【作者】 李玲；

【导师】 苏淑钗；

【作者基本信息】 北京林业大学 ， 森林培育， 2020， 硕士

【摘要】 板栗(Castanea mollissima BL.)隶属于壳斗科(Fagaceae)栗属(Castanea),因其种仁富含丰富的营养物质被誉为“干果之王”。我国板栗的单产相对较低,这与雌雄花比例失调、授粉受精不良和胚败育等因素有关。受精且胚胎发育是保证板栗产量的关键环节,这些过程中任一时期发生败育均会降低其结实率,从而严重影响产量。本研究以板栗‘遵化短刺’为试材通过对授粉后子房与胚珠进行细胞学观察,确定受精和胚发育关键期,对受精和种仁发育关键期胚珠进行转录组测序,筛选出与受精及胚发育相关的关键基因,进一步为基因功能研究提供参考依据。主要结果如下:(1)从解剖结构看,7 DAP(授粉后天数)板栗子房显著膨大,胚珠形成。15DAP胚囊发育成熟,但未完成受精。18 DAP授粉胚珠内珠心组织逐渐解体且胚乳核开始分裂,可见双受精作用完成,因此,16-17 DAP完成双受精作用;而未授粉胚珠的胚囊发育不成熟。21 DAP同一子房内发育和败育胚珠形态开始出现极显著差异,发育胚珠内胚乳核大量分裂。27 DAP发育胚珠内形成球形胚与大量胚乳细胞,而败育胚珠呈现空腔胚囊。(2)对18 DAP未授粉胚珠vs授粉胚珠转录组分析得到7023个差异表达基因,上调基因2915个,下调基因4108个。筛选出与受精作用相关的关键基因主要有乙醛脱氢酶家族2成员B7、糖载体蛋白C、磷脂酶Dα4、转录因子WRKY40、壁相关的受体激酶1、富含甘氨酸的DOT1蛋白和MYB108等,这些结果表明在授粉胚珠中表达的关键基因有利于提高抗病性、为完成受精作用提供能量以及雌花簇快速增长。(3)对21 DAP发育胚珠vs败育胚珠转录组分析得到1615个差异表达基因,上调基因530个,下调基因1085个。筛选出的与胚发育相关的关键基因主要有含B3域转录因子LEC2、转录因子MYB118、B型谷蛋白2、钠/钙交换因子NCLI、核仁蛋白、真核翻译起始因子、聚半乳糖醛酸酶、富含甘氨酸的细胞壁结构蛋白、扩展蛋白A23和AHP2等。以上结果表明在发育胚珠中表达的关键基因调节胚胎发生、细胞壁可塑性和细胞增殖生长等,并参与DNA复制和淀粉与蔗糖代谢通路进而促进雌花簇膨大。(4)对27 DAP发育胚珠vs败育胚珠转录组分析得到4370个差异表达基因,上调基因2151个,下调基因2219个。筛选出的与胚发育相关的关键基因主要有果胶酯酶2、扩展蛋白A23、AGL61、MYB115、AHP2、两组分应答调节剂ARR21、SAUR21、CNR6和蔗糖合酶2等。这些结果表明在发育胚珠表达的关键基因调控胚与胚乳发育、细胞分裂与膨大和细胞壁可塑性等,促进雌花簇进一步膨大变硬。本研究揭示了雌花簇授粉后胚珠的发育进程、双受精时期以及胚发育过程,并提供了大量关于板栗受精与胚发育的转录组数据,为提高其结实率及研究胚发育的分子机理提供了理论基础。更多还原

【Abstract】 Chinese chestnut(Castanea mollissima BL.),belongs to the Fagaceae family,is known as the "king of dried fruits" because of rich nutrients in its seed kernel.However,the yield per unit area of Chinese chestnut is relatively low,which is closely related to the imbalance of male-female flower ratio,poor pollination and fertilization,and embryo abortion.Fertilization and embryonic development are the key links of ensuring higher yield of Chinese chestnut,but abortion at any stage of these processes will reduce its seed setting rate and thus seriously affecting the yield.In this study,using the chestnut cultivar ‘Zunhuaduanci’ as test materialthe,and then the key stages of fertilization and embryo development were determined by cytological observation of ovaries and ovules after pollination,and the key genes related to these processes were selected by transcriptome sequencing of ovule at the critical stages of fertilization and seed development,which will provide a reference for the study of gene function.The main results are as follows:(1)From the anatomical structure,in the 7th day after pollination,chestnut had significantly enlarged ovaries,and ovules had been formed.In the 15 th day after pollination,the embryo sac was mature,but fertilization had not been completed.In the pollinated ovule of 18 th day after pollination,the nucellus tissue gradually disintegrated and the endosperm nucleus began to divide,indicating the completion of double fertilization.Therefore,the pollinated ovules completed double fertilization in 16-17 th day after pollination,however,the embryo sac of the unpollinated ovule was immature.In the same ovary of 21 st day after pollination,there were significant differences in morphology between developed and abortive ovules,and the massive internal endosperm nuclei were divided.In the 27 th day after pollination,the globular embryo and a large number of endosperm cells had been formed in the developed ovules,while the vacuolar embryo sac was observed in abortive ovules.(2)Transcriptome analysis of the unpollinated ovules and pollinated ovules of 18 th day after pollination yielded 7023 differentially expressed genes,including 2915 up-regulated genes and 4108 down-regulated genes.The key genes related to fertilization mainly include aldehyde dehydrogenase family 2 member B7,sugar carrier protein C,phospholipase D alpha 4,transcription factor WRKY40,wall related receptor kinase 1,glycine-rich DOT1 protein,and MYB108,etc.These results indicate that the key genes expressed in the pollinated ovules are beneficial to the improvement of disease resistance,the supply of energy for the completion of fertilization,and the rapid growth of female flower clusters.(3)Transcriptome analysis of the developed ovules and abortive ovules of 21 st day after pollination yielded 1615 differentially expressed genes,including 530 up-regulated genes and 1085 down-regulated genes.The selected key genes related to embryo development mainly include B3 domain-containing transcription factor LEC2,transcription factor MYB118,glutelin type-B 2,sodium/calcium exchanger NCL1,nucleolin,eukaryotic translation initiation factor,polygalacturonidase,glycine-rich cell wall structural protein 1,expansin-A23,and histidine-containing phosphotransfer protein 2,etc.These results indicate that the key genes expressed in the developed ovules regulate embryogenesis,cell wall plasticity,cell proliferation and growth to promote the enlargement of female flower clusters.(4)Transcriptome analysis of the developed ovules and abortive ovules of 27 th day after pollination yielded 4370 differentially expressed genes,including 2151 up-regulated genes and 2219 down-regulated genes.The key genes related to embryo development were pectinesterase 2,expansin-A23,AGL61,MYB115,AHP2,two-component response regulator ARR21,SAUR21,cell number regulator 6,and sucrose synthase 2,etc.These results indicated that the key genes expressed in developed ovules regulated embryo and endosperm development,cell division and expansion,and cell wall plasticity,promoting the further enlargement and hardening of female flower clusters.This study reveals the ovule development process,double fertilization period and embryo development process after female flower cluster pollinated,and provids a large amount of transcriptome data on fertilization and embryonic development,which could offer a theoretical basis for improving seed setting rate of Chinese chestnut and further research on molecular mechanism of embryonic development.更多还原