茄子花柱异型遗传分析及基因差异表达研究

发布时间：2018-03-06 16:51

本文选题：茄子　切入点：花柱异型　出处：《华南农业大学》2016年博士论文　论文类型：学位论文

【摘要】：花柱异型是一种特有的雌雄隔离和多态性特征,在被子植物中十分常见,包括茄子(Solanum melongena L.)。通常,茄子长柱花(L-morph)类型坐果能力强、生境适宜性广,而短柱花(S-morph)类型易落花落果。虽然关于茄子长柱花和短柱花坐果能力差异,以及对光、温等环境适应性的报道较多,但茄子花柱异型发育调控的分子机制和品种改良尚未见报道。我国华南地区主栽的紫长茄属于兼生长短柱花类型,坐果数量少,不宜设施栽培,生产上亟需对紫长茄进行改良。本文对完全长柱花品系和兼生长短柱花品系雌蕊发育、坐果能力、遗传规律及分子调控机制进行研究,主要结果如下:1.将茄子从现蕾到开花时期可划6个时期:现蕾期、幼蕾膨大期、露冠期、花冠膨大期、松蕾期和开花期。从花蕾长度来判断,在0~10mm短柱花雌蕊发育与长柱花保持一致,到花冠膨大期(约10mm)后短柱花雌蕊基本停止发育。花发育期间,L-morph花花柱长度与花蕾长度呈线性关系。花柱长度与花蕾长度、子房高度相关性达极显著水平,与花梗粗度达到显著水平,而与子房直径以及花药长度相关性则不显著。通过荧光显微观察,L-morph花的柱头和花粉均具有可授性,而S-morph花仅花粉具有活力。光学显微观察完全长柱花和兼生长短柱花胚囊中的卵细胞、中央细胞、助细胞和反足细胞结构完整。2.短柱花自交坐果率仅为5%左右,而长柱花自交坐果率均在94%以上。杂交试验表明对S-morph花柱头进行授粉坐果率极显著低于L-morph花的坐果率。3.完全长柱花类型与兼生长短柱花进行杂交,F1代植株全部为完全长柱花类型,B3-3×56和01×119两组材料的F2代完全长柱花类型和兼生长短柱花类型的比值分别为12.14:1和11.85:11,在两个组合BC1群体中完全长柱花类型和兼生长短柱花类型的分离比例分别为3.64:1和2.56:1。经χ2检测,完全长柱花类型相对兼生长短柱花类型为显性遗传,为两对基因共同作用产生基因重叠效果;Shapiro-Wilk检验F2完全长柱花柱头长度也表明花柱长度由多基因控制的数量遗传,存在主效基因作用。4.基于高通量miRNA测序、微阵列及降解组测序,我们鉴定了兼生长短柱花茄子雌蕊发育过程中miRNA的表达及其靶基因。miRNA测序鉴定了55个已知的茄子miRNA和35个与miRBase数据库中已知miRNA相同序列的miRNA,594个新的miRNA,而大部分miRNA与其他植物相比有着高度的保守性。在微阵列芯片上的686个miRNA中,鉴定到10个差异表达的miRNA,其中,仅2个miRNA在L-morph雌蕊发育中显著下调。同时,我们利用qRT-PCR对差异表达的miRNA进行验证。此外,我们也预测并注释了差异表达miRNA的靶基因。5.通过微阵列对紫长茄S-morph花和千指茄L-morph花雌蕊miRNA的表达谱进行了鉴定。在微阵列芯片上的686个miRNA中,我们发现35个表达差异的miRNA,在L-morph花雌蕊中下调的9个miRNA,PC-5p-1408812_1和PC-5p-3215765_1下调最显著;而上调的26个miRNA中,PC-3p-1358_405 and PC-5p-132923_7表达差异最显著。利用了qRT-PCR对通过微阵列检测获得的表达特征进行了验证,结果基本与芯片数据相一致。此外,我们还进行了差异表达miRNA的靶基因预测,并对推测的靶基因进行了GO功能分析。在差异表达的miRNA的靶基因中,基因编码转录因子bHLH、MYB以及AP2等,重要的酶或功能蛋白例如三磷酸水解酶超家族蛋白、蛋白激酶、磷酸酶以及ATP酶等,这些可能对于花柱异型发育起重要调控作用。6.利用iTRAQ技术对兼生长短柱花茄子雌蕊发育过程中蛋白质组表达分析,共鉴定了300个差异表达的蛋白,其中在花发育期,S-morph花与L-morph花雌蕊相比有26个蛋白质表达量下调,23个蛋白质表达量上调。在表达下调的蛋白中,甲硫氨酸硫氧化物还原酶A3和DNA损伤修复/耐受性蛋白表达差异最大。在表达上调的蛋白中,甲硫氨酸硫氧化物还原酶和肌氨酸氧化酶的表达差异最大。这一时期差异表达蛋白主要参与能量储备以及对外界应激反应、抵御病原体感染等方面。在花成熟期,97个蛋白表达上调和157个蛋白表达下调。在表达下调的蛋白中,富亮氨酸重复蛋白和性器官表达蛋白的表达差异最大。在表达上调的蛋白中,LAT52和ntp201的表达差异最大。这一时期差异表达蛋白主要参与生物合成过程、生物分子转运和代谢过程。此外,花成熟期S-morph花雌蕊中乙醇NADP+氧化还原酶、乙醇脱氢酶、半胱氨酸蛋白酶和过氧化物酶等表达上调,这些酶参与细胞死亡过程,表达的上调可能与S-morph花不能坐果有关。而qRT-PCR结果并未完全与iTRAQ鉴定的结果一致,这可能是花形态特异性转录后调控的原因所致。本研究通过对茄子花柱异型细胞形态学特征和miRNA、蛋白差异表达进行了研究,这些结果将会为揭开被子植物花柱异型发育模式及其分子机制的研究提供有用信息,同时也将对基础理论研究以及农业改良、育种等方面提供十分重要的参考价值。
[Abstract]:Heterostyly is a unique female isolation and polymorphism characteristic, is very common in angiosperms, including eggplant (Solanum melongena L.). Usually, the eggplant flower long column (L-morph) type of fruit setting ability, habitat suitability is wide, and short column flowers (S-morph) type to drop flowers FruitDrop. While on the difference of Eggplant long column and short column flower flowering and fruit setting ability, as well as the light, temperature and other environmental adaptability are reported, but the molecular mechanism and improved varieties of eggplant heterostyly developmental regulation has not been reported in China. The Southern China area planted purple eggplant and belongs to the growth of short column type flowers, fruit number, not greenhouse on the need for improved production of eggplant. The