砀山酥梨PbMADS12和PbMADS14基因克隆与功能分析

发布时间：2018-09-07 08:59

【摘要】：砀山酥梨(Pyrus bretschneideri Rehd.cv.‘Dangshansuli’)在园艺学分类上介于白梨和砂梨之间,在我国安徽省砀山地区有着悠久的栽培历史,是我国栽培面积最大的梨品种之一。近年来,在对砀山酥梨种质资源的持续调查过程中,我们发现了一株酥梨的高糖芽变品系,该品系果实总糖含量显著提高,甜度较高的果糖为主要糖组分,果实表面较非变异果实粗糙、皮孔密集、果锈重,而非变异果实为翠绿色,且变异性状表现十分稳定。课题组进一步通过对变异果实和非变异果实的代谢组学和转录组学比较研究发现,在变异果实发育过程中,乙烯代谢发生显著变化,推测与上游的两个MADS转录因子的调控有关。为此,本论文对两个MADS转录因子进行基因克隆和功能分析,以验证MADS对砀山酥梨果实发育过程中乙烯代谢的调控作用,为阐释砀山酥梨高糖芽变果实糖积累的分子机制提供试验依据。同时,有关果实成熟的研究多集中在呼吸跃变型果实上,对于非跃变型果实的成熟调控研究较少,因此,本论文对于揭示MADS转录因子调控非跃变型果实成熟机理研究也有着重要意义。本试验以砀山酥梨的变异型和非变异型果实为试材,根据前期转录组和代谢组的整合分析结果,克隆2个MADS基因PbMADS12和PbMADS14,并进行相关生物信息学分析。采用实时定量PCR技术,对PbMADS12和PbMADS14以及注释为乙烯代谢相关功能基因进行表达模式分析。构建超表达载体PC1301-MADS12和PC1301-MADS14,并成功转化Micro-Tom番茄,并鉴定得到若干转基因植株。本试验取得以下主要结果:1.以采集的花后100 d(DPA100)变异和非变异果实为试材,克隆得到两个MADS基因PbMADS12和PbMADS14;测序结果显示,变异和非变异果实中克隆的PbMADS14基因序列相同,PbMADS12基因序列有一个碱基差异,翻译出氨基酸序列无差异。PbMADS12的ORF框由720个碱基组成,编码239个氨基酸,PbMADS14的ORF框由750个碱基组成,编码249个氨基酸。结果表明,砀山酥梨的高糖芽变果实不是因为MADS基因的序列差异造成的。2.MADS基因进行序列分析及进化树分析结果表明,本试验所克隆得到的两个MADS基因序列与NCBI数据库中发布的基因序列同源性较高,都含有两个保守程度较高的结构域MADS-box和K-box。3.对砀山酥梨转录组分析中注释为乙烯代谢的相关功能基因以及PbMADS12和PbMADS14进行了实时定量PCR分析,结果表明,非变异果实的供试基因表达量峰值均为花后130 d(DPA130),变异果实的供试基因表达量峰值均为花后160 d(DPA160)。4.成功构建超表达载体PC1301-MADS12和PC1301-MADS14,转化Micro-Tom番茄,分别以PbMADS12和PbMADS14基因和潮霉素基因序列设计引物进行PCR扩增,鉴定得到转基因阳性番茄植株5个。
[Abstract]:Dangshan pear (Pyrus bretschneideri Rehd.cv.'Dangshansuli') is between white pear and sand pear in horticultural classification. It has a long cultivation history in Dangshan area of Anhui Province and is one of the largest pear varieties in China. In recent years, during the continuous investigation on the germplasm resources of Dangshanshu pear, we found a high sugar budding strain of Dangshanshu pear. The total sugar content of the fruit of this strain was significantly increased, and the fructose with high sweetness was the main sugar component. The surface of the fruit was rougher, the lenticels were dense, the fruit rust was heavy, but the non-variant fruit was emerald green, and the variation character was very stable. Through the comparative study of metabolomics and transcriptome of mutant fruit and non-variant fruit, it was found that ethylene metabolism changed significantly during the development of variant fruit, which was related to the regulation of two MADS transcription factors upstream. Therefore, two MADS transcription factors were cloned and analyzed in order to verify the role of MADS in regulating ethylene metabolism during fruit development of Dangshan pear. To provide experimental basis for explaining the molecular mechanism of sugar accumulation in high sugar budding fruit of Dangshan pear. At the same time, the researches on fruit ripening are mostly focused on the respiration type fruit, and there are few researches on the ripening regulation of non-climacteric fruit, so, This paper is also of great significance for the study of the mechanism of MADS transcription factors regulating the ripening of non-jumping fruits. In this experiment, two MADS genes, PbMADS12 and PbMADS14, were cloned from the mutant and non-variant fruits of Dangshanshu pear. According to the results of integrative analysis of transcriptional and metabolic groups, two MADS genes were cloned and analyzed by bioinformatics. The expression patterns of PbMADS12, PbMADS14 and genes related to ethylene metabolism were analyzed by real-time quantitative PCR. The superexpression vectors PC1301-MADS12 and PC1301-MADS14, were constructed and transformed into Micro-Tom tomato successfully, and some transgenic plants were identified. The main results of this experiment are as follows: 1. 1. Two MADS genes, PbMADS12 and PbMADS14;, were cloned from DPA100 and non-variant fruits collected after anthesis. The results showed that the sequence of PbMADS14 gene was the same as that of PbMADS12 gene, and there was a difference in the sequence of PbMADS12 gene between mutated and unmutated fruits. The ORF frame of PbMADS12 consists of 720 bases, and the ORF frame encoding 239 amino acids, PbMADS14, consists of 750 bases and encodes 249 amino acids. The results showed that the high sugar budding fruit of Dangshanshu pear was not caused by the sequence difference of MADS gene. 2. The results of sequence analysis and phylogenetic tree analysis of MADS gene showed that: 1. The two MADS gene sequences cloned in this study have high homology with those published in the NCBI database, and both contain two conserved domains, MADS-box and K-box.3. Real-time quantitative PCR analysis of functional genes related to ethylene metabolism and PbMADS12 and PbMADS14 in Dangshan pear transcriptome analysis was carried out. The peak value of gene expression in unmutated fruit was 130d after anthesis (DPA130), and the peak value of gene expression in mutant fruit was 160d after anthesis (DPA160) .4. The superexpression vector PC1301-MADS12 and PC1301-MADS14, were successfully constructed to transform Micro-Tom tomato. Five transgenic tomato plants were identified by PCR amplification with primers designed for PbMADS12 and PbMADS14 genes and hygromycin gene sequence, respectively.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S661.2

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