Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components.Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression.In this study,eight candidate reference genes were selected from our previously published RNA-seq data,and primers for each candidate reference gene were designed and evaluated.The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples,including 12 subgroups of developing or abiotic-stressed tissues.Different combinations of stable reference genes were screened according to a comprehensive rank,which was synthesized from the results of four algorithms,including the geNorm,NormFinder,BestKeeper andΔCt methods.The screened reference genes were verified by normalizing EjLGA1 in each subgroup.The obtained suitable combinations of reference genes for accurate normalization were GAPDH,EF1αand ACT for floral development;GAPDH,UBCE and ACT for fruit setting;EF1α,GAPDH and eIF2B for fruit ripening;ACT,EF1αand UBCE for leaves under heat stress;eIF2B,UBCE and EF1αfor leaves under freezing stress;EF1α,TUA and UBCE for leaves under salt stress;ACT,EF1αand eIF2B for immature pulp under freezing stress;ACT,UBCE and eIF2B for immature seeds under freezing stress;EF1α,eIF2B and UBCE for both immature pulp and seeds under freezing stress;UBCE,TUB and TUA for red-fleshed fruits under cold-storage stress;eIF2B,RPS3 and TUB for white-fleshed fruits under coldstorage stress;and eIF2B,UBCE and RPS3 for both red-and white-fleshed fruits under cold-storage stress.This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat.To our knowledge,this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in E.japonica.The use of the three most stable reference genes could increase the reliability of future quantification experiments.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect on Phenolic Profile of Loquat (<i>Eriobotrya japonica</i>) Fruit

The objective of this research was investigated the effect of polyphenol oxidase microwave treatment on phenolic composition, antioxidant activity and microstructure of loquat fruit. Phenolic profile of methanolic extracts prepared from fresh, and microwave-treated samples were analyzed. Antioxidant activity was also evaluated by 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS?+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH+) methods. In addition, polyphenol oxidase inactivation was carried out using a response surface methodology to establish the optimal conditions of treatment. The phenolic content of fresh mesocarp was 311 ± 0.60 mg gallic acid equivalents (GAE)/100g dry weight (DW) and that of microwave-treated mesocarp was 1230 ± 0.36 mg GAE/100g DW. Total phenolic content of water/ methanol extract significantly increases after microwave treatment rather than methanolic extract of fresh loquat. Five glycoside phenolics were identified by HPLC-DAD-MS as 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, 5-caffeoylquinic acid and quercetin-3-O-sambubioside. Methanolic extract of microwave-treated mesocarp showed higher antioxidant activity than that of fresh mesocarp. Thus, polyphenol oxidase inactivation by microwave energy preserved the integrity of phenolic compounds as well as antioxidant activity in mesocarp extracts prepared from loquat fruit. It was also noted that phenolics were more abundant in the microwaved samples than in the fresh samples.

Seed Germination Traits of Loquat (Eriobotrya japonica Lindl.) as Affected by Various Pre-Sowing Treatments (Cutting of Cotyledons, Removal of Perisperm, Moist Chilling and/or Exogenous Application of Gibberellin)

The purpose of this study was to investigate the effects of various presowing treatments on the germinability(final germination percentage)and germination rate of loquat seeds in order to increase seedling production in nurseries(applied research)as well as provide answers for important physiological issues related to loquat seeds and their seed coat(basic research).Three experiments were carried out with various pre-sowing treatments.These treatments included full or partial removal of seed coat(perisperm),partial cutting of cotyledons as well as moist chilling at 5℃ for 13 days and/or soaking the seeds in water or 250 ppm gibberellic acid(GA_(3))solution for 24 h.According to the results,cotyledons excision resulted in delayed germination,regardless of the presence or absence of the seed coat in comparison with the decoated seeds that demonstrated the highest germination rate amongst them.In addition,even the partial excision of seed coats affected positively both the germinability and the germination rate,compared to the control-intact seeds.Furthermore,control-intact seeds had a higher germination percentage when exposed to moist chilling independently of the application or not of gibberellin;while the combination of gibberellin application and moist chilling improved both the percentage and the rate of germination of decoated seeds.In conclusion,the role of perisperm(seed coat)in the germination procedure of loquat seeds seems to be important,indicating the existence of seed coat-imposed dormancy on loquat seeds.Finally,the existence of a mild endogenous embryo-dormancy on loquat is also discussed.

