果树学报2017，Vol.34Issue(6)：660-669,10.DOI:10.13925/j.cnki.gsxb.20160129
‘翠冠’梨大果型芽变的细胞学及相关基因表达研究
Studies on cytology and related gene expression pattern of a large-fruited bud mutant from ‘ Cuiguan’ pear (Pyrus pyrifolia)
摘要
Abstract
[Objective] Fruit size at harvest is determined by both cell number and cell size of flesh which result from cell division and cell expansion processes,respectively.Cell division is regulated by cell cycle.Some important regulatory proteins of cell cycle have been studied,such as cyclins (CYCs) and cyclin-dependent kinases (CDKs).CDKs control the key transitions in the plant cell cycle.CDKA plays a pivotal role in both the G1-to-S and the G2-to-M transitions,while a reduction in CDKB1 activity results in a block at the G2-to-M transition.Plants contain much more CYCs than previously described in other organisms,some of them have been found to associate with CDKs.D-type cyclins (CYCD) are thought to regulate the Gl-to-S transition and function at the G2-to-M transition.A-type cyclins regulate the S-to-M phase,and B-type cyclins control both the G2-to-M transition and the intra-M-phase.To investigate cellular and molecular mechanism related to fruit size in pear,‘ Cuiguan’ pear (Pyrus pyrifolia Nakai.) and its spontaneous mutant with larger fruit size (named ‘Panzhuang Dacuiguan’) were used as materials in this study.[Methods] AFLP (amplified fragment length polymorphism) was used to analyze the genomic differences between ‘Cuiguan’ and ‘Panzhuang Dacuiguan’.128 EcoR Ⅰ/Mse Ⅰ selective primer pairs were adopted in AFLP analysis.To minimize the effect of low-intensity background peaks(noise),threshold value for fragment selection were set towards average signal intensity and fragment frequency.FCM (flow cytometry) analysis was carried out to record the chromosome ploidy.Genome size was measured using the Otto method.The filtered fluid was then centrifuged at a speed of 5 000 r· min-1 for 2min.The system's light source of FCM was 488 nm argon lasers.Seasonal changes in longitudinal and transverse diameters were measured at regular intervals during the course of fruit development.Fresh weight,longitudinal diameter and transverse diameter of both ‘Cuiguan’ and ‘Panzhuang Dacuiguan’fruits were measured at harvest.The flesh width was calculated from the difference between the largest width of the transverse section of the fruits and core diameter.Fruit (flower) samples were collected for cytology and gene expression analysis during 0-28 d after full bloom (DAFB).Paraffin section was used for microscopy observation of cell number and size of the fruits.Cell number was determined by counting the number of cell layers.Cell size was determined by measuring the average diameter of seven contiguous cells.Ten observation zones per paraffin section were measured.Quantitative real-time PCR (Q-PCR) was performed to test the gene expression.Each Q-PCR reaction mixture contained SYBR Premix Ex TaqTM (10.0μL),both primer (0.4 μL,10 μmol·L-1),cDNA (2 μL),and RNase-free H2O (7.2 μL) in a total volume of 20 μL.The reaction started with a preliminary step of 95 ℃ for 30 s,followed by 40 cycles of 95 ℃ for 5 s and 60 ℃ for 20 s.The Q-PCR primers were designed using Primier 3 according to obtained sequences of cDNA fragments.[Results] A total of 116 polymorphic primer pairs were chosen from 128 selective primer pairs for AFLP analysis which amplified 8 620 DNA fragments.The polymorphic rate and Neips association coefficient between them were 8.18％ and 0.957 4,respectively,confirming that ‘Panzhuang Dacuiguan’ was a sport of ‘Cuiguan’.FCM analysis showed that both ‘Cuiguan’ and ‘Panzhuang Dacuiguan’were diploid.Therefor,the larger fruit size in ‘Panzhuang Dacuiguan’ was not the result of the chromosome doubling.At maturity stage,‘Panzhuang Dacuiguan’ had a 17％ larger fruit diameter and a 64％ heavier fruit weight than those of ‘Cuiguan’,while no difference in internal quality was observed between two cultivars.Cell division started at 0 DAFB (days after full bloom) and continued till 24 DAFB in ‘Panzhuang Dacuiguan’,longer than that of ‘Cuiguan’ by 4 d.Cell number (cell layers) in the floral-tube tissue was nine cell layers more in ‘Panzhuang Dacuiguan’ than in ‘Cuiguan’ at 28 DAFB.The average areas of the cell and cell nucleus at full bloom stage were larger in ‘Panzhuang Dacuiguan’ than those in ‘Cuiguan’.QPCR analysis indicated CYCD3 expression of ‘Panzhuang Dacuiguan’ fruit during early fruit development was 1.2 times as high as that of ‘Cuiguan’.In other cyclin-related genes including CYCA2、CDKA1 and CDKB2,different expressions were also observed between ‘Panzhuang Dacuiguan’ and ‘Cuiguan’.[Conclusion] The larger fruit size of ‘Panzhuang Dacuiguan’ pear,the spontaneous mutant of ‘Cuiguan’ pear,could be contributed to the increased cell number in the fruit flesh which was related to extended period of cell division.Our work verified the important role of cell division in regulating pear fruit size.关键词
‘翠冠’梨/大果型芽变/细胞数目/细胞大小/AFLPKey words
‘Cuiguan’ pear/Large-fruited mutant/Cell number/Cell size/AFLP分类
农业科技引用本文复制引用
舒莎珊,李鑫,骆军,白松龄,滕元文..‘翠冠’梨大果型芽变的细胞学及相关基因表达研究[J].果树学报,2017,34(6):660-669,10.基金项目
浙江省园艺作物育种先进技术团队项目(2013TD05) （2013TD05）
