Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

Genomic selection of eight fruit traits in pear

Genomic selection (GS) has the potential to improve selection efficiency and shorten the breeding cycle in fruit tree breeding. In this study,we evaluated the effect of prediction methods, marker density and the training population (TP) size on pear GS for improving its performance and reducing cost. We evaluated GS under two scenarios:(1) five-fold cross-validation in an interspecific pear family;(2) independent validation. Based on the cross-validation scheme, the prediction accuracy (PA) of eight fruit traits varied between 0.33 (fruit core vertical diameter)and 0.65 (stone cell content). Except for single fruit weight, a slightly better prediction accuracy (PA) was observed for the five parametrical methods compared with the two non-parametrical methods. In our TP of 310 individuals, 2 000 single nucleotide polymorphism (SNP) markers were sufficient to make reasonably accurate predictions. PAs for different traits increased by 18.21%-46.98%when the TP size increased from 50to 100, but the increment was smaller (-4.13%-33.91%) when the TP size increased from 200 to 250. For independent validation, the PAs ranged from 0.11 to 0.45 using rrBLUP method. In summary, our results showed that the TP size and SNP numbers had a greater impact on the PA than prediction methods. Furthermore, relatedness among the training and validation sets, and the complexity of traits should be considered when designing a TP to predict the test panel.

PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Isolation and Characterization of a Cinnamoyl CoA Reductase Gene(CCR) in Pear(Pyrus pyrifolia)

Pear is a popular and commercially important fresh fruit, and its texture is related to the presence of sclereid formatted by parenchyma cell with lignification in vascular plants. Previous studies have demonstrated that content of lignin may be regulated by cinnamoyl CoA reductase(CCR) in various plants. However, the function of CCR in pears remains very limited. In the present study, we isolated a cDNA encoding CCR(PpCCR, GenBank accession No. KF999958) and its promoter(proPpCCR) from Whangkeumbae pear to investigate the function of CCR in lignin biosynthesis. PpCCR-GFP expressed in rice mesophyll protoplast demonstrated that PpCCR-GFP was localized in the cytoplasm, indicating that CCR may function in cytoplasm without localization signals. In transgenic plants carrying PpCCR, we observed higher lignin content compared with that in wild type plants, further suggesting that PpCCR can affect the lignin contents through regulating lignin biosynthesis in Arabidopsis thaliana. More studies in other plants are needed to confirm our conclusion.

The HY5 transcription factor negatively regulates ethylene production by inhibiting ACS1 expression under blue light conditions in pear

Ethylene is the main factor controlling fruit ripening of pear(Pyrus ussuriensis).Ethylene production rate is negatively correlated with fruit shelf life;therefore,it is important to decrease the ethylene levels for optimal fruit storage.Here,we observed that blue light treatment could inhibit ethylene production and promote the expression of ELONGATED HYPOCOTYL 5(PuHY5),a basic leucine zipper domain(bZIP)transcription factor.The following studies showed that PuHY5 could bind to the promoter of ACC synthase 1(PuACS1),a rate-limiting enzyme in ethylene biosynthesis,and inhibit its expression.For pears in which Pu HY5 was silenced,the ethylene production and PuACS1 expression were much higher than those in the control fruit.These results demonstrated that blue light inhibited ethylene production through the induction of Pu HY5 in pear.Our finding provides a new method for prolonging fruit shelf life.

异质性SPS措施对中国鲜梨出口贸易生存的影响

利用2002—2019年HS6分位鲜梨出口贸易数据,采用生存分析法探讨鲜梨出口生存状态,进一步根据制约内容研究异质性SPS措施对其产生的影响效应。结果表明:中国鲜梨出口有58.89%的贸易段在3年内发生中断,且风险率存在负时间依存性;出口贸易段长度的平均数为6.79年,与全球主要鲜梨出口国相比不具优势;整体上,贸易国SPS措施的实施显著地增加了中国对其出口的风险率,且措施的严厉程度与提出频率具有相关性;异质性分析表明,“地域资格限制”等SPS措施对存续性具有抑制作用;“基于SPS原因的进口产品授权要求”等SPS措施对存续性具有促进作用。基于此,应提升出口鲜梨品质,实时监测分析异质性SPS措施,助力鲜梨出口可持续发展。