Effects of Lanthanum and Europium on Rooting of Plantlet Eriobotrya Japonica Lindl. in vitro

The effect of La 3+ and Eu 3+ on the rooting of Eriobotrya japonica Lindl. plantlet in vitro was studied with adding La 3+ and Eu 3+ to the rooted medium. The rooting rate, the number of root and the length of root were studied after transplanting 20 d. The activity of peroxidase, nitrate reductase and fresh weight of roots were determined after transplanting 44 d. The results show that the optimum concentration range of La 3+ (1.0～3.0 μmol·L -1), Eu 3+ (2.0～3.0 μmol·L -1) in the rooted medium can increase the rooting rate and the fresh weight of roots, and promote the length of root and raise the activities of peroxidase and nitrate reductase significantly. La 3+ has more effect in improving the rooting rate, root length and the activities of peroxidase and less effect in promoting root fresh weight and the activities of nitrate reductase than Eu 3+.

Occurrence, Prevention and Control of Cryphalus eriobotryae, a New Devastating Pest of Loquat

A devastating trunk beetle has been found in Baisha loquat in Dongshan Town, Wuzhong District, Suzhou City in recent years. It is iden-tified as a new insect species, Cryphalus eriobotryae, belonging to Scolytinae, Curculionidae, Coleoptera. So far, loquat is known as the unique host of C. eriobotryae, and trees of 20-30 years old or 3-4 years old are all attacked by the insect. Holes drilled by adults with the diameter of about0.8 mm and sores(ate by adults) with the diameter of 1 mm are found on bark and trunk of damaged trees. Three-dimensional channels in 3-4 layers are formed in cortex, which are filled with insect dung. The phloem turns black-brown in late stage, and loses transport function, resulting in plant death and a great impact on loquat production and industrial development. During the occurrence period of C. eriobotryae from April to November,the damaged trunks and branches should be sprayed with 48% chlorpyrifos EC or 5% imidacloprid EC 100 times dilution once every 10 d for con-tinuous 2-3 times, and various states of the insect could be killed. If the phloem is damaged in the early and middle stages and does not turn black and brown completely, the plant could grow new cortex to realize recovery.

腐植酸水溶肥对枇杷幼苗生长的影响

为研究叶面喷施腐植酸水溶肥对枇杷[Eriobotrya japonica(Thunb.)Lindl.]幼苗生长的影响,以大五星枇杷幼苗为材料,叶面喷施不同稀释浓度(700、1 000、1 300、1 600倍)的腐植酸水溶肥,以喷施等量清水为对照(CK),测定腐植酸水溶肥对枇杷幼苗生物量、光合色素含量和抗氧化酶活性的影响。结果表明,叶面喷施不同浓度的腐植酸水溶肥提高了枇杷幼苗茎杆和叶片生物量、光合色素含量,且均以稀释1 000倍效果最好。稀释1 000、1 300倍腐植酸水溶肥处理提高了枇杷幼苗过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)活性。枇杷幼苗茎杆、叶片生物量与叶绿素a、叶绿素b、类胡萝卜素含量、CAT活性、POD活性和SOD活性呈正相关。因此,叶面喷施腐植酸水溶肥能够促进枇杷幼苗生长,且以稀释1 000倍最佳。

