MdDGK3-like as a negative regulator participates in ALA-induced PP2AC to promote stomatal opening in apple leaves

5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple(Malus domestica Borkh.cv.‘Fuji’)leaves.With the promoter of MdPP2AC as bait,a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid(Y1H)from the cDNA library of the epidermis of apple leaves treated by exogenous ALA.Additional to binding the promoter of MdPP2AC,MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter,while ALA significantly eliminated the role of MdDGK3-like.In tobacco leaves,MdDGK3-like was localized in the nucleus of stomatal guard cells.Therefore,MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure.To further identify MdDGK3-like functions,several transiently transgenic apple leaves(including overexpression and interference)were established.The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca^(2+)and H_(2)O_(2)and decreasing flavonol levels in the guard cells.Conversely,MdDGK3-like(i)led the stomatal opening with lower levels of Ca^(2+)and H_(2)O_(2)but higher flavonols.Based on these,we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity,which improved the stomatal aperture.Since it was the first time that MdDGK3-like was showed to act as a transcription factor,the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.

Basic helix-loop-helix(bHLH)transcription factor MdbHLH3 negatively affects the storage performance of postharvest apple fruit

The storage period of fleshy fruits greatly affects their quality and selection,and is largely controlled by genetic factors.Therefore,it is imperative to elucidate how genetic factors affect fruit ripening and its storage.Here,we evaluated the postharvest storage properties of the basic helix-loop-helix(bHLH)transcription factor MdbHLH3-overexpressing transgenic Royal gala apple fruits.During storage,the contents of starch,malic acid,fructose,glucose,and sucrose in fruits of three MdbHLH3 transgenic lines were always higher than those of the wild-type(WT)control.Interestingly,the sugar-acid ratio also showed the same trend during fruit storage.Additionally,the fruit firmness decreased with increasing storage time,and the contents of cell wall components such as water-soluble pectin and cellulose in transgenic fruits were higher than those in control fruits,while the firmness of transgenic fruits was lower than that in WT control fruits.Though the ethylene release rate in both showed the same trend(firstly increasing,then decreasing,and finally peaking)in 90-day stored fruits,transgenic apples had higher ethylene levels than the WT control throughout storage.Furthermore,the activities of membrane peroxidase,antioxidant enzymes,and fruit ripening enzymes in all transgenic fruits were significantly higher than those in the WT control.Thus,our findings show how MdbHLH3 negatively regulates and reduces apple storage time.This may prove useful for not only developing biotechnological strategies,but also support traditional breeding programs,to help improve the storage time of fleshy fruits.

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino acids.Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri.The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain.qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous NaCl.Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions.Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29.The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

KNO_(3)对NaCl胁迫下苹果砧木T337幼苗生理特性的影响

【目的】研究100 mmol/L NaCl胁迫下不同浓度KNO_(3)对T337幼苗生理特性的影响。【方法】以盆栽2年生苹果砧木T337为试验材料,本试验设置6个处理,即CK1(浇灌清水)、盐胁迫(CK2)以及盐胁迫+喷施4种浓度KNO_(3)[0.1(T_(1))、0.2(T_(2))、0.3(T_(3))、0.4 mg/L(T_(4))],测定各处理叶片的叶绿素含量、光合荧光参数、抗氧化酶活性及渗透调节物质的含量,并利用主成分分析对其进行综合评价。【结果】随盐胁迫(CK2)时间的延长,T337叶片光合色素(Chl a、Chlb、Chla/b、Chla+b),气孔导度(Gs)、蒸腾速率(Tr)、净光合速率(Pn)、PSⅡ最大光化学效率(Fv/Fm)、光化学淬灭系数(qP)含量均呈逐渐下降的趋势,非光化学淬灭系数(qN)、初始荧光(Fo)、胞间二氧化碳浓度(Ci)、相对电导率(REC)、丙二醛(MDA)含量、脯氨酸(Pro)均呈上升趋势,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)呈先升后降的趋势。与CK1相比,盐胁迫+外源KNO_(3)(T_(1)-T_(4))处理后T337幼苗叶片各指标均呈现不同幅度变化,且存在明显的浓度效应,并以T_(3)(0.3 mg/L KNO_(3))处理叶片叶绿素含量、Gs、Tr、Pn、Fv/Fm、qP降幅最小,Ci、REC、MDA、qN、Fo升幅最小,Pro及SOD、POD、CAT升幅最大。综合评价表明,各处理对T337幼苗特性的效应依次为:CK1>T_(3)>T_(1)>T_(2)>T_(4)>CK2。【结论】综上,施用适宜浓度外源KNO_(3)可有效改善盐胁迫下T337幼苗光合能力,显著提高抗氧化酶活性和渗透调节物质含量,降低细胞膜透性,从而达到缓解盐胁迫的作用,且以0.3 mg/L KNO_(3)处理缓解盐胁迫对T337造成的伤害效果最好。

