枇杷花期调控的分子生物学研究

发布时间：2018-09-02 09:44

【摘要】：枇杷(Eriobotrya Lindl.)为蔷薇科枇杷属植物,该属包含二三十种,不同种间花期差异明显,并且有一些枇杷种类的花期是可变的,或称可塑的。例如,普通枇杷在秋冬季节开花;台湾枇杷为春季开花;而台湾枇杷恒春变型在原产地台湾春季开花,引种到广州华南农业大学枇杷种质资源圃以及广州市果树所后却变为秋冬开花。然而,迄今为止,关于枇杷花期的分子生物学方面的研究甚少,并且未见涉及野生枇杷及花期差异的研究。本研究一方面从枇杷成花的基础方面入手,进行了枇杷花期相关基因的克隆、表达分析和功能验证的初步探索;另一方面对花期不同的枇杷属材料花芽分化前及花芽分化期间的叶片进行转录组测序,深入研究枇杷花芽分化起始过程中基因转录的动态变化,为花期调控的分子生物学研究提供了大量信息,希望为揭示枇杷花期调控的分子机理奠定基础,为生产实践中人工调控花果发育,以及培育不同成熟期新品种提供理论依据,并为其它木本果树的成花机理研究提供借鉴。主要结果如下:(1)对两种不同花期野生枇杷台湾枇杷(E.deflexa Nakai)和台湾枇杷恒春变型(E.deflexa f.koshunensis Nakai)的茎顶端进行切片观察,结果显示台湾枇杷恒春变型在9月底至10月初就已经开始花芽分化,而台湾枇杷11月才开始,表明不同枇杷种或变型之间花期不同是因为它们花芽分化开始的时间即已有不同。同时对普通枇杷开花时间不同的两个品种‘早钟6号’和‘解放钟’的茎顶端在不同发育阶段的观察,发现‘早钟6号’和‘解放钟’开花时间差异则不是因为花芽分化起始时间的不同,而主要是因为它们花序发育快慢不同。(2)从台湾枇杷恒春变型中成功得到FT、CO、GI、SOC1和PIF4同源基因各一个,及FD和SVP同源基因各两个,分别命名为EdFT、EdCO、EdGI、EdSOC1、EdPIF4、EdFD1、EdFD2、EdSVP1和EdSVP2。对这些基因预测的氨基酸序列进行生物信息学分析,表明它们都具有各基因所特有的保守氨基酸残基或结构域。(3)亚细胞定位显示EdFT、EdSVP1和EdSVP2在细胞核和细胞质中均有分布,而EdFD1、EdFD2、EdCO、EdGI、EdSOC1和EdPIF4只定位于细胞核。(4)在拟南芥中过表达EdFT、EdFD1、EdFD2、EdCO、EdGI和EdSOC1均表现出早花表型,说明它们具有保守的促进开花的功能。昼夜节律表达分析表明EdGI、EdCO和EdFT的表达均随昼夜变化而变化,受光周期影响。BiFC实验证明EdFT可以和EdFD1/2在体内发生蛋白水平上的互作。这些都进一步证明这些基因是拟南芥相应基因的同源基因,可能具有类似的功能。(5)时空表达分析发现,EdFD1、EdFD2和EdSOC1可能对台湾枇杷恒春变型花芽分化起正调控作用,EdSVP1起负调控作用;而在普通枇杷‘解放钟’和‘早钟6号’中,除了EdFD1和EdSOC1,EdCO和EdFT可能也对花芽分化起正调控作用,而EdFD2却没有明显影响。(6)对台湾枇杷和台湾枇杷恒春变型花芽分化前及花芽分化期间的叶片进行转录组测序,总计产出74314777320 nt数据,组装得到总unigene 116674个。共有94459个unigene获得注释信息。注释结果有39221个unigene匹配到COG分类的25个功能类别,有66514个unigene注释到GO类别的55个功能组。共预测得到CDS 88481条。这为枇杷花期调控进一步研究提供了丰富的序列和生物学信息。(7)差异表达基因分析发现不同样品间差异表达基因数量较多。采用WGCNA构建基因共表达网络,构建了28个模块。对lightcyan模块进行KEGG功能分析,发现该模块显著富集的代谢途径包括:昼夜节律-植物,戊糖和葡萄糖醛酸相互转化,苯丙氨酸代谢,次生代谢物生物合成,倍半萜和三萜生物合成,苯丙烷生物合成,类胡萝卜素生物合成,半胱氨酸和甲硫氨酸代谢。其中植物昼夜节律和类胡萝卜素生物合成代谢通路可能在枇杷花期调控中起重要作用,为后续功能和调控机制研究打开通道。(8)与拟南芥开花相关基因进行同源性比对,鉴定了56个同源基因,共193条序列。其中包括大量参与光周期途径,以及一些参与热感应途径和自主途径的基因。对鉴定的光周期同源基因进行表达分析,发现其中一些基因在台湾枇杷及台湾枇杷恒春变型花芽分化不同阶段的表达趋势较一致,均随着叶芽向花芽的转化呈上升趋势,暗示这些基因可能对台湾枇杷及台湾枇杷恒春变型的花期调控起重要作用。
[Abstract]:Eriobotrya Lindl. (Eriobotrya Lindl.) is a member of the genus Eriobotrya of the Rosaceae. There are twenty or thirty species in the genus Eriobotrya. The florescence varies significantly among different species, and some loquat species have variable or plastic florescence. For example, common loquat blossoms in autumn and winter; Taiwanese loquat blossoms in spring; and Taiwanese loquat floresses in spring. Flowers, introduced to the loquat germplasm resources nursery of Guangzhou South China Agricultural University and Guangzhou Fruit Tree Institute, become autumn and winter flowering. The cloning, expression analysis and functional verification of loquat florescence-related genes were studied. On the other hand, the transcriptome of loquat leaves before and during florescence differentiation was sequenced to study the dynamic changes of gene transcription during florescence initiation, which was a molecular organism regulated by florescence. The results are as follows: (1) Two different flowering stages of wild loquat were studied in this paper. The stem apices of E. deflexa f. koshunensis Nakai and E. deflexa f. koshunensis Nakai were observed by sectioning. The results showed that the flower buds of loquat in Taiwan began to differentiate from the end of September to the beginning of October. At the same time, the difference of flowering time between'Zaozhong 6'and'Jiefangzhong' was found not because of the difference of