千岛湖地区不同蜜蜂传粉对枇杷增产提质效果的评价

应用中华蜜蜂和意大利蜜蜂为浙江省千岛湖地区的枇杷传粉,评估2种蜜蜂传粉对枇杷果实品质和产量的影响。结果表明,枇杷花期不同时间2种蜜蜂访花密度差异较大,其中9:30~9:50时,中华蜜蜂的访花密度(10.35±2.15只/100朵花)显著大于意大利蜜蜂的访花密度(5.00±1.39只/100朵花)(P<0.05),其余时段2种蜂的访花密度差异不显著。经2种蜜蜂传粉的枇杷果实维生素C含量、总糖、糖酸比、单果重及果形指数等指标均显著优于隔离对照区的枇杷果实(P<0.05),在改善枇杷果实品质方面2种蜜蜂传粉效果基本一致。大田亩产量调查结果显示,中华蜜蜂传粉区的平均枇杷亩产量略高于意大利蜜蜂传粉区,但二者之间差异不显著(P>0.05)。本研究认为2种蜜蜂传粉均达到枇杷增产提质的效果,其中中华蜜蜂更适合为山区冬季开花的枇杷传粉。

低温胁迫下油菜素内酯对枇杷幼果生理特性的影响

对枇杷(Eriobotrya japonica LindL.)幼果使用0、0.5、1.0、1.5、2.0 mg/L外源油菜素内酯预处理后,于-3℃条件下低温处理24 h,研究低温胁迫下油菜素内酯对枇杷幼果相对电导率、抗氧化酶活性和渗透调节物质含量等生理指标的影响。结果表明,低温处理使枇杷幼果的相对电导率显著升高,冻害指数升高至90.5,但随油菜素内酯浓度的增加呈下降趋势;与0 mg/L相比,枇杷幼果的SOD、POD、CAT、APX酶活性和可溶性糖、可溶性蛋白质、脯氨酸含量在油菜素内酯的作用下得到不同程度的提高,并均与其相对电导率呈极显著负相关。这表明在低温胁迫下油菜素内酯可以通过调控抗氧化酶活性,提高渗透调节物质含量,增强枇杷幼果的耐寒性,并在油菜素内酯浓度为2.0 mg/L时枇杷幼果的耐寒性最佳。

枇杷PYL基因家族鉴定及表达分析

脱落酸(ABA)是调节植物生长发育与非生物胁迫的重要激素之一,ABA受体Pyrabactin Resistance 1(PYR)-like(PYL)于信号通路的顶端发挥核心调控作用,但枇杷Eriobotrya japonica的PYL基因家族尚未鉴定。本研究通过生物信息学分析鉴定出枇杷12个PYL基因家族成员,枇杷PYL蛋白分子量为10.47~29.12 kDa,且主要定位于细胞质。枇杷PYL基因家族成员分布在8条染色体上,进化树分析显示PYL基因家族包含三个亚家族,且枇杷EjPYL1与拟南芥AtPYL13亲缘关系较近。枇杷PYL蛋白含有1~6个motif,其中motif3为家族成员共有。枇杷PYL基因家族成员在物种内存在共线性,与拟南芥的物种间共线性有2对。顺式作用元件分析发现枇杷PYL基因启动子含有低温、干旱、光以及激素响应元件。枇杷幼果转录组分析表明,果肉和种子中EjPYL6和EjPYL8基因受低温诱导上调表达。该研究结果将为枇杷ABA信号转导途径的解析和抗冻新品种培育提供理论依据。

Gas exchange and water relations of young potted loquat cv.Algerie under progressive drought conditions

Relationships between plant water status and gas exchange parameters at increasing levels of water stress were determined in Algerie loquats which grown in 50 I pots. Changes in soil water content and stem water potential and their effects on stomatal conductance （Gs ） and net photosynthesis （Pn） rate were followed in control plants and in plants without irrigation until the latter reached near permanent wilting point and some leaf abscission took place. Then, the irrigation was restarted and the comparison repeated. Soil water content and stem water potential gradually diminished in response to drought reaching the minimum values of 0.9 mm and -5.0 MPa, respectively, 9 days after watering suspension. Compromised plant water status had drastic effects on Gs values that dropped by 97％ in the last day of the drought period. Pn was diminished by 80％ at the end of the drought period. The increasing levels of water stress did not cause a steady increase in leaf temperature in non-irrigated plants. Non-irrigated plants wilted and lost some leaves due to the severity of the water stress. However, all non-irrigated plants survived and reached similar Pn than control plants just a week after the irrigation was restarted, confirming drought tolerance of loquat and suggesting that photosynthesis machinery remained intact.

