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.

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。

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.

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.

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.

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.

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.

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。

铅对‘早酥’梨花粉萌发及花粉管生长的影响

【目的】为揭示重金属污染影响梨花粉管生长发育的特点及作用机理提供一定的理论依据。【方法】利用含有浓度为0 mg/L、0.1 mg/L、1 mg/L、10 mg/L、50 mg/L、100 mg/L Pb的培养基进行梨花粉的培养,观察不同浓度Pb对梨花粉萌发、花粉管长度及细胞壁主要成分的影响。【结果】Pb抑制梨花粉萌发和花粉管生长,且随着浓度升高,抑制作用越强。此外,Pb改变了梨花粉管细胞壁成分的分布情况:在对照组中,梨花粉管上纤维素均匀分布于花粉管壁表面,胼胝质分布向花粉管顶端逐渐减少,酸性果胶均匀分布于花粉管,酯化果胶富集于花粉管顶端与亚顶端;0.1 mg/L的Pb处理没有明显影响花粉管细胞壁成分分布;100 mg/L Pb处理后,纤维素和胼胝质分别积累于花粉管基部与顶端,酸性果胶含量减少,酯化果胶仅在顶端分布。【结论】低浓度Pb对梨花粉萌发和花粉管生长影响不显著,但是100 mg/L的Pb对花粉萌发和花粉管伸长生长有显著的抑制作用,对花粉管细胞壁成分分布影响也较大。

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。

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。

我国近30a梨育种研究进展与今后工作建议

阐述了新中国梨资源与育种工作成就。包括梨种质资源收集、保存、评价、利用、起源、演变;引种、芽变选种、实生选种、杂交育种、诱变育种、、砧木育种、遗传规律研究;以及组织培养、基因工程、分子标记、遗传图谱构建等。提出了今后应加强对核心种质的挖掘评价,培育具有自主知识产权、品质优良、抗病虫、抗逆性强、自花结实、栽培省工、外观鲜艳的新品种;还有整合育种资源、加强分工协作,创新育种技术,提高育种效率。

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。

苹果梨分类地位的RAPD鉴定

利用RAPD技术对15个梨品种及类型的遗传变异进行了研究。从100个10bp随机引物中筛选出12个多态性引物用于正式扩增,共扩增出89条DNA带,其中多态性DNA带84条,占93 6%,平均每个引物扩增的DNA带的数目为8条。利用DNA扩增结果进行聚类分析,初步认为延边苹果梨应该归属于白梨系统。

梨树CDPK基因家族进化和表达分析

钙依赖蛋白激酶(CDPK或CPK)在植物生长和发育、逆境信号刺激及其对病原物的防御反应过程中发挥着非常重要的作用。本研究利用梨树全基因组数据,采用生物信息学的方法,在全基因组水平上对梨树CDPK基因家族进行系谱进化关系、基因结构、共线性关系及表达情况等分析。结果表明,梨树基因组中共存在31个CDPK基因,命名为PbCDPK1~PbCDPK31。系谱分析结果表明,这些CDPK基因归属于4个亚家族。共线性分析检测到12对CDKP基因间存在显著的共线性关系,其中10对由最近一次发生的全基因组复制事件形成。非同义/同义置换率的比率(dN/dS)说明CDPK基因在功能上进化得非常保守。荧光定量PCR结果发现,5个梨树CDPK基因对干旱逆境有响应。试验结果可为进一步开展梨树CDPK基因家族的功能鉴定和分子进化机制的研究提供参考。

梨果实品质评价因子的选择

测定了‘清香’、‘新雅’等26个梨(Pyrus pyrifolia)选育品种(系)的单果重、果形指数、果肉硬度、可滴定酸、可溶性固形物、可溶性糖、维生素C、糖酸比等8个主要品质因子,采用多元统计主成分分析法将上述8个相关的随机变量压缩成5个综合变量,再通过系统聚类分析,确定单果重、可溶性固形物、糖酸比、可滴定酸、果肉硬度为5个具有代表性的品质评价因子。

生理性缺铁对库尔勒香梨叶片解剖结构的影响

为了解生理性缺铁对库尔勒香梨叶片解剖结构的影响,利用透射电镜和石蜡切片法,比较研究了库尔勒香梨在缺铁状态下不同发育期的叶片解剖结构。结果表明,与正常叶相比,缺铁黄化叶片厚度、叶片栅栏组织厚度均极显著降低(P<0.01),叶片上表皮与下表皮厚度均极显著增加(P<0.01)。缺铁黄化叶片维管束细胞排列凌乱,导管严重变形,口径变小,生长后期表现更为突出。黄化叶片叶肉栅栏细胞中叶绿体变形且明显变小,片层系统不能堆垛形成基粒而使基粒片层大部分消失;海绵组织细胞排列松散,细胞内结构解体,甚至整个细胞空泡化。研究认为,香梨叶片外部表现黄化症状是由叶片内部及组织结构变化控制的,对比缺铁导致的标志性显微结构异常以及亚显微结构中特定细胞器的损伤,可以初步区分不同缺素或多重缺素对香梨叶片解剖结构造成的影响。