Control), N (240 kg N ha^‐1), H (soya bean straw mulching at half rate 700 kg ha^‐1 with 240 kg N ha^‐1) and F (soya bean straw mulching at full rate 1,400 kg ha^‐1 with 240 kg N ha^‐1). Soil organic carbon (SOC), soil labile organic carbon (LOC), soil available N (AN), available P (AP) and enzyme activities were analysed after wheat harvesting in 2016 and 2017. Results show that straw amounts had positive effects on the soil fertility indices being higher for treatment F. The SOC, LOC, AN, AP and enzyme activities (i.e. saccharase, urease and alkaline phosphatase) were in the order of F > H > N > CK. High wheat grain yield and WUE were observed for F treatment. A total of six years mulching along with 240 kg ha^‐1 nitrogen fertilizer application is sufficient for wheat yield stability and improving soil properties except urease activities in the semi‐arid condition of China. However, the straw mulching amount should be further studied with minimum nitrogen fertilizer for an environment‐friendly and effective approach for improving the soil biological properties with adequate crop production on a sustainable basis in the semi‐arid region of China.     

饥饿和重摄食对鲤鱼脂蛋白脂肪酶活性的影响

采用对硝基苯酚法测定正常摄食、饥饿(14 d)和重摄食(3 d)状态下鲤鱼不同组织和器官中脂肪酶及脂蛋白脂肪酶(lipoproteinlipase,LPL)的活性。结果显示:与正常摄食相比,饥饿鲤鱼肌肉、脂肪和心脏中脂肪酶比活力显著下降(P0.05),但脂肪和心脏中LPL比活力显著升高(P0.05);除脂肪中LPL外,重摄食3d的鲤鱼各组织和器官中脂肪酶和LPL比活力比正常摄食和饥饿鲤鱼均显著升高(P0.05),有明显的补偿作用。