Removal of the main inflorescence to induce reflowering of loquat

Loquat(Eriobotrya japonica Lindl.)is an evergreen fruit tree species of the Rosaceae,and its unique flowering time greatly hinders its production.To explore the artificial regulation of loquat flowering time,we removed the main inflorescence(by cutting it)to induce reflowering.For different loquat tree cultivars with different stages,the inflorescence was removed by cutting the main floral axis at two alternative positions:the upper or the lower position beneath the inflorescence,and it was found that the proportion of reflowering resulting from removing the upper position of the main floral axis of yellow-flesh loquat cultivars during the full-bloom stage was the highest.In addition,compared with those of the normal-growing panicles,the number of flower buds and branch axes of the reflowering panicles decreased significantly after cutting.Importantly,these newly produced inflorescences flowered 2–4 months later than normal-growing inflorescences did,effectively prolonging both the flowering and fruiting time.In addition,qRT-PCR results showed that EjFT1,EjFT2,EjAP1–1 and EjAP1–2 were highly expressed in the floral axis.These findings highlighted a new method for extending the production cycle of loquat and provided a reference for the flowering regulation of loquat and other economically important fruit tree species.

基于熵权TOPSIS法综合评价不同产地枇杷叶质量

目的 对不同产地枇杷叶的质量进行综合评价。方法 以不同产地的30份枇杷叶为样品,采用热浸法测定其醇溶性浸出物含量,采用紫外分光光度法分别测定其总黄酮含量和总三萜酸含量,采用高效液相色谱法测定其5个三萜酸类成分(野鸦椿酸、山楂酸、科罗索酸、齐墩果酸及熊果酸)的含量。采用熵权逼近理想排序法(TOPSIS)综合评价不同产地枇杷叶的质量;运用SPSS22.0软件对枇杷叶片重、综合评价值、醇溶性浸出物含量、总黄酮含量、总三萜酸含量及5个三萜酸类成分含量进行双变量相关性分析。结果 30份不同产地枇杷叶中醇溶性浸出物含量为(24.56±0.08)%~(34.85±0.13)%,总黄酮含量为(4.69±0.11)~(14.23±0.27) mg/g,总三萜酸含量为(27.58±0.59)~(63.95±1.27) mg/g,野鸦椿酸、山楂酸、科罗索酸、齐墩果酸、熊果酸的含量分别为(0.728±0.011)~(6.064±0.063)、(0.526±0.013)~(3.245±0.022)、(1.222±0.025)~(8.807±0.094)、(0.856±0.021)~(2.931±0.075)、(4.704±0.087)~(11.806±0.283) mg/g;熵权TOPSIS法分析结果显示,综合评价值排名前3位的样品依次为S14(福建漳州云霄县火田镇产)、S19(广西钦州钦南区产)及S29(安徽亳州涡阳县产)。枇杷叶的综合评价值与野鸦椿酸、山楂酸、科罗索酸、齐墩果酸和熊果酸这5个三萜酸类成分的含量均呈正相关(P<0.01),且枇杷叶片重与综合评价值呈正相关(P<0.01)。结论 不同产地枇杷叶质量存在较大差异,其中以福建漳州云霄县火田镇产、广西钦州钦南区产和安徽亳州涡阳县产枇杷叶的质量相对较优;枇杷叶片重与其品质呈正相关。

