森林草莓短果突变体鉴定及差异表达基因分析

发布时间：2018-07-03 19:24

本文选题：森林草莓 + 赤霉素　；参考：《沈阳农业大学》2016年博士论文

【摘要】：二倍体森林草莓(Fragaria vesca)是自花授粉植物,基因纯合度高,基因组小,遗传基础单纯,用理化因素处理突变率高,容易获得各种代谢功能的缺陷型,是改良现代栽培草莓的重要基因资源,同时也成为果树分子生物学研究的模式植物。果形指数是外观品质的一个重要指标,草莓果形的种类多样,最常见的是圆锥形,也有一些品种是长圆锥形、短圆锥形。为了探究草莓果实形状形成的分子机制,本研究以二倍体森林草莓'Yellow wonder'和其短果突变体为试材,研究分析野生型和突变体表型差异、遗传规律以及与赤霉素的相关性。应用转录组测序对野生型与突变体进行差异表达基因分析,并对赤霉素合成途径的候选基因进行克隆鉴定,研究结果如下：1.将二倍体森林草莓野生型和突变体进行了形态比较、组织细胞分析和赤霉素含量分析,发现突变体果实和叶片变短,叶色深绿。突变体的花粉萌发率低,存在一定程度的育性下降现象。突变体的花托明显短于野生型的花托,突变体的短果实形状在花蕾时期已经形成。野生型赤霉素水平明显高于突变体的赤霉素水平,对突变体外施赤霉素,短果突变体的果实形状能够恢复到野生型的果实形状,因此该突变体为对赤霉素敏感型的赤霉素缺陷突变体。遗传分析数据表明该突变性状为单基因控制的隐性性状。2.分别从花蕾和小绿果提取总RNA,利用Illumina高通量测序技术进行转录组测序。对野生型和突变体转录组数据进行比较,并利用qRT-PCR技术对45个差异表达基因进行验证。qRT-PCR结果与转录组数据结果相一致,表明转录组测序数据准确可靠。鉴定出一些赤霉素合成途径的差异表达基因。其中,在花蕾阶段鉴定出29个下调表达赤霉素合成相关基因,包括赤霉素合成关键基因GA3ox3(gene10124), GA3ox4 (gene02231), GA20ox1(geneO1062)和CYP450 714C2-like (gene16769)。在小绿果阶段鉴定出28个下调表达赤霉素合成相关基因,包括赤霉素合成关键基因GA3ox3(gene02611), GA20ox1(gene31924), CYP450 734A1-like (gene01176)和CYP450 714C2-like (gene 16769)。在草莓花蕾和小绿果两个阶段的差异表达基因中,赤霉素合成相关基因CYP450714C2-like(gene 16769)在两个阶段均差异表达。3.利用PCR技术,从草莓野生型和突变体中分别克隆了FvCYP714C2基因DNA序列,序列全长为1940 bp。将野生型和突变体进行比较,FvCYP714C2基因在核苷酸序列上没有差异,并且与NCBI上公布的Hawaii4的CYP714C2同源性达到100%。FvCYP714C2有5个外显子,4个内含子,其中内含子的长度分别为80bp、92bp、134 bp和95 bp。草莓FvCYP714C2 CDS全长1539 bp,共编码512个氨基酸,含有细胞色素P450s的最主要特征的保守氨基酸序列FxxGxRxCxG。通过理化性质推测该基因编码的蛋白为亲水性差的不稳定蛋白质。4. FvCYP714C2基因在野生型和突变体的根、茎、叶、花梗、花蕾、花、小绿果中均表达。除了在茎中的表达没有差异外,在根、叶、花梗、花蕾、花、小绿果实中,FvCYP714C2基因在野生型(YW)中的表达水平均明显高于在突变体(sf)中的表达量。在同一株系的表达方面,在FvCYP714C2叶片中表达量最高,其次为在花梗中,在根和果实中最少。5.构建了FvCYP714C2基因的植物过量表达载体pRI 101-ANFvCYP714C2。利用农杆菌介导浸染拟南芥,获得5株宿有FvCYP714C2基因的过表达拟南芥转化植株。FvCYP714C2转基因株系的赤霉素GA1+3含量均极显著高于野生型的赤霉素GA1+3含量。FvCYP714C2对拟南芥的营养生长有明显的促进作用。6.草莓短果突变体的形成可能是由于FvCYP714C2基因下调表达所导致。
[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.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S668.4

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