两个秋子梨果实品质分析

为探究两个秋子梨品种差异,更好地对其进行开发利用,以红丰和早黄成熟期果实为试材,利用显著性分析、相关性分析、主成分分析和聚类分析等方法对两个不同品种秋子梨果实品质进行分析。结果表明,两种梨的果实在果实外观、果肉风味、果实香气、果汁含量上有所不同,在外观品质上红丰梨单果重为212.96 g、横径为75.49 mm、纵径为70.25 mm、果梗长度为36.48 mm,这4个指标分别达到了早黄梨的171.13%、119.33%、121.29%和113.19%;红丰梨的果实硬度显著低于早黄梨,为早黄梨的79.37%。因此红丰梨相较于早黄梨果形较大、果实较重、果肉较软,适合喜好大果、软果的消费者选择;在营养成分上红丰梨的可溶性固形物、可溶性糖、矿质元素含量均显著高于早黄梨,其中可溶性固形物含量为11.10%、矿质元素含量为3748.49 mg·kg^(-1),而可溶性糖含量为124.61 mg·g^(-1),达到了早黄梨可溶性糖含量的206.89%;果实的单果重、果实横径、果实纵径、果实硬度4项形态指标与可溶性糖、可溶性固形物、维生素C、矿质元素呈现出显著相关性,与可滴定酸呈极显著相关;在矿质元素含量上,红丰梨果肉和果皮中矿质元素Mg、P、K、Ca、Fe的含量以及12种矿质元素的总含量均高于早黄梨。对两个品种果实品质综合比较得出,红丰梨果实品质优于早黄梨,更适合在生产上开发利用。

‘巴梨’和‘早红考密斯’梨汁品质特性及其抗氧化活性分析

该研究以冷藏的‘巴梨’和‘早红考密斯’梨为材料,采用去皮和带皮的果实为原料制备梨汁,测定梨汁的理化性质、抗氧化活性和香气品质等指标。结果表明,同冷藏15 d梨果所制梨汁相比,冷藏90 d梨果所制梨汁的可溶性固形物含量、果糖和葡萄糖含量增加,可滴定酸含量、柠檬酸和苹果酸含量降低;主要多酚组分——熊果苷和绿原酸含量、总酚含量(total phenolics content,TPC)和总黄酮含量(total flavonoids content,TFC)以及DPPH自由基和羟自由基清除活性降低;酯类、醇类、酮类和烯类化合物的含量增加,醛类物质含量降低。同去皮榨汁相比,带皮压榨的梨汁中熊果苷和绿原酸含量、TPC和TFC以及抗氧化活性显著增加;糖类、有机酸化合物以及香气物质的变化规律不明显。电子鼻技术可以有效区分不同梨汁,W5S和W1W传感器响应值有明显区别;电子舌分析结果显示,不同梨汁的甜味和酸味强度值差异较明显,并分别与梨汁的糖类和有机酸含量呈显著正相关。因此,较长时间冷藏和带皮榨汁可改善梨汁的理化性质、抗氧化性能和风味品质。

砂梨(Pyrus pyrifolia)过敏原蛋白基因(Ppmal)的克隆与表达分析

【目的】分离和克隆梨过敏原蛋白基因Ppmal(Gen Bank登录号为KP008110),探讨其在梨果实发育过程中的功能。【方法】以砂梨品种‘翠冠’[Pyrus pyrifolia(Burm.f.)Nakai]为试材,在盛花期后30 d对植株果柄进行涂抹赤霉素处理,利用同源克隆和RACE方法克隆目的基因全长序列,并通过实时荧光定量技术和半定量技术分析该基因在梨不同组织以及梨果实发育过程中的表达变化。【结果】Ppmal的全长是906 bp,含有480 bp的开放阅读框(ORF),编码159个氨基酸,预测的蛋白质相对分子质量为17.56 k D,等电点为5.62。生物信息学分析显示该基因没有信号肽序列,没有跨膜结构域,具亲水性。与其他物种的过敏原蛋白基因核苷酸序列同源性超过75%,与苹果和西洋梨编码的氨基酸序列同源性分别高达97.48%和96.23%,进化树分析表明该基因与苹果的进化关系最近。同时,定量结果显示经过赤霉素处理后的果实中该基因的表达量随着果实的生长发育呈现先降低后升高的趋势,并且具有组织差异表达的特点,其表达量在叶片中最大,其次是嫩叶,再次是花,枝条最低。【结论】获得梨Ppmal基因全长,对该基因在砂梨品种‘翠冠’发育过程中的表达分析显示,Ppmal受到赤霉素的调控,并在砂梨果实膨大期发生上调表达。

