本文關(guān)鍵詞：柿花性別分化調(diào)控的關(guān)鍵基因篩選及表達(dá)模式分析　出處：《中國(guó)林業(yè)科學(xué)研究院》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 柿 花性別分化 轉(zhuǎn)錄組 關(guān)鍵基因 表達(dá)模式分析

【摘要】：柿(Diospyros kaki Thunb.)為柿科(Ebenaeeae)柿屬(Diospyros)植物,果實(shí)營(yíng)養(yǎng)豐富、風(fēng)味香甜,樹(shù)姿優(yōu)美,葉色澤艷麗,是良好的果用、葉用及觀賞樹(shù)種。我國(guó)擁有豐富的柿種質(zhì)資源,這些資源除‘羅田甜柿’外,其余皆為澀柿。澀柿富含單寧,脫澀后方能食用,各澀柿品種在脫澀難易、耐貯性、果實(shí)風(fēng)味、活性成分含量等性狀的表現(xiàn)上存在顯著差異。甜柿雖不需脫澀即可鮮食,但其風(fēng)味與澀柿不盡相同,因而其不能完全替代澀柿。目前,選育兼具多個(gè)優(yōu)良性狀(如易脫澀、耐貯存、風(fēng)味好、活性成分高等)的優(yōu)異種質(zhì)是推動(dòng)柿產(chǎn)業(yè)發(fā)展的首要任務(wù)。雜交育種目的性強(qiáng),在引種和選擇育種創(chuàng)新潛力下降的情況下,它是種質(zhì)創(chuàng)新最經(jīng)典、最有效的手段。然而,柿雄性資源極度缺乏的現(xiàn)實(shí)嚴(yán)重限制了雜交育種工作的開(kāi)展。要培育雄性資源,首先需要探索柿花性別分化的調(diào)控機(jī)理。因此,本研究在雌雄同株的六倍體柿‘禪寺丸’(Diospyros kaki Thunb.)花性別分化形態(tài)學(xué)關(guān)鍵時(shí)期的前四天(4月13日,分子調(diào)控早于形態(tài)建成,因此用于轉(zhuǎn)錄組分析的樣品在形態(tài)學(xué)關(guān)鍵時(shí)期前采集)采集雌、雄花芽進(jìn)行Illumina HiSeq轉(zhuǎn)錄組測(cè)序,對(duì)測(cè)序數(shù)據(jù)進(jìn)行質(zhì)量評(píng)估和分析統(tǒng)計(jì),并篩選柿SSR和SNP分子標(biāo)記,做多態(tài)性分析。同時(shí)從柿雌、雄花芽轉(zhuǎn)錄組測(cè)序的差異基因中篩選出調(diào)控柿花性別分化的關(guān)鍵基因,然后篩選不同實(shí)驗(yàn)條件下柿qRT-PCR分析所需的適宜內(nèi)參基因,用于驗(yàn)證關(guān)鍵基因的表達(dá)量并分析其在雌雄花芽各發(fā)育階段的表達(dá)模式。主要研究結(jié)果有:(1)對(duì)‘禪寺丸’柿雌雄花芽進(jìn)行轉(zhuǎn)錄組測(cè)序得到54.47Gb clean reads,序列組裝拼接獲得154741條unigene,平均長(zhǎng)度為663bp。有38.49%(59570條)的unigene在七大數(shù)據(jù)庫(kù)中得到注釋。Nr數(shù)據(jù)庫(kù)注釋到的unigene數(shù)量最多(47435條,30.65%),其次是SwissProt數(shù)據(jù)庫(kù)(33320條,21.53%),KO數(shù)據(jù)庫(kù)注釋到的unigene數(shù)量最少,為14813條(9.57%)。注釋到GO數(shù)據(jù)庫(kù)中的差異基因可歸為細(xì)胞組分、分子功能和生物過(guò)程3大類(lèi)別56個(gè)小類(lèi)。KOG數(shù)據(jù)庫(kù)注釋到的unigene可以分為26個(gè)類(lèi)別。KEGG數(shù)據(jù)庫(kù)的代謝途徑富集分析表明,所有unigene可分為129個(gè)代謝通路。(2)對(duì)柿轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行SSR和SNP標(biāo)記多態(tài)性進(jìn)行研究。利用MISA軟件進(jìn)行SSR位點(diǎn)的搜索,共得到44304個(gè)SSR,包含83種重復(fù)基元,其中以A/T類(lèi)型為主的單核苷酸重復(fù)所占的比例最高(20006個(gè),占47.63%),其次是以AG/CT類(lèi)型為主的二核苷酸重復(fù)(16055個(gè),占38.23%)。在轉(zhuǎn)錄組得到的unigene中共發(fā)現(xiàn)SNP 405685個(gè),發(fā)生頻率為1/253bp。6種單核苷酸變異中,轉(zhuǎn)換類(lèi)型發(fā)生頻率顯著高于顛換類(lèi)型。本研究開(kāi)發(fā)出了大量的SSR和SNP標(biāo)記,可為柿遺傳圖譜構(gòu)建、遺傳多樣性和親緣關(guān)系的研究提供豐富的基礎(chǔ)數(shù)據(jù)信息。