本文選題：森林草莓 + 赤霉素　； 參考：《沈陽農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：二倍體森林草莓(Fragaria vesca)是自花授粉植物,基因純合度高,基因組小,遺傳基礎(chǔ)單純,用理化因素處理突變率高,容易獲得各種代謝功能的缺陷型,是改良現(xiàn)代栽培草莓的重要基因資源,同時也成為果樹分子生物學(xué)研究的模式植物。果形指數(shù)是外觀品質(zhì)的一個重要指標,草莓果形的種類多樣,最常見的是圓錐形,也有一些品種是長圓錐形、短圓錐形。為了探究草莓果實形狀形成的分子機制,本研究以二倍體森林草莓'Yellow wonder'和其短果突變體為試材,研究分析野生型和突變體表型差異、遺傳規(guī)律以及與赤霉素的相關(guān)性。應(yīng)用轉(zhuǎn)錄組測序?qū)σ吧团c突變體進行差異表達基因分析,并對赤霉素合成途徑的候選基因進行克隆鑒定,研究結(jié)果如下：1.將二倍體森林草莓野生型和突變體進行了形態(tài)比較、組織細胞分析和赤霉素含量分析,發(fā)現(xiàn)突變體果實和葉片變短,葉色深綠。突變體的花粉萌發(fā)率低,存在一定程度的育性下降現(xiàn)象。突變體的花托明顯短于野生型的花托,突變體的短果實形狀在花蕾時期已經(jīng)形成。野生型赤霉素水平明顯高于突變體的赤霉素水平,對突變體外施赤霉素,短果突變體的果實形狀能夠恢復(fù)到野生型的果實形狀,因此該突變體為對赤霉素敏感型的赤霉素缺陷突變體。遺傳分析數(shù)據(jù)表明該突變性狀為單基因控制的隱性性狀。2.分別從花蕾和小綠果提取總RNA,利用Illumina高通量測序技術(shù)進行轉(zhuǎn)錄組測序。對野生型和突變體轉(zhuǎn)錄組數(shù)據(jù)進行比較,并利用qRT-PCR技術(shù)對45個差異表達基因進行驗證。qRT-PCR結(jié)果與轉(zhuǎn)錄組數(shù)據(jù)結(jié)果相一致,表明轉(zhuǎn)錄組測序數(shù)據(jù)準確可靠。鑒定出一些赤霉素合成途徑的差異表達基因。其中,在花蕾階段鑒定出29個下調(diào)表達赤霉素合成相關(guān)基因,包括赤霉素合成關(guān)鍵基因GA3ox3(gene10124), GA3ox4 (gene02231), GA20ox1(geneO1062)和CYP450 714C2-like (gene16769)。在小綠果階段鑒定出28個下調(diào)表達赤霉素合成相關(guān)基因,包括赤霉素合成關(guān)鍵基因GA3ox3(gene02611), GA20ox1(gene31924), CYP450 734A1-like (gene01176)和CYP450 714C2-like (gene 16769)。在草莓花蕾和小綠果兩個階段的差異表達基因中,赤霉素合成相關(guān)基因CYP450714C2-like(gene 16769)在兩個階段均差異表達。3.利用PCR技術(shù),從草莓野生型和突變體中分別克隆了FvCYP714C2基因DNA序列,序列全長為1940 bp。將野生型和突變體進行比較,FvCYP714C2基因在核苷酸序列上沒有差異,并且與NCBI上公布的Hawaii4的CYP714C2同源性達到100%。FvCYP714C2有5個外顯子,4個內(nèi)含子,其中內(nèi)含子的長度分別為80bp、92bp、134 bp和95 bp。草莓FvCYP714C2 CDS全長1539 bp,共編碼512個氨基酸,含有細胞色素P450s的最主要特征的保守氨基酸序列FxxGxRxCxG。通過理化性質(zhì)推測該基因編碼的蛋白為親水性差的不穩(wěn)定蛋白質(zhì)。4. FvCYP714C2基因在野生型和突變體的根、莖、葉、花梗、花蕾、花、小綠果中均表達。除了在莖中的表達沒有差異外,在根、葉、花梗、花蕾、花、小綠果實中,FvCYP714C2基因在野生型(YW)中的表達水平均明顯高于在突變體(sf)中的表達量。在同一株系的表達方面,在FvCYP714C2葉片中表達量最高,其次為在花梗中,在根和果實中最少。5.構(gòu)建了FvCYP714C2基因的植物過量表達載體pRI 101-ANFvCYP714C2。利用農(nóng)桿菌介導(dǎo)浸染擬南芥,獲得5株宿有FvCYP714C2基因的過表達擬南芥轉(zhuǎn)化植株。FvCYP714C2轉(zhuǎn)基因株系的赤霉素GA1+3含量均極顯著高于野生型的赤霉素GA1+3含量。FvCYP714C2對擬南芥的營養(yǎng)生長有明顯的促進作用。6.草莓短果突變體的形成可能是由于FvCYP714C2基因下調(diào)表達所導(dǎo)致。
[Abstract]:The diploid forest Strawberry (Fragaria vesca) is a self pollinated plant with high gene homozygosity, small genome, simple genetic basis, high mutation rate and easy to obtain various metabolic defects with physicochemical factors. It is an important gene resource for improving modern cultivation of strawberry, and also a model plant in the molecular biology of fruit trees. Shape index is an important index of appearance quality. The variety of strawberry fruit shape is the most common cone-shaped, and some varieties are long conical and short conical. In order to explore the molecular mechanism of the shape formation of strawberry fruit, this study used the diploid forest strawberry'Yellow Wonder'and its short fruit mutants as test materials to study and analyze the wild type. Phenotypic differences, genetic rules and correlation with gibberellin. The transcriptional group was sequenced to analyze the differentially expressed genes of the wild type and the mutant, and the candidate genes of the gibberellin synthesis pathway were cloned and identified. The results were as follows: 1. the morphological comparison of the wild and mutants of the diploid forest raspberry was compared. It was found that the fruit and leaf of the mutant were shorter and the leaf color was dark green. The pollen germination rate of the mutant was low and there was a certain degree of