菌虫复合技术在梨火疫病疫木生物转化中的应用

林果疫木枝条的无害化处理和资源化利用,是当前农林废弃物处理以及植物疫病防控的难点,也是农业环境科学与植物保护科学交叉研究的新方向。本试验以库尔勒香梨(Pyrus sinkiangensis Yu)梨火疫病疫木为靶标,采用菌-虫(白星花金龟,Protaetia brevitarsis)联合,开展菌剂、牛粪香梨枝条配比、酵化时间的三因素五水平正交试验,明确疫木发酵关键技术参数,以及白星花金龟最佳取食组合。试验结果表明,5种腐解菌+牛粪处理最高发酵温度均已超过55℃,其中EM菌剂腐解效果最好,LK菌剂次之。发酵产物经菌虫复合转化12 d后,幼虫质量增加5.32~8.38 g,虫砂量可达36.93~83.55 g,虫砂转化率最高可达94.26%。综合考虑降低牛粪添加比例、酵化时间及物料处理成本,LK菌剂、添加40%的牛粪及酵化25 d为白星花金龟幼虫最佳取食组合方案,且发酵产物以及白星花金龟转化产物虫体和虫砂中均未检测出梨火疫病菌(Erwinia amylovora)。研究表明,微生物与白星花金龟相结合对香梨疫木枝条有较好的转化率,通过白星花金龟实现了疫木枝条的资源化利用,且阻断了疫木枝条传播病源的途径。

基于HS-SPME-GC-MS方法的不同倍性库尔勒香梨果实风味相关代谢物质的变化

以普通二倍体库尔勒香梨及其自然发生的三倍体为试验材料,利用顶空固相微萃取法(HS-SPME)结合气相色谱-质谱联用技术(GC-MS),对果皮和果肉中的挥发性成分及果肉中的初生代谢物质进行评价,并分析比较其种类和含量上的差异。对挥发性物质代谢谱的分析显示,与普通库尔勒香梨相比,三倍体库尔勒香梨果皮中挥发性物质总含量增加33%,但种类减少,主要挥发性物质中醛类总比例从69.74%升高到74.40%;在果肉中,挥发性物质含量无显著变化,但种类增加了9种。初生代谢产物对比结果显示,二倍体与三倍体香梨的蔗糖和果糖含量相对稳定,但三倍体中D-(-)-呋喃果糖、D-果糖、D-(-)-果糖和蔗糖的占比分别升高了4.62%、1.59%、1.28%和0.68%;苹果酸、奎宁酸、柠檬酸含量均低于二倍体库尔勒香梨且游离氨基酸减少了4种。因此,三倍体香梨果皮中挥发性物质及初生代谢物质代谢谱都发生了较大变化,可导致香味品质发生变化。

6株拮抗酵母对梨毛霉菌(Mucor sp.)和青霉菌(Penicillium sp.)防效测定

采用稀释涂布平板法从拉萨市城关区的12种市售水果中分离酵母菌,并采用平板划线法对酵母菌菌株进行纯化;采用病斑法对皿内抑菌效果较强的6株拮抗酵母进行活体防效测定,并采用26S r DNA D1/D2区域序列测定法对酵母菌进行了分子鉴定。结果表明,193和233号两株酵母对梨毛霉菌(Mucor sp.)有明显的抑菌作用,其发酵液防效强于菌悬液;193号菌株对梨青霉菌(Penicillium sp.)有明显防效,其菌悬液抑菌作用大于发酵液。分子鉴定结果显示,6株酵母菌归为5个种,48号菌株为隐球酵母(Cryptococcus adeliensis),77号为葡萄有孢汉逊酵母(Hanseniaspora uvarum),193号为汉逊德巴利酵母(Debaryomyces hansenii),230和235号为禾本红酵母(Rhodotorula graminis),233号为胶红酵母(Rhodotorula mucilaginosa)。