(3)本研究共篩選出1228個(gè)差異表達(dá)基因,其中,849個(gè)基因在雄花芽中上調(diào),379個(gè)基因在雌花芽中上調(diào)。通過(guò)GO分析和KEGG代謝途徑分析,可以將差異表達(dá)的unigene分為6個(gè)GO類(lèi)別和81個(gè)pathway代謝途徑。從中篩選出27個(gè)參與植物激素合成、信號(hào)轉(zhuǎn)導(dǎo)途徑或與生殖器官發(fā)育相關(guān)的關(guān)鍵基因。在雄花芽中上調(diào)表達(dá)的基因有赤霉素合成關(guān)鍵酶基因GA20OX2、乙烯生物合成酶基因S-腺苷甲硫氨酸合酶SAM基因、參與玉米素降解的CKX6、CKX5和CKX3、C/D類(lèi)基因PMADS2、C類(lèi)基因AG、WUS基因和能夠誘導(dǎo)生殖器官形成的E類(lèi)基因SEP2、HEC1、與性別分化相關(guān)的Beta-amyrin28-oxidase-like、絨氈層發(fā)育轉(zhuǎn)錄因子DYT1、細(xì)胞凋亡相關(guān)的Metacapase家族基因AMC9和雄性減數(shù)分裂關(guān)鍵基因MMD1。在雌花芽中上調(diào)的基因有編碼生長(zhǎng)素響應(yīng)蛋白AUX/IAA的基因IAA3和IAA32、ABA信號(hào)轉(zhuǎn)導(dǎo)途徑負(fù)調(diào)控因子蛋白磷酸酶2C(PP2C)At2g29380、乙烯生物合成相關(guān)的關(guān)鍵酶基因ACO、乙烯信號(hào)轉(zhuǎn)導(dǎo)途徑中的關(guān)鍵酶EBF1、類(lèi)AP2乙烯響應(yīng)因子ANT、ERF034、ERF115等轉(zhuǎn)錄因子和雌性化基因MeGI�；ㄍ吹鞍譇PETALA 2-LIKE AP2(c107926_g2)和SAP(c105715_g1)在柿雄花芽中上調(diào)表達(dá),另外2個(gè)WRKY轉(zhuǎn)錄因子:WRKY轉(zhuǎn)錄因子44(c108839_g2)和WRKY轉(zhuǎn)錄因子71(c102065_g1)也在柿雄花中上調(diào)表達(dá)。(4)對(duì)6個(gè)常用內(nèi)參基因在柿不同器官、不同發(fā)育時(shí)期的花芽和不同柿品種花芽中的表達(dá)穩(wěn)定性進(jìn)行評(píng)估,篩選出相對(duì)穩(wěn)定的內(nèi)參基因。結(jié)果發(fā)現(xiàn),各內(nèi)參基因在不同的試驗(yàn)材料中穩(wěn)定性存在差異。當(dāng)以不同品種花芽為材料時(shí)EF1-α的穩(wěn)定性最好,而以不同組織器官和不同發(fā)育時(shí)期的花芽為材料時(shí),GAPDH的穩(wěn)定性最好。分析基因在雌、雄花芽各發(fā)育階段的表達(dá)模式時(shí),以GAPDH作為內(nèi)參基因更為合適。(5)選取HEC1、SAM、Beta-amyrin 28-oxidase-like、AMC9、AG、GA20OX2、MeGI、EBF1、ERF034、ACO、IAA32基因進(jìn)行qRT-PCR表達(dá)量驗(yàn)證,驗(yàn)證結(jié)果與RNA-Seq相符。對(duì)6個(gè)性別分化關(guān)鍵基因MeGI、GA20OX2、ACO、IAA32、Beta-amyrin28-oxidase-like和HEC1進(jìn)行雌雄花芽發(fā)育過(guò)程中表達(dá)模式分析。結(jié)果表明,生長(zhǎng)素響應(yīng)蛋白AUX/IAA家族基因IAA32、轉(zhuǎn)錄因子Beta-amyrin 28-oxidase-like和HEC1從階段1到階段4(6月初到次年3月下旬)在雄花芽中的表達(dá)量顯著高于雌花芽,表明它們?cè)诨ㄑ堪l(fā)育早期可能具有促進(jìn)雄花芽分化的作用;赤霉素(GA)合成關(guān)鍵基因GA20OX2從階段7到階段11(4月中下旬到五月初)在雄花芽中表達(dá)量顯著高于雌花芽,表明此時(shí)高含量的GA可能對(duì)雄花芽分化有促進(jìn)作用。柿性別決定因子MeGI和乙烯合成基因ACO從階段6到階段10(4月初到5月初)在雌花芽中表達(dá)量顯著升高,表明這一時(shí)期是MeGI基因促進(jìn)雌花芽分化的關(guān)鍵時(shí)期,且此時(shí)高釋放量乙烯具有促雌作用。相關(guān)性分析表明,在雌花芽和雄花芽中,ACO的表達(dá)量分別與IAA32和GA20OX2呈顯著負(fù)相關(guān)。而IAA32、Beta-amyrin 28-oxidase-like和HEC1的表達(dá)量均呈顯著正相關(guān)。這說(shuō)明各基因所調(diào)控的生理過(guò)程能夠相互影響,共同調(diào)控柿花性別分化。
[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.

