翠冠梨大果芽变果实组织切片、激素变化及转录组分析  

Tissue sections,hormone changes,and transcriptomes analysis of the large-fruited bud mutation of Pyrus pyrifolia‘Cuiguan’

作　　者：蒋爽[1] 骆军[1] 王晓庆 李水根 周博强 JIANG Shuang;LUO Jun;WANG Xiaoqing;LI Shuigen;ZHOU Boqiang(Forestry and Fruit Tree Research Institute/Shanghai Key Laboratory of Protected Horticultural Technology,SAAS,Shanghai 201403,China)

机构地区：[1]上海农业科学院林木果树研究所·上海市设施园艺技术重点实验室,上海201403

出　　处：《果树学报》2022年第10期1737-1747,共11页Journal of Fruit Science

基　　金：上海市科技兴农项目(沪农科推字2019-1-3)。

摘　　要：【目的】芽变材料是果树育种中的重要资源。前期发现1个翠冠梨的大果芽突变体,果实明显大于普通翠冠,对其变异来源和分子机制进行探究。【方法】通过石蜡切片、内源激素测定以及转录组测序解析大果芽变机制。【结果】石蜡切片结果显示,大翠冠果肉细胞横切面积大于翠冠,说明大翠冠的细胞体积增大。大翠冠中反式玉米素和赤霉素A3的含量分别在开花后10和20 d高于翠冠。花后40 d内分5个时间点取样,利用RNA-seq高通量测序评估幼果发育期间的基因表达水平,共鉴定出2015个差异表达基因,其中302个基因在2个以上时间点发生差异表达。在花后0和10 d时,一些植物激素信号转导相关基因发生差异表达。在大翠冠中3个细胞分裂素相关基因(AHP1、ORR10和ARR17)花后10 d上调表达,在大翠冠中赤霉素负调控相关基因GA3ox1和GA2ox1分别在花后0和10 d下调表达。在大翠冠中发现1个长链非编码lncRNA在所有时间点均下调表达。在大翠冠中晚期胚胎富集蛋白基因D29、锌指蛋白基因dof2.1和伸展蛋白基因extensin等在一些时间点发生表达上调,表明这些基因可能在控制果实细胞大小中起重要作用。【结论】该研究结果从生理生化和分子水平揭示了翠冠梨大果芽变可能的变异原因,细胞分裂素和赤霉素可能参与调控果实的大小,转录组分析为解析翠冠梨大果芽变的分子机制奠定了基础。【Objective】Fruit size is a very important agronomic trait,and large fruit cultivars are more appreciated in the market.Genetic background,cultivation measures,and chemicals could regulate fruit size.A large-fruited bud mutant of the Cuiguan pear(Pyrus pyrifolia)was found previously.The mechanism of mutation of this mutant were detected by studying the physiology,biochemistry and gene expression of the mutant.【Methods】A large-fruited bud mutant from Cuiguan pear(BCG)and normal Cuiguan pear(CG)were studied.All trees were grafted on seedling rootstocks.The fruit flesh of CG and BCG were sampled on 0,10,20,30,40 days after blossoming(DAB).Three independent biological replicates were analyzed per treatment(each replicate was composed of a pool of 5 fruits).The histological observation of the pulp tissue of the cross section was obtained from fruit samples.Three pears in each sample were used for slicing.The anatomical images were observed using a microscopic imaging system of Eclipse Ti-S(Nikon).The plant hormones were determined by liquid chromatography-mass spectrometry.A total of 100 mg of sample were extracted by ethyl acetate.The supernatants were evaporated to dryness.The dry matter at the bottom of the tube was dissolved with methanol.The super natant was injected into a LC-MS(AB Qtrap5500).The content of trans-zeatin(tZT)(Parent ion:m/z=218.0;quantitative ion:m/z=134.0)and gibberellin A_(3)(GA_(3))(Parent ion:m/z=345.2;quantitative ion:m/z=239.1)were analyzed.The standards were tZT(Z0876)and GA_(3)(48880)(Sigma-Aldrich).The calibration curves were plotted using 0,2.5,5.0,10,12.5,25 and 50 ng·mL^(-1) of each standard.The total RNA was extracted using the CTAB method.The RNA purity and concentration were assessed using a NanoDrop 2000(Thermo).The cDNA libraries were constructed using NEBNext Ultra RNA Library Prep Kit(NEB)according to the manufacturers instructions.The cDNA fragments of preferentially 250-300 bp in length were purified.Subsequently,USER Enzyme(NEB)was used with size-selected,adaptor-lig

关 键 词：翠冠梨 芽变 果实大小 发育 转录 

分 类 号：S661.2[农业科学—果树学]