flowering time between'Zaozhong 6'and'Jiefangzhong', but mainly because of the difference of flowering time between'Zaozhong 6' and'Jiefangzhong'. Two homologous genes of FT, CO, GI, SOC1 and PIF4, and two homologous genes of FD and SVP, named EdFT, EdCO, EdGI, EdSOC1, EdPIF4, EdFDFD1, EdFD2, EdSVP1 and EdSVP2, were successfully obtained from the Taiwan loquat Hengchun variant. All of them have their own conserved amino acid residues or domains. (3) Subcellular localization showed that EdFT, EdSVP1 and EdSVP2 were distributed in the nucleus and cytoplasm, while EdFD1, EdFD2, EdCO, EdGI, EdSOC1 and EdPIF4 were only localized in the nucleus. (4) Overexpression of EdFT, EdFD1, EdFD2, EdCO, EdGI and EdPIF4 in Arabidopsis showed early flowering appearance. The expression of EdGI, EdCO and EdFT varied with the day and night, and were affected by the photoperiod. BiFC experiment showed that EdFT and EdFD 1/2 could interact at the protein level in vivo. All these further proved that these genes were the same as the corresponding genes in Arabidopsis. (5) Spatio-temporal expression analysis showed that EdFD1, EdFD2 and EdSOC1 may play a positive role in regulating flower bud differentiation of loquat var. perennialis var. perennialis, while EdSVP1 may play a negative role in regulating flower bud differentiation. In common loquat'Jiefang Zhong'and'Zaozhong 6', besides EdFD1 and EdSOC1, EdCO and EdFT may also play a positive role in regulating flower bud differentiation. (6) Transgenomic sequencing was performed on the leaves of loquat and Loquat Cultivars before and during flower bud differentiation, resulting in a total of 74314777320 NT data. A total of 116674 unigenes were assembled. A total of 94459 unigenes obtained annotation information. Among the 25 functional classifications, 65 514 unigenes were annotated into 55 functional groups of GO. A total of 88481 CDS were predicted. This provided abundant sequence and biological information for further study on Florescence regulation of loquat. (7) Differentially expressed genes were found to be more abundant among different samples. WGCNA was used to construct gene co-tables. The lightcyan module was analyzed by KEGG function. The metabolic pathways of significant enrichment included circadian rhythm-plant, pentose and glucuronic acid conversion, phenylalanine metabolism, secondary metabolite biosynthesis, sesquiterpene and triterpene biosynthesis, phenylpropane biosynthesis, and carotenoid biosynthesis. The circadian rhythm and carotenoid biosynthetic pathways in plants may play an important role in the regulation of loquat flowering, opening up a pathway for further functional and regulatory mechanisms. (8) By homology comparison with Arabidopsis Flowering-related genes, 56 homologous genes with 193 sequences were identified. Some genes involved in photoperiodic pathway, thermosensitive pathway and autonomous pathway were identified. The expression of some genes in different stages of flower bud differentiation of Taiwanese loquat and Taiwanese loquat were found to be consistent with the transformation from leaf bud to flower bud. These results suggest that these genes may play an important role in the florescence regulation of Taiwanese loquat and Taiwanese loquat.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S667.3

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