Strategies for timing nitrogen fertilization of pear trees based on the distribution, storage, and remobilization of ^(15)N from seasonal application of (^(15)NH_4)_2SO_4

In order to improve the management of nitrogen(N) fertilization in pear orchards, we investigated the effects of application timing on the distribution, storage, and remobilization of N in mature pear trees in a field experiment at Jingtai County, Gansu Province, China. Nine trees were selected for the experiment and each received equal aliquots of 83.33 g N in the autumn, spring, and summer, with ^(15)N-labeled(NH_4)_2SO_4 used in one of the aliquots each season. Results showed that the(^(15)NH_4)_2SO_4 applied in the autumn remained in the soil during the winter. In the following spring this N was absorbed and rapidly remobilized into each organ, especially new organs(leaves, fruit and new shoots). The ^(15)N supplied in spring was rapidly transported to developing fruit between the young fruit and fruit enlargement stages. ^(15)N from the summer application of fertilizer was mainly stored in the coarse roots over the winter, then was mobilized to support growth of new organs in spring. In conclusion, for pear trees we recommend that the autumn application of N-fertilizer be soon after fruit harvest in order to increase N stores in fine roots. Spring application should be between full bloom and the young fruit stages to meet the high N demands of developing fruit. Summer application of fertilizer at the fruit enlargement stage does not contemporaneously affect the growth of pears, but increases the N stored in coarse roots, and in turn the amount available for remobilization in spring.

Genome-wide identification and co-expression analysis of GDSL genes related to suberin formation during fruit russeting in pear

Glycine-aspartic acid–serine-leucine(GDSL)type lipases/esterases genes play critical roles in plant development and are related to the responses to abiotic and biotic stress.However,little is known about the GDSL family in pear(Pyrus spp.).Studies have shown GDSL-domain proteins play key roles in suberin deposition.Suberin deposition in the fruit epidermis,also called russeting,is an important defect that negatively affects consumer's appeal in some fruit species,such as pear,apple and grapevine.Fruit russeting is mainly associated with cuticle microcracking and suberin accumulation in the inner part of the epidermal cell walls.To gain insight into the role of the GDSL gene family in suberin deposition and russet development in pear,we performed a genome-wide characterization of the GDSL family,including their identification,chromosomal localization,phylogenetic relationships,and expression patterns,in different tissues/organs in pear.One hundred and thirteen GDSL-type lipases/esterases genes were identified in the pear genome,and a phylogenetic analysis revealed that GDSL family can be classified into four distinct groups.Thirty GDSL genes were co-expressed with five homolog pear genes of three well-known suberin biosynthesis Arabidopsis genes(AtGPAT5,AtASFT,and AtCYP86B1)in the transcriptional co-expression network during pear fruit development.Among the 30 co-expressed GDSL genes,twelve genes were further analyzed by quantitative Real-time PCR,and the results showed the expression levels of the 12 genes were different between the russet exocarp and green exocarp of sand pear at different fruit development stages.Our study provides a detailed overview of the GDSL gene family and lays the foundation for future functional characterization of GDSL genes in P.bretschneideri.

梨火疫病菌在库尔勒香梨枝条中的侵染定殖和扩展特征研究

【目的】示踪梨火疫病菌Erwinia amylovora在库尔勒香梨(Pyrus sinkiangensis Yu)(简称香梨)枝条组织中侵染定殖和扩展特征,为梨火疫病的有效防治提供依据。【方法】以分离获得梨火疫病强致病力菌株E.a001为材料,采用热激法将携带有绿色荧光蛋白基因(green fluorescent protein,GFP)质粒phc60-gfp转化E.a001,获得了能发出强烈绿色荧光的标记菌株E.a001-gfp。利用该标记菌株接种香梨枝条,检测、观察其在香梨枝条组织中的侵染定殖和扩展动态。【结果】针刺接种和喷雾接种E.a001-gfp引起枝条发病的最低浓度分别为106cfu·mL^(-1)和107cfu·mL^(-1)。相同浓度的病原菌接种,喷雾接种较针刺接种后发病显症时间延迟2~3 d。梨火疫病菌侵染枝条产生的坏死病斑的扩展速率随病原菌浓度提高而升高;病菌在1年生枝条上的生长扩展速度显著快于在2年生枝条内的扩展速度。梨火疫病菌入侵后主要存在于枝条维管系统中,大量分布于皮层薄壁细胞和韧皮部薄壁细胞间隙,少量存在于木质部导管中。在香梨发病枝条上,病健交界处的活菌数量最高,可达到108cfu·g^(-1),未显症部位0~10 cm处病原菌为105cfu·g^(-1),但在香梨枝条病斑外未显症部位10~15 cm处均未分离出病原菌。【结论】研究明确了梨火疫病菌在香梨枝条组织中的扩展距离、迁移时间及速率,以及分布及定殖量,为病枝的准确修剪和制定病害的综合防控措施提供了科学依据。