fertility decline. The mutant was obviously shorter than the wild type, and the short fruit shape of the mutant had been formed in the bud period. At the level of gibberellin in the mutant, the shape of the fruit of the mutant was restored to the shape of the wild type. Therefore, the mutant was a gibberellin sensitive mutant of gibberellin. The genetic analysis data showed that the mutation was a single based recessive trait controlled by.2. from buds and small green. The total RNA was extracted and the transcriptional group was sequenced using Illumina high throughput sequencing technology. The data of the wild type and the mutant transcriptional group were compared, and the qRT-PCR technique was used to verify the results of the 45 differentially expressed genes. The results of the.QRT-PCR were consistent with the results of the transcriptional data, indicating that the sequence data of the transcriptional group were accurate and reliable. In the bud stage, 29 down regulated gibberellin synthesis related genes, including GA3ox3 (gene10124), GA3ox4 (gene02231), GA20ox1 (geneO1062) and CYP450 714C2-like (gene16769), were identified in the bud stage, and 28 down-regulated expression of gibberellin was identified in the small green fruit stage. Genes, including the key genes of gibberellin synthesis GA3ox3 (gene02611), GA20ox1 (gene31924), CYP450 734A1-like (gene01176) and CYP450 714C2-like (gene 16769). In the differential expression genes of two stages of strawberry buds and small green fruit, the gibberellin synthesis correlation group expressed the difference in CYP450714C2-like (gene 16769) at two stages. The FvCYP714C2 gene DNA sequence was cloned from the wild and mutants of Strawberry by PCR technique. The total length of the FvCYP714C2 gene was 1940 bp., the wild type and the mutant were compared. There was no difference in the nucleotide sequence of the FvCYP714C2 gene. And the CYP714C2 homology of Hawaii4 on NCBI had reached 100%.FvCYP714C2 with 5 exons and 4 contents. The length of the intron is 80bp, 92bp, 134 BP and 95 bp. strawberry FvCYP714C2 CDS full length 1539 BP, which encodes 512 amino acids, and the conservative amino acid sequence containing the most important characteristic of cytochrome P450s is that the protein encoded by the gene is a poor hydrophilic unstable protein.4. FvCYP714C2 basis. In the root, stem, leaf, flower stalk, flower bud, flower, and small green fruit, the expression level of FvCYP714C2 gene in the root, leaf, flower, flower, and small green fruit is higher than that in the mutant (YW). The expression level of the FvCYP714C2 gene in the same line, in the same line, is in F. The highest expression in the vCYP714C2 leaves is the next in the pedicel, and the least.5. in the root and fruit has the least expression vector of the FvCYP714C2 gene, pRI 101-ANFvCYP714C2., using Agrobacterium tumefaciens to impregnate Arabidopsis thaliana, and to obtain 5 strains of FvCYP714C2 gene of the Arabidopsis transgenic plant.FvCYP714C2 transgenic line of gibberellin GA1 The content of +3 was significantly higher than that of wild type gibberellin GA1+3 content.FvCYP714C2 significantly promoted the nutritional growth of Arabidopsis thaliana. The formation of.6. strawberry short fruit mutants may be caused by the downregulation of FvCYP714C2 gene.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S668.4

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