【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S665.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 秦力;陳景麗;潘長(zhǎng)田;葉蕾;盧鋼;;植物性染色體進(jìn)化及性別決定基因研究進(jìn)展[J];植物學(xué)報(bào);2016年06期

2 劉滔;朱維;李春美;;我國(guó)柿子加工產(chǎn)業(yè)的現(xiàn)狀與對(duì)策[J];食品工業(yè)科技;2016年24期

3 安俊;鄭彩霞;姚陽(yáng);陳彬麗;;油松胚珠轉(zhuǎn)錄組微衛(wèi)星特征分析[J];北京林業(yè)大學(xué)學(xué)報(bào);2016年05期

4 李加茹;孫鵬;韓衛(wèi)娟;李芳東;傅建敏;刁松鋒;;不完全甜柿‘禪寺丸’花性別分化形態(tài)學(xué)關(guān)鍵時(shí)期的研究[J];園藝學(xué)報(bào);2016年03期

5 吳起順;;被子植物的性別決定機(jī)制分析[J];黑龍江農(nóng)業(yè)科學(xué);2015年12期

6 馬秋月;廖卓毅;張得芳;戴曉港;陳贏男;李淑嫻;;碧桃花瓣轉(zhuǎn)錄組微衛(wèi)星特征分析[J];南京林業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2015年03期

7 王金星;張利軍;廖資億;張運(yùn)根;邱乾棟;孫鵬;孫宇涵;胡瑞陽(yáng);盧楠;李云;;刺槐實(shí)時(shí)定量PCR分析中內(nèi)參基因的選擇[J];林業(yè)科學(xué);2014年09期

8 許俊強(qiáng);孫梓健;劉智宇;楊樸麗;湯青林;王志敏;宋明;王小佳;;結(jié)球甘藍(lán)雌蕊發(fā)育調(diào)控因子SPT與HEC1的基因克隆及相互作用[J];作物學(xué)報(bào);2014年06期

9 艾呈祥;秦志華;辛力;;2013年中國(guó)柿產(chǎn)業(yè)發(fā)展報(bào)告[J];中國(guó)果菜;2014年02期

10 傅建敏;梁晉軍;周道順;;柿葉有效成分研究綜述[J];中南林業(yè)科技大學(xué)學(xué)報(bào);2013年11期

相關(guān)會(huì)議論文 前3條

1 宿福園;姚延興;李長(zhǎng)林;裴_，

本文編號(hào)：1432269