采收期对烟富3号苹果果实品质的影响

以15年生烟富3号苹果为试材,研究了甘肃省陇东旱塬区6个采收期(2022年10月5日、10月11日、10月17日、10月23日、10月29日、11月5日)果实低温贮藏时品质生理变化规律及特性。结果表明:随着采收期的推迟,果形指数无显著变化,果实纵径、横径、可溶性固形物含量、可溶性糖含量及固酸比、糖酸比、糖心率均显著增加,硬度及可滴定酸含量、总酚含量显著降低,10月23日采收的果实单果重最大、着色最好、维生素C含量最高,可溶性固形物含量达15.13%。低温贮藏期间,不同采收期果实硬度及可滴定酸含量、维生素C含量均不断下降,可溶性固形物含量先升高后下降,总酚含量先下降后升高,果实呼吸强度、乙烯释放速率均在贮藏120 d时出现峰值;10月17日采收的果实硬度及可滴定酸含量、维生素C含量下降幅度较小,果面光洁度保持稳定,贮藏180 d后,果实硬度、可溶性固形物含量、维生素C含量、L^(*)值均最高,果实呼吸强度、乙烯释放速率峰值、失重率、腐烂率均最低。综合分析,10月23日可作为甘肃省陇东旱塬区烟富3号果实鲜食的最适宜采收期,10月17日可作为长期低温贮藏的最佳采收期。

Apple（苹果）iPod、iPod nano、iPod shuffle MP3播放机

Apple公司最近推出了全新的第6代iPod和铝合金外壳iPodnano、iPodshuffle三款MP3播放机，并为其配备了新款的耳机。其中第6代iPod有30GB和60GB两种容量和黑、白两种颜色供选择，采用了与第5代iPod同样大小的6．35cm（2．5英寸）彩色液晶屏幕，分辨率同样为320×240像素，

负载量对首富3/M9T337苹果树体生长结果的影响

以6年生首富3/M9T337为试材,研究不同留果量(干截面积的2、4、6、8、10、12倍留果)对树体生长发育的影响。结果表明,不同负载量对当年树高、干径的增长影响不显著;随着负载量的增大,冠径、新梢生长量减小;叶片的叶绿素含量变化为先升后降趋势,4、6倍负载量时净光合速率最高,分别达到19.63、20.16μmol/mol;平均单果重在8~12倍负载量时显著下降;可溶性固形物含量在12倍负载量时显著下降,其他处理间无显著差异。666.7 m^(2)产量随负载量增大表现先增后降趋势,10倍负载量时最高,12倍时最低,比10倍负载量时降低3.86%。从当年数据看,6~8倍干截面积负载量较为适宜。

Linkage map and QTL mapping of red flesh locus in apple using a R1R1×R6R6 population

The red flesh in apple fruit is a desired trait by consumers and it is associated to the anthocyanin content,which is mainly controlled by MdMYB10 with a R6 promoter.In this study,a high-density linkage group was constructed using the‘Fuji’x‘Red3’population which contained homozygous alleles R1R1 and R6R6,respectively.The linkage group consists of 7630 SNPs along 17 linkage groups,spanning 2270.21 cM,with an average density of 0.30 cM permarker.The cyanidin-3-galactoside concentration was used as the phenotypic data in QTL analysis.Moreover,one QTL peak which was flaked by two markers,marker2187260 to marker2173766,with LOD scores of 4.49 was detected.This QTL ranged from 0 to 40.79 cM on the top of linkage group(LG16).In addition one candidate molecular marker(marker2175442)in this QTL was identified,which was significant correlated with the flesh cyanidin-3-galactoside concentration.These genetic findings enrich the breeding basis of fruit flesh coloration in apple.

Genome-wide identification and functional characterization of the MdCLE peptide family in apple(Malus×domestica)

The CLAVATA3/EMBRYO SURROUNDING REGION-related(CLE)peptides are critical for stem cell homeostasis in plant shoot and root apical meristem.Although CLE genes have been reported in numerous plants,there is limited information for apple.Here,twenty-five MdCLE genes were identified fromapple genome(Apple Genome V1.0 predicted peptides).Analysis of chromosomal location showed that the 25 MdCLE genes were located on 12 of 17 apple chromosomes.Genetic structure analysis showed that 21 of 25 the MdCLE genes were intron-free.Expression patterns showed thatmost of the MdCLE genes showed higher expression levels in leaves and root compared with the other tissues.In addition,expression analysis demonstrated that MdCLE genes had different gene expression patterns for abiotic stress treatment,suggesting their potential roles in acclimation of apple to adverse environments.The 25 MdCLE genes encoded 19 different CLE peptides,and they were divided into two groups depending on their effects on the inhibition of Arabidopsis root growth.Our results suggest that MdCLE genesmay have an important but redundant function in regulating plant growth and development,and this work provides valuable references for further investigation of the biological functions of MdCLE genes.