枇杷外泌体及细胞液基于体外水平的护肤功效研究

探索枇杷(Eriobotrya japonica L.)外泌体及细胞液对体外人皮肤细胞的生物活性,为其在护肤品上的开发提供科学依据。采用超高速离心法和机械破碎法分别获得枇杷外泌体和细胞液,以CCK8法检测不同浓度枇杷叶来源外泌体、枇杷细胞悬浮培养来源的外泌体及其细胞液对人皮肤成纤维细胞(HSF)存活率的影响;对HSF细胞采用脂多糖(lipopolysaccharide,LPS)处理进行炎症造模,选取显著促进细胞存活率的浓度,后续开展添加外泌体或细胞液处理,进行细胞凋亡及迁移能力检测。对黑色素瘤细胞(A375)开展外泌体或细胞液处理,进行黑色素含量及酪氨酸酶活性检测。结果表明:每克枇杷叶含8.54×10^(9)个外泌体颗粒,粒径86.78 nm;每毫升(约1 g)枇杷悬浮细胞上清液中含8.67×10^(8)个外泌体颗粒,粒径80.39 nm。浓度为20μg/mL时,枇杷叶外泌体、枇杷悬浮细胞外泌体、枇杷悬浮细胞液均显著提高体外HSF细胞存活率(P<0.05)。与阳性对照(LPS处理)相比,枇杷叶外泌体浓度为20μg/mL,枇杷悬浮细胞外泌体浓度为20μg/mL和枇杷悬浮细胞液浓度为10μg/mL时,均极显著降低LPS处理引起的细胞凋亡(P<0.01),均显著促进细胞迁移(P<0.05)。浓度为5μg/mL的枇杷悬浮细胞外泌体和枇杷悬浮细胞液均可显著降低体外A375细胞黑色素含量,枇杷细胞液还显著降低酪氨酸酶活性(P<0.05)。

枇杷叶片性状与单果质量的遗传规律研究

为了解枇杷(Eriobotrya japonica)叶片性状和单果质量的遗传多样性及其相关性,对‘宁海白’与‘大房’杂交组合的F1群体(123株)的7个叶片性状与单果质量进行相关分析。结果表明,叶片的长度、宽度、厚度和叶柄长度及单果质量5个性状在后代中均呈现连续性较好的正态分布,其中单果质量、叶片的长度、宽度和厚度呈趋小遗传趋势,叶柄长度呈趋中变异趋势。F_(1)杂交群体叶面形态主要以“稍皱”为主,叶片形状以“椭圆形”为主,叶基形状以“楔形”为主。单果质量与叶柄长度、叶片长度、叶片宽度、叶片厚度均表现出极显著的正相关性。因此,叶柄长度可考虑作为早期筛选大果优株的参考指标之一。

1-MCP处理结合自发气调包装对枇杷贮藏品质的影响

为探索不同浓度的1-甲基环丙烯(1-methylcyclopropene,1-MCP)熏蒸结合自发气调包装对普通枇杷在常温下贮藏品质的效果,以“五星枇杷”为试验材料,研究不同浓度的1-MCP(0.08、0.10、0.12、0.14 g/kg)结合自发气调包装对9成熟枇杷室温下贮藏期品质(失重率、腐烂指数、可滴定酸、可溶性性固形物、过氧化物酶、纤维素酶)的变化。结果表明,室温(25±0.5)℃条件下,与对照相比,4种浓度的1-MCP处理均可以在一定程度上抑制采后枇杷果实失水皱缩,延缓了可滴定酸含量的下降速度;遏制了贮藏中期过氧化物酶活性的升高;0.08、0.10、0.12 g/kg浓度的1-MCP处理均显著降低了采后枇杷果实在贮藏期间病害的发生;0.10 g/kg 1-MCP处理组在贮藏中后期可以延缓纤维素酶活性的上升速度。可见,0.10 g/kg1-MCP处理结合自发气调包装可延缓采后普通枇杷的软化进程和维持果实的贮藏品质。

枇杷抑制α-葡萄糖苷酶和α-淀粉酶活性部位的筛选及其酶动力学

比较研究枇杷不同药用部位(根、茎、叶、花、果肉、种子)醇提取物对α-葡萄糖苷酶和α-淀粉酶抑制活性,并探究最强活性部位及其总黄酮的酶促反应动力学特征。采用95%乙醇超声提取制备枇杷不同药用部位醇提取物,超声辅助浸提并经AB-8大孔树脂制备总黄酮,利用紫外光谱法测定α-葡萄糖苷酶和α-淀粉酶抑制活性,通过酶促动力学方法与Lineweaver-Burk曲线推断酶抑制类型。结果表明,枇杷不同药用部位醇提取物均具有一定的α-葡萄糖苷酶和α-淀粉酶抑制活性,α-葡萄糖苷酶抑制活性强弱依次为花>茎>根>叶>果肉>种子,α-淀粉酶抑制活性强弱依次为根>茎>花>叶>果肉>种子。枇杷花醇提取物、枇杷花总黄酮对α-葡萄糖苷酶抑制活性半抑制浓度(half inhibitory concentration,IC_(50))值分别为(4.65±0.35)、(0.0174±0.0035)g/L,均为可逆非竞争性抑制类型;对α-淀粉酶抑制活性IC_(50)值分别为(14.41±0.59)、(1.57±0.03)g/L,均为可逆非竞争性抑制类型。枇杷根对α-淀粉酶抑制活性最强,IC_(50)值为(1.51±0.24)g/L,为可逆竞争性抑制类型。该研究结果为枇杷作为降血糖食品药品的开发利用提供了科学依据。