entire cylindrical flowers and growth lines and short column lines flower pistil development, fruit setting ability, to study inheritance and molecular regulation mechanism, the main results are as follows: 1. the eggplant from budding to flowering The period can be divided into 6 periods: bud, bud growth stage, exposed crown, corolla enlargement period, pine flower budding and flowering. Judging from bud length, 0~10mm in short and long column column flower pistil flower consistent to the corolla enlargement period (about 10mm) after a short column flower stop the basic development. During the flower development, flower bud length and the length of the L-morph column. There is a linear relationship between the style length and bud length, ovary height correlation reached extremely significant level, and pedicel degree reached significant level, and the diameter of ovary and anther length correlation is not significant. The fluorescence microscopy, stigma and pollen L-morph flowers have receptivity, and only S-morph flower pollen has vitality. Observe the entire cylindrical flowers and flower growth and short column embryo sac egg cells, micro optical central cell, synergid and antipodal cell structural integrity of.2. short column pollination fruit rate is only about 5%, and Long column pollination fruit rate was above 94%. Hybridization experiments indicated that the S-morph flower stigma pollinated fruit rate was significantly lower than that of L-morph flower fruit rate.3. complete flower type and long column and short column growth flowers hybridization F1 generation plants complete long column flower type, B3-3 * 56 and 01 * 119 two group of materials F2 generation completely long column type and flower growth and short column flower type ratio were 12.14:1 and 11.85:11, the two combinations in the BC1 group completely long column type and flower growth and flower type short column separation ratio were 3.64:1 and 2.56:1. by 2 types of detection, completely and spend a relatively long column students spend the length of column type is dominant, which is the common effect of two gene overlapping effect on gene; Shapiro-Wilk test F2 complete long column flower stigma length also showed that the number of genetic style length controlled by many genes, some major genes based on high-throughput.4. MiRNA sequencing, microarray and Degradome sequencing, we identified the expression of its target gene.MiRNA sequencing and miRNA growth of short column flower pistil development in eggplant eggplant known 55 known miRNA and 35 with miRBase miRNA database in the same sequence of miRNA were identified, 594 new miRNA, and most of the miRNA compared with the other plants are highly conserved. In 686 miRNA of microarray, identified 10 differentially expressed miRNA, among them, only 2 miRNA in L-morph is significantly down regulated in pistil development. At the same time, we use qRT-PCR to verify the differential expression of miRNA. In addition, we also predicted and annotated expression target the.5. miRNA gene by microarray of eggplant