'玉露香’与’新世纪’杂交后代果实性状遗传研究

以’玉露香’与’新世纪’及94株杂交后代F1为材料,研究了8个果实重要性状的遗传倾向。结果表明:杂交后代平均单果重小于亲中值,遗传传递力为79.47%;果实硬度、可溶性固形物和可溶性糖大于亲中值,呈增大趋势,遗传传递力分别为110.48%、101.15%,110.2%;平均果形指数大于亲中值,与'玉露香'果形指数相接近;后代果皮颜色表现为4种,黄色、绿色、黄绿色与绿黄色,以黄色和绿色为主;杂交后代小果心的比率最大,占总数的56.4%;梨果点的遗传比较复杂,杂交后代出现比较明显的性状分离,果点明显的比率较大,占总数的44.7%。

内蒙古西南地区南果梨无公害栽培技术

南果梨(Pyrus ussuriensis‘Nanguo Pear’)是中国优良梨地方品种,营养丰富,风味好,适应性强。通过实地调查和多年经验积累,结合内蒙古西南地区南果梨的生长特点,提出了南果梨无公害栽培管理措施,以期为各地生产栽培南果梨提供借鉴。

降尘对塔里木盆地香梨叶片光合特性及叶绿素含量的影响

试验对塔里木盆地生长的不同树龄香梨(Pyrus bretschneideri Rehd.cv.Fragrant pear)树的叶片在受降尘及不受降尘影响下的光合特性及叶绿素含量进行了比较分析。结果表明,香梨叶片受降尘影响后在各个生育期的净光合速率、气孔导度、胞间CO2浓度均降低。除新梢生长期外,各树龄的香梨叶片受降尘影响后蒸腾速率均下降。40年生的香梨树叶片受降尘影响后,各个生育期的叶绿素含量SPAD值均显著降低;而10年生及20年生的香梨树叶片受降尘影响后,各个生育期的叶绿素含量SPAD值均升高。

梨不同品种果实香气成分的GC-MS分析

采用顶空萃取和气相色谱质谱联用技术分析了3个白梨品种与3个砂梨品种商熟期果实香气成分。结果表明,参试的6个梨品种主要香气成分种类数及其质量分数、主要香气类别质量分数与香气总量均存在一定差异,其中3个白梨品种香气总量、醛类物质质量分数及其在香气总量中的比例均明显高于3个砂梨品种;己醛为6个参试梨品种共有的质量分数均为最高的香气成分;己醛、1-己醇及乙酸己酯是6个梨品种共有的特征香气成分,参试的6个梨品种特征香气成分及其香气值均存在一定差异,其中3个白梨品种己醛和乙酸己酯的香气值均明显高于3个砂梨品种。

梨属植物分类的历史回顾及新进展

一般相信梨属植物的原种起源于第三纪的我国西部和西南部的山区。根据其原生分布,分为东方梨和西方梨。由于梨属植物分布范围广,加上梨属种间很容易发生自然杂交,梨属植物的分类至今仍存在很多问题。被大多数植物分类学家所认可的种大约在30个左右。我国是东方梨种的主要起源地,我国分类学家相信有13个种起源于我国。不仅如此,我国也是世界上梨品种类型最多的国家。主栽系统有砂梨、白梨、秋子梨和新疆梨等。最新的分子生物学证据表明,白梨、砂梨和日本梨系统可能起源于共同的祖先:分布于我国长江流域的野生砂梨。考虑到白梨在我国梨栽培中的重要地位,建议用Pyruspyrifoliavar.sinensis(Lindley)TengetTanabe来表示白梨系统。