基于DIVA-GIS和MaxEnt的我国枇杷适生区预测和生态适宜性研究

枇杷是原产于我国的秋冬季观花植物和重要经济果树,但其野生群体的生存现状不容乐观。基于枇杷在中国的320个有效自然分布点和不同时间段的19个生物气候变量,运用地理信息系统软件DIVA-GIS和最大熵模型MaxEnt进行适生区预测,采用受试者工作特征(ROC)曲线对预测结果进行检验和评价,同时利用刀切法(Jackknife)检验评估其潜在地理分布的影响因子。结果表明,枇杷的适生区主要在南方省份以及山东、陕西等少数北方省份,DIVA-GIS的ROC值和Maxent模型的ROC曲线面积(AUC)值都表明预测结果准确。最干季度降水量、最干月降水量、年均降水量、最冷月最低气温和温度季节性变化是影响枇杷适生区分布的主要因子。在未来气候变化情景下,枇杷的潜在地理分布总体上在南北方向有所扩张,整体面积有所减少,少部分适生区破碎化。

枇杷叶多糖的提取及抑菌活性研究

目的:优化枇杷叶中多糖提取的工艺条件,并考察其抑菌活性。方法:以干燥的枇杷叶粉末为原料,采用水提法对枇杷叶中的多糖进行提取,通过正交实验筛选出最佳工艺参数;考察枇杷叶多糖对金黄色葡萄球菌,枯草芽孢杆菌和大肠杆菌的抑菌效果。结果:通过实验表明,枇杷叶多糖最佳提取温度为70℃,提取时间为2h,料液比为1∶40,此时,枇杷叶多糖提取量为16.224mg/g。枇杷叶多糖的抑菌实验结果显示,枇杷叶多糖对大肠杆菌抑制作用最强,对金黄色葡萄球菌和枯草芽孢杆菌抑制作用较弱。结论:枇杷叶中多糖成分丰富,有一定抑菌作用,该研究为枇杷叶活性成分筛选及进一步的扩展应用提供了理论依据。

正交试验法优选蜜枇杷叶炮制工艺

以齐墩果酸和熊果酸总量、浸出物及标准汤剂出膏率为综合评价指标,采用正交试验法考察闷润时间、蜜水比、炒制温度及炒制时间4个因素,优选蜜枇杷[Eriobotrya japonica(Thunb.)Lindl.]叶的加工炮制工艺。结果表明,蜜枇杷叶的加工炮制工艺为取净枇杷叶丝,加入20%的蜂蜜(蜜∶水=3∶1,m∶m)拌匀,闷润1.0 h,置于140℃的炒药机中炒制15 min,取出,晾凉筛去碎屑即可。该优选的蜜枇杷叶工艺稳定可行,可为蜜枇杷叶的扩大生产提供参考。

‘早钟6号’枇杷主要营养元素含量的年周期变化

‘早钟 6号’枇杷叶片中较高的N、P营养水平有利于花芽分化 ;叶片中营养元素含量次序为Ca >N >K >Mg >P ,花穗为K >N >Ca >Mg >P ,果实为K >N >Ca >P >Mg ;认为早钟 6号枇杷属喜Ca、喜K果树 ,生产上应重视Ca肥和K肥的施用 ;枇杷叶片营养分析采样以第一次夏梢叶片为宜 ,初步确定叶片营养诊断的采样适期为 9月。