3种树形对苹果幼树内源激素及成花的影响

[目的]富士苹果(Malus domestica Borkh.)成花难,大小年严重,制约我国苹果产业发展,本文通过研究3种不同树形对富士苹果树短枝顶芽内源激素含量和激素、成花相关基因表达的影响,比较这3种树形在花芽生理分化阶段对成花的影响,探讨促进富士苹果花芽分化、提高产量的最佳树形结构。[方法]以3年生‘烟富3号’苹果树为试材,采用高纺锤形、Y字形、古优特形3种树形,对花芽分化期苹果幼树树体生长指标、短枝顶芽赤霉素(GA3)、生长素(IAA)、脱落酸(ABA)和玉米素核苷(ZR)含量、激素相关基因及成花相关基因表达量进行测定。[结果]树形结构的不同严重影响着树体的枝类组成,古优特形短枝占比最高,Y字形次之,高纺锤形最低;对内源激素含量及其相关基因表达量的测定表明,古优特形、Y字形短枝顶芽中ABA、ZR含量显著高于高纺锤形,IAA、GA3含量显著低于高纺锤形,激素相关基因MdARF1、MdABF3、MdGA20ox1在不同时期有不同程度的表达;且和高纺锤形相比,古优特形和Y字形上调了短枝顶芽中成花促进基因(AFL1、FT1、SOC1、AP1、LFY1)在不同时期的表达量,但成花抑制基因(TFL1)在3种树形之间差异不显著。[结论]研究认为古优特形和Y字形均可以提高短枝顶芽花芽分化促进类激素ZR、ABA含量,降低花芽分化抑制类激素GA3、IAA含量,抑制枝条旺长,提高短枝比例,促进成花,在生产上利用可以一定程度上解决富士苹果难成花的问题。且古优特形独特的二维平面结构,将更适用于省力化机械修剪,有利于提高矮化密植果园的管理效率,有一定的应用前景。

苹果14-3-3基因家族的鉴定与MdGRF13的功能分析

【目的】通过鉴定和分析苹果14-3-3基因家族,并在苹果愈伤组织中过表达MdGRF13,研究逆境胁迫下其对苹果愈伤组织生长的影响,为探究该家族基因功能提供理论参考。【方法】利用生物信息学方法对苹果14-3-3基因家族进行鉴定和表达分析,构建MdGRF13过表达载体并转化苹果愈伤组织。【结果】共鉴定出36个苹果14-3-3基因家族成员。系统进化分析将该家族成员分为ε类和非ε类,每个亚族基因结构相似,且高度保守。共线性分析发现片段重复是该基因家族扩张的主要原因。基因组织特异性表达分析显示,苹果14-3-3家族基因在茎中多数上调表达。亚细胞定位表明MdGRF13蛋白定位在细胞核、细胞质和细胞膜上。qRT-PCR分析发现,在PEG和NaCl处理下分别有14和13个基因在不同时间点上调表达。过表达型(OE)愈伤组织在PEG和NaCl处理下长势优于野生型(WT)。【结论】鉴定出36个苹果14-3-3基因家族成员,且各亚族成员结构高度保守。过表达MdGRF13增强了苹果愈伤组织的抗旱性和耐盐性。

miR-483-3p靶向APPL1对脂多糖诱导的小鼠Ⅱ型肺泡上皮细胞损伤的影响

目的探讨miR-483-3p对脂多糖(LPS)诱导的小鼠Ⅱ型肺泡上皮细胞损伤的影响及可能机制。方法LPS诱导小鼠Ⅱ型肺泡上皮细胞后,实时荧光定量PCR(RT-qPCR)检测细胞中miR-483-3p和磷酸酪氨酸衔接蛋白1(APPL1)mRNA表达水平,酶联免疫吸附法检测细胞中丙二醛(MDA)和超氧化物歧化酶(SOD)水平,流式细胞术检测细胞凋亡,蛋白印迹(Western Blot)法检测细胞中APPL1、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bax)蛋白表达水平。转染miR-483-3p抑制剂或APPL1过表达载体构建miR-483-3p表达干扰或APPL1过表达小鼠Ⅱ型肺泡上皮细胞。LPS诱导干扰miR-483-3p表达或过表达APPL1的小鼠Ⅱ型肺泡上皮细胞后,上述相同方法检测细胞中MDA和SOD水平、细胞凋亡及Bcl-2和Bax蛋白表达水平。双荧光素酶报告基因实验验证miR-483-3p与APPL1调控关系。结果LPS诱导细胞后,细胞中miR-483-3p和MDA表达、细胞凋亡率和Bax蛋白表达升高(P<0.05),APPL1的mRNA和蛋白、SOD活性及Bcl-2蛋白表达降低(P<0.05)。干扰miR-483-3p表达或过表达APPL1后,LPS诱导的细胞中MDA含量、凋亡率和Bax蛋白表达降低(P<0.05),SOD活性升高(P<0.05)。miR-483-3p在小鼠Ⅱ型肺泡上皮细胞中靶向负调控APPL1表达。抑制APPL1表达逆转了干扰miR-483-3p对LPS诱导的细胞中MDA和SOD水平、细胞凋亡及Bcl-2和Bax蛋白表达的影响。结论干扰miR-483-3p表达可能通过负调控抑制APPL1表达抑制LPS诱导的小鼠Ⅱ型肺泡上皮细胞氧化应激和凋亡,减轻细胞损伤。

Apple iPod mini

在MP3播放器的发展史上，苹果公司推出的iPod无疑扮演着非常重要的角色。它时尚的外形、创新的设计理念，海量的存储空间，使其在数字随身听领域一炮而红，时至今日。