S-morph thousand flowers and flower pistil expression refers to eggplant L-morph spectra of miRNA were identified. In 686 miRNA of microarray, we found 35 differentially expressed miR In NA, L-morph spent 9 downregulated miRNA. PC-5p-1408812_1 and PC-5p-3215765_1, most significantly down regulated; while the 26 miRNA increase in the expression of PC-3p-1358_405 and PC-5p-132923_7 the most significant difference. Using the qRT-PCR to validate the expression obtained by microarray detection, results consistent with the microarray data. In addition, we also the differential expression of the predicted target gene of miRNA, and the putative target genes of GO. Functional analysis of target genes in the differential expression of miRNA gene, encoding the transcription factor bHLH, MYB and AP2, enzyme or functional protein important for example three Phosphohydrolase superfamily protein, protein kinase, phosphatase and ATP enzyme so, these may be for heterostyly development plays an important role in the regulation of.6. based on iTRAQ technology and the growth of short column flower pistil development process of eggplant proteome analysis identified 3 00 differentially expressed proteins, which developed in the flowers, flowers and flower S-morph L-morph compared the expression of 26 proteins were down regulated, 23 proteins up-regulated. The downregulated protein, methionine sulfoxide reductase A3 and DNA damage repair / tolerance protein expression differences in the expression. The differentially expressed protein, methionine sulfoxide reductase and sarcosine oxidase. The differentially expressed proteins are mainly involved in energy reserves and external stress response against pathogens and other aspects. In flower maturity, 97 up-regulated and 157 protein expression. The downregulated protein, rich leucine rich repeat protein expression and organ protein. In the differentially expressed proteins in the differential expression of LAT52 and ntp201. The protein expression differences during this period the main parameters With the biosynthesis, biological molecular transport and metabolism. In addition, flower maturity S-morph flower NADP+ oxidation of ethanol in ethanol dehydrogenase reductase, pistil, upregulate the expression of cysteine proteinase and peroxidase, the enzyme involved in the cell death process, the expression of S-morph increased with fruit. Flowers can not be the result of qRT-PCR and iTRAQ is not fully identified results, this may be caused by flower morphology specific post transcriptional regulation. Through the study on Eggplant heterostyly cell morphology and miRNA protein expression differences were studied. These results will provide useful information to uncover the research model and the molecular mechanism of angiosperm heterostyly development, but also on the basis of theoretical research and agricultural improvement, provide very important reference value in breeding and other aspects.

【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】：S641.1

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