【摘要】：枇杷(Eriobotrya Lindl.)為薔薇科枇杷屬植物,該屬包含二三十種,不同種間花期差異明顯,并且有一些枇杷種類的花期是可變的,或稱可塑的。例如,普通枇杷在秋冬季節(jié)開花;臺(tái)灣枇杷為春季開花;而臺(tái)灣枇杷恒春變型在原產(chǎn)地臺(tái)灣春季開花,引種到廣州華南農(nóng)業(yè)大學(xué)枇杷種質(zhì)資源圃以及廣州市果樹所后卻變?yōu)榍锒_花。然而,迄今為止,關(guān)于枇杷花期的分子生物學(xué)方面的研究甚少,并且未見涉及野生枇杷及花期差異的研究。本研究一方面從枇杷成花的基礎(chǔ)方面入手,進(jìn)行了枇杷花期相關(guān)基因的克隆、表達(dá)分析和功能驗(yàn)證的初步探索;另一方面對(duì)花期不同的枇杷屬材料花芽分化前及花芽分化期間的葉片進(jìn)行轉(zhuǎn)錄組測(cè)序,深入研究枇杷花芽分化起始過程中基因轉(zhuǎn)錄的動(dòng)態(tài)變化,為花期調(diào)控的分子生物學(xué)研究提供了大量信息,希望為揭示枇杷花期調(diào)控的分子機(jī)理奠定基礎(chǔ),為生產(chǎn)實(shí)踐中人工調(diào)控花果發(fā)育,以及培育不同成熟期新品種提供理論依據(jù),并為其它木本果樹的成花機(jī)理研究提供借鑒。主要結(jié)果如下:(1)對(duì)兩種不同花期野生枇杷臺(tái)灣枇杷(E.deflexa Nakai)和臺(tái)灣枇杷恒春變型(E.deflexa f.koshunensis Nakai)的莖頂端進(jìn)行切片觀察,結(jié)果顯示臺(tái)灣枇杷恒春變型在9月底至10月初就已經(jīng)開始花芽分化,而臺(tái)灣枇杷11月才開始,表明不同枇杷種或變型之間花期不同是因?yàn)樗鼈兓ㄑ糠只_始的時(shí)間即已有不同。同時(shí)對(duì)普通枇杷開花時(shí)間不同的兩個(gè)品種‘早鐘6號(hào)’和‘解放鐘’的莖頂端在不同發(fā)育階段的觀察,發(fā)現(xiàn)‘早鐘6號(hào)’和‘解放鐘’開花時(shí)間差異則不是因?yàn)榛ㄑ糠只鹗紩r(shí)間的不同,而主要是因?yàn)樗鼈兓ㄐ虬l(fā)育快慢不同。(2)從臺(tái)灣枇杷恒春變型中成功得到FT、CO、GI、SOC1和PIF4同源基因各一個(gè),及FD和SVP同源基因各兩個(gè),分別命名為EdFT、EdCO、EdGI、EdSOC1、EdPIF4、EdFD1、EdFD2、EdSVP1和EdSVP2。對(duì)這些基因預(yù)測(cè)的氨基酸序列進(jìn)行生物信息學(xué)分析,表明它們都具有各基因所特有的保守氨基酸殘基或結(jié)構(gòu)域。(3)亞細(xì)胞定位顯示EdFT、EdSVP1和EdSVP2在細(xì)胞核和細(xì)胞質(zhì)中均有分布,而EdFD1、EdFD2、EdCO、EdGI、EdSOC1和EdPIF4只定位于細(xì)胞核。(4)在擬南芥中過表達(dá)EdFT、EdFD1、EdFD2、EdCO、EdGI和EdSOC1均表現(xiàn)出早花表型,說明它們具有保守的促進(jìn)開花的功能。晝夜節(jié)律表達(dá)分析表明EdGI、EdCO和EdFT的表達(dá)均隨晝夜變化而變化,受光周期影響。BiFC實(shí)驗(yàn)證明EdFT可以和EdFD1/2在體內(nèi)發(fā)生蛋白水平上的互作。這些都進(jìn)一步證明這些基因是擬南芥相應(yīng)基因的同源基因,可能具有類似的功能。(5)時(shí)空表達(dá)分析發(fā)現(xiàn),EdFD1、EdFD2和EdSOC1可能對(duì)臺(tái)灣枇杷恒春變型花芽分化起正調(diào)控作用,EdSVP1起負(fù)調(diào)控作用;而在普通枇杷‘解放鐘’和‘早鐘6號(hào)’中,除了EdFD1和EdSOC1,EdCO和EdFT可能也對(duì)花芽分化起正調(diào)控作用,而EdFD2卻沒有明顯影響。(6)對(duì)臺(tái)灣枇杷和臺(tái)灣枇杷恒春變型花芽分化前及花芽分化期間的葉片進(jìn)行轉(zhuǎn)錄組測(cè)序,總計(jì)產(chǎn)出74314777320 nt數(shù)據(jù),組裝得到總unigene 116674個(gè)。共有94459個(gè)unigene獲得注釋信息。注釋結(jié)果有39221個(gè)unigene匹配到COG分類的25個(gè)功能類別,有66514個(gè)unigene注釋到GO類別的55個(gè)功能組。共預(yù)測(cè)得到CDS 88481條。這為枇杷花期調(diào)控進(jìn)一步研究提供了豐富的序列和生物學(xué)信息。(7)差異表達(dá)基因分析發(fā)現(xiàn)不同樣品間差異表達(dá)基因數(shù)量較多。采用WGCNA構(gòu)建基因共表達(dá)網(wǎng)絡(luò),構(gòu)建了28個(gè)模塊。對(duì)lightcyan模塊進(jìn)行KEGG功能分析,發(fā)現(xiàn)該模塊顯著富集的代謝途徑包括:晝夜節(jié)律-植物,戊糖和葡萄糖醛酸相互轉(zhuǎn)化,苯丙氨酸代謝,次生代謝物生物合成,倍半萜和三萜生物合成,苯丙烷生物合成,類胡蘿卜素生物合成,半胱氨酸和甲硫氨酸代謝。其中植物晝夜節(jié)律和類胡蘿卜素生物合成代謝通路可能在枇杷花期調(diào)控中起重要作用,為后續(xù)功能和調(diào)控機(jī)制研究打開通道。(8)與擬南芥開花相關(guān)基因進(jìn)行同源性比對(duì),鑒定了56個(gè)同源基因,共193條序列。其中包括大量參與光周期途徑,以及一些參與熱感應(yīng)途徑和自主途徑的基因。對(duì)鑒定的光周期同源基因進(jìn)行表達(dá)分析,發(fā)現(xiàn)其中一些基因在臺(tái)灣枇杷及臺(tái)灣枇杷恒春變型花芽分化不同階段的表達(dá)趨勢(shì)較一致,均隨著葉芽向花芽的轉(zhuǎn)化呈上升趨勢(shì),暗示這些基因可能對(duì)臺(tái)灣枇杷及臺(tái)灣枇杷恒春變型的花期調(diào)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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S667.3

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