柿花性别分化调控的关键基因筛选及表达模式分析

发布时间：2018-01-16 08:12

本文关键词：柿花性别分化调控的关键基因筛选及表达模式分析　出处：《中国林业科学研究院》2017年硕士论文　论文类型：学位论文

【摘要】：柿(Diospyros kaki Thunb.)为柿科(Ebenaeeae)柿属(Diospyros)植物,果实营养丰富、风味香甜,树姿优美,叶色泽艳丽,是良好的果用、叶用及观赏树种。我国拥有丰富的柿种质资源,这些资源除‘罗田甜柿’外,其余皆为涩柿。涩柿富含单宁,脱涩后方能食用,各涩柿品种在脱涩难易、耐贮性、果实风味、活性成分含量等性状的表现上存在显著差异。甜柿虽不需脱涩即可鲜食,但其风味与涩柿不尽相同,因而其不能完全替代涩柿。目前,选育兼具多个优良性状(如易脱涩、耐贮存、风味好、活性成分高等)的优异种质是推动柿产业发展的首要任务。杂交育种目的性强,在引种和选择育种创新潜力下降的情况下,它是种质创新最经典、最有效的手段。然而,柿雄性资源极度缺乏的现实严重限制了杂交育种工作的开展。要培育雄性资源,首先需要探索柿花性别分化的调控机理。因此,本研究在雌雄同株的六倍体柿‘禅寺丸’(Diospyros kaki Thunb.)花性别分化形态学关键时期的前四天(4月13日,分子调控早于形态建成,因此用于转录组分析的样品在形态学关键时期前采集)采集雌、雄花芽进行Illumina HiSeq转录组测序,对测序数据进行质量评估和分析统计,并筛选柿SSR和SNP分子标记,做多态性分析。同时从柿雌、雄花芽转录组测序的差异基因中筛选出调控柿花性别分化的关键基因,然后筛选不同实验条件下柿qRT-PCR分析所需的适宜内参基因,用于验证关键基因的表达量并分析其在雌雄花芽各发育阶段的表达模式。主要研究结果有:(1)对‘禅寺丸’柿雌雄花芽进行转录组测序得到54.47Gb clean reads,序列组装拼接获得154741条unigene,平均长度为663bp。有38.49%(59570条)的unigene在七大数据库中得到注释。Nr数据库注释到的unigene数量最多(47435条,30.65%),其次是SwissProt数据库(33320条,21.53%),KO数据库注释到的unigene数量最少,为14813条(9.57%)。注释到GO数据库中的差异基因可归为细胞组分、分子功能和生物过程3大类别56个小类。KOG数据库注释到的unigene可以分为26个类别。KEGG数据库的代谢途径富集分析表明,所有unigene可分为129个代谢通路。(2)对柿转录组数据进行SSR和SNP标记多态性进行研究。利用MISA软件进行SSR位点的搜索,共得到44304个SSR,包含83种重复基元,其中以A/T类型为主的单核苷酸重复所占的比例最高(20006个,占47.63%),其次是以AG/CT类型为主的二核苷酸重复(16055个,占38.23%)。在转录组得到的unigene中共发现SNP 405685个,发生频率为1/253bp。6种单核苷酸变异中,转换类型发生频率显著高于颠换类型。本研究开发出了大量的SSR和SNP标记,可为柿遗传图谱构建、遗传多样性和亲缘关系的研究提供丰富的基础数据信息。(3)本研究共筛选出1228个差异表达基因,其中,849个基因在雄花芽中上调,379个基因在雌花芽中上调。通过GO分析和KEGG代谢途径分析,可以将差异表达的unigene分为6个GO类别和81个pathway代谢途径。从中筛选出27个参与植物激素合成、信号转导途径或与生殖器官发育相关的关键基因。在雄花芽中上调表达的基因有赤霉素合成关键酶基因GA20OX2、乙烯生物合成酶基因S-腺苷甲硫氨酸合酶SAM基因、参与玉米素降解的CKX6、CKX5和CKX3、C/D类基因PMADS2、C类基因AG、WUS基因和能够诱导生殖器官形成的E类基因SEP2、HEC1、与性别分化相关的Beta-amyrin28-oxidase-like、绒毡层发育转录因子DYT1、细胞凋亡相关的Metacapase家族基因AMC9和雄性减数分裂关键基因MMD1。在雌花芽中上调的基因有编码生长素响应蛋白AUX/IAA的基因IAA3和IAA32、ABA信号转导途径负调控因子蛋白磷酸酶2C(PP2C)At2g29380、乙烯生物合成相关的关键酶基因ACO、乙烯信号转导途径中的关键酶EBF1、类AP2乙烯响应因子ANT、ERF034、ERF115等转录因子和雌性化基因MeGI。花同源蛋白APETALA 2-LIKE AP2(c107926_g2)和SAP(c105715_g1)在柿雄花芽中上调表达,另外2个WRKY转录因子:WRKY转录因子44(c108839_g2)和WRKY转录因子71(c102065_g1)也在柿雄花中上调表达。(4)对6个常用内参基因在柿不同器官、不同发育时期的花芽和不同柿品种花芽中的表达稳定性进行评估,筛选出相对稳定的内参基因。结果发现,各内参基因在不同的试验材料中稳定性存在差异。当以不同品种花芽为材料时EF1-α的稳定性最好,而以不同组织器官和不同发育时期的花芽为材料时,GAPDH的稳定性最好。分析基因在雌、雄花芽各发育阶段的表达模式时,以GAPDH作为内参基因更为合适。(5)选取HEC1、SAM、Beta-amyrin 28-oxidase-like、AMC9、AG、GA20OX2、MeGI、EBF1、ERF034、ACO、IAA32基因进行qRT-PCR表达量验证,验证结果与RNA-Seq相符。对6个性别分化关键基因MeGI、GA20OX2、ACO、IAA32、Beta-amyrin28-oxidase-like和HEC1进行雌雄花芽发育过程中表达模式分析。结果表明,生长素响应蛋白AUX/IAA家族基因IAA32、转录因子Beta-amyrin 28-oxidase-like和HEC1从阶段1到阶段4(6月初到次年3月下旬)在雄花芽中的表达量显著高于雌花芽,表明它们在花芽发育早期可能具有促进雄花芽分化的作用;赤霉素(GA)合成关键基因GA20OX2从阶段7到阶段11(4月中下旬到五月初)在雄花芽中表达量显著高于雌花芽,表明此时高含量的GA可能对雄花芽分化有促进作用。柿性别决定因子MeGI和乙烯合成基因ACO从阶段6到阶段10(4月初到5月初)在雌花芽中表达量显著升高,表明这一时期是MeGI基因促进雌花芽分化的关键时期,且此时高释放量乙烯具有促雌作用。相关性分析表明,在雌花芽和雄花芽中,ACO的表达量分别与IAA32和GA20OX2呈显著负相关。而IAA32、Beta-amyrin 28-oxidase-like和HEC1的表达量均呈显著正相关。这说明各基因所调控的生理过程能够相互影响,共同调控柿花性别分化。
[Abstract]:Persimmon (Diospyros kaki Thunb.) (Ebenaeeae) for Ebenaceae Diospyros (Diospyros) plants, fruit nutrient rich, sweet flavor, beautiful tree appearance, leaf bright color, good fruit, leaf and ornamental trees. China has rich Persimmon Germplasm resources, these resources in "Luotian persimmon". The rest are astringent persimmon persimmon. Rich in tannins, Deastringency before being consumed, the astringent persimmon varieties in Deastringency of difficulty, storability, fruit flavor, there are significant differences in traits of active ingredient content as well. Although no Deastringency persimmon can be fresh, but its flavor and astringent persimmon vary. So it can not completely replace the astringent persimmon. At present, both the breeding of many excellent properties (such as easy deastringented, resistant storage, good flavor, high active ingredient) excellent germplasm is the primary task of promoting the development of persimmon industry. Breeding strong purpose, in the introduction and selection of breeding innovation potential decline, it Is the most classic germplasm innovation, the most effective means. However, the reality of the lack of male persimmon resources are extremely seriously limits the breeding work. To cultivate the male resources, first need to explore the regulation mechanism of persimmon flower sex differentiation. Therefore, in this study, monoecious was six times as much as the persimmon 'Zenjimaru' (Diospyros kaki Thunb.) four days spent a critical period of sexual differentiation morphology (April 13th, built early in the form of molecular regulation, therefore for transcriptome analysis of samples in the critical period before the acquisition of morphology acquisition) female, male flower bud of Illumina HiSeq transcriptome sequencing, quality evaluation and statistical analysis of sequencing data, and SSR and SNP molecular screening of persimmon do marker polymorphism analysis. At the same time from persimmon female, key genes regulating persimmon Floral Sex Differentiation out screening of differentially expressed genes in male flower buds transcriptome sequencing, then screened under different experimental conditions of persimmon Q Suitable reference genes required for RT-PCR analysis, the expression of key genes to verify and analyze its expression pattern in different developmental stages of female and male flower. The main results are: (1) on 'Zenjimaru' persimmon female and male flower transcriptome sequencing of 54.47Gb clean reads, sequence assembly get 154741 UniGene, the average length of 663bp. 38.49% (59570) of the UniGene.Nr database to the UniGene annotation annotation number seven in the database (47435, 30.65%), followed by the SwissProt database (33320, 21.53%), KO data base notes to the number of at least UniGene, 14813 (9.57%). Notes to differences in GO database the gene can be classified into cellular component, molecular function and biological process of UniGene 3 categories of 56 kinds of.KOG database notes can be divided into 26 categories of.KEGG database metabolic pathway enrichment analysis showed that all u Nigene can be divided into 129 metabolic pathways. (2) on persimmon transcriptome data of SSR and SNP polymorphism of the SSR locus. Using MISA software to search, get a total of 44304 SSR, contains 83 kinds of repeat motifs, which were the main types of A/T mononucleotide repeat of the proportion of the highest (20006, accounting for 47.63%), followed by the AG/CT main types of dinucleotide repeats (16055, 38.23%). The Communist Party of China in the transcriptome of UniGene SNP 405685, the frequency of occurrence of 1/253bp.6 single nucleotide variation in frequency conversion type is significantly higher than transversion type. This study developed the SSR and SNP a large number of markers, can be constructed for persimmon genetic map, provide basic data rich information on genetic diversity and genetic relationship. (3) this study screened 1228 differentially expressed genes, among them, 849 genes up-regulated in male flower buds, 379 genes Rise in the female flower bud. Through the GO analysis and KEGG analysis of metabolic pathways, can be differentially expressed UniGene GO divided into 6 categories and 81 metabolic pathways of pathway. Screened from 27 involved in the synthesis of plant hormones, signal transduction and reproductive organ development and key genes related. In male flower bud expression the gene of gibberellin biosynthesis key enzyme genes GA20OX2, ethylene biosynthetic enzyme gene S- of S-adenosylmethionine synthase SAM gene involved in zeatin degradation of CKX6, CKX5 and CKX3, C/D gene PMADS2, C gene AG, WUS gene can induce the formation of genital organs and E genes SEP2, HEC1, and related to sex differentiation Beta-amyrin28-oxidase-like, tapetum transcription factor DYT1 gene and apoptosis of Metacapase family genes AMC9 and male meiosis key gene MMD1. was up-regulated in the female flower bud in the growth hormone response encoding Protein AUX/IAA gene IAA3 and IAA32, ABA signal transduction pathway negative regulator of protein phosphatase 2C (PP2C) At2g29380, ethylene biosynthesis key enzyme genes related to ACO, EBF1 is the key enzyme in ethylene signal transduction, AP2 ethylene response factor ANT, ERF034, ERF115 transcription factor gene MeGI. and female flower homologous protein APETALA 2-LIKE AP2 (c107926_g2) and SAP (c105715_g1) expression in male flower bud of persimmon, the other 2 WRKY transcription factor: WRKY transcription factor 44 (c108839_g2) and WRKY transcription factor 71 (c102065_g1) was also up-regulated in the persimmon male flower expression. (4) of the 6 commonly used reference genes in different organs of persimmon, expression stability flower buds at different developmental stages and different persimmon varieties in flower buds were evaluated and screened relatively stable reference genes. The results showed that the reference gene in different test materials in stability are different. When using different products A flower bud for stability of EF1- alpha best material, and to the flower bud in different organs and different developmental stages as materials, GAPDH has the best stability. In the analysis of gene expression patterns in different developmental stages of female, male flower buds, with GAPDH as the reference gene is more appropriate. (5) were HEC1, SAM, Beta-amyrin 28-oxidase-like, AMC9, AG, GA20OX2, MeGI, EBF1, ERF034, ACO, IAA32 gene expression of qRT-PCR verification, the verification results consistent with the RNA-Seq. The 6 sex differentiation of key genes MeGI, GA20OX2, ACO, IAA32, Beta-amyrin28-oxidase-like and HEC1 expression pattern analysis of female and male flower in the development process. The results showed that auxin responsive protein the AUX/IAA family of transcription factor Beta-amyrin gene IAA32, 28-oxidase-like and HEC1 from stage 1 to stage 4 (early June to late March) expression in male flower bud was significantly higher than that of female flower bud, suggesting that they spend in Early bud development may promote the male flower bud differentiation; gibberellin (GA) synthesis of key gene GA20OX2 from stage 7 to stage 11 (mid to late 4 to early May) was significantly higher than that of female flower bud expression in male flower bud, it shows that high levels of GA may contribute to male flower bud differentiation. Persimmon sex determining factor MeGI and ethylene biosynthesis gene ACO from stage 6 to stage 10 (from early April to early May) was significantly higher expressed in the female flower bud, that this period is the key period of MeGI gene promotes the differentiation of female flower bud, and the high ethylene production can promote the female role. The correlation analysis showed that in female flower buds and flower buds in the expression of ACO and IAA32 and GA20OX2 respectively. There was a negative correlation and IAA32 were positively related to expression of Beta-amyrin and HEC1 are 28-oxidase-like. This shows that the physiological process regulation of each gene can influence each other, common Regulate the sex differentiation of persimmon flowers.

【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S665.2

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