本文選題：巴西橡膠樹　切入點：MADS-box基因　出處：《海南大學》2017年碩士論文　論文類型：學位論文

【摘要】：巴西橡膠樹俗稱橡膠樹,是我國熱帶地區(qū)重要的經(jīng)濟作物,其生產(chǎn)的天然橡膠是關(guān)系到國計民生的重要戰(zhàn)略物資。隨著國內(nèi)天然橡膠消費量的逐年增加,受植膠區(qū)環(huán)境條件的限制,在難以通過擴大種植規(guī)模的前提下,加大橡膠樹高產(chǎn)新品種選育力度,進一步提高單位面積產(chǎn)量是有效緩解國內(nèi)天然橡膠生產(chǎn)量與消耗量之間矛盾的有效途徑之一。然而,橡膠樹屬多年生喬木,有性世代周期長,在橡膠樹新品種選育中存在花多果少,座果率偏低等問題,導致許多雜交組合不能成功組配,使得橡膠樹選育種效率受到很大限制。因此,深入研究橡膠樹花發(fā)育及生殖發(fā)育機理,旨在改變橡膠樹"繁花少實"的現(xiàn)象,提高其坐果率,可為橡膠樹優(yōu)良雜交組合的配置提供理論基礎(chǔ)與技術(shù)手段。在植物花器官及生殖發(fā)育中,MADS-box基因作為一類重要的轉(zhuǎn)錄因子基因家族,它們在植物的生長發(fā)育和信號轉(zhuǎn)導中起著關(guān)鍵性的調(diào)控作用。如前人研究已證實MADS-box基因在調(diào)控開花時間、決定花器官發(fā)育、影響果實成熟、調(diào)節(jié)營養(yǎng)器官發(fā)育、以及對外界信號的響應中起著重要作用。然而,到目前為止,關(guān)于橡膠樹中MADS-box基因的種類、表達特征及調(diào)控功能仍然未知。本研究通過對不同發(fā)育時期的橡膠樹花器官進行轉(zhuǎn)錄組測序分析,鑒定出多個參與橡膠樹花器官發(fā)育的MADS-box基因,進一步對這些參與橡膠樹花器官發(fā)育的MADS-box基因進行生物信息學分析、進化樹分析、時空表達模式分析、亞細胞定位、以及轉(zhuǎn)基因功能驗證,獲得如下結(jié)果:1、利用IluminaHiseq平臺對橡膠樹早期發(fā)育的花序、雄蕊及雌蕊進行轉(zhuǎn)錄組測序,共測得16.23 Gb數(shù)據(jù)。組裝并去冗余后得到109411個Unigene,平均長度1044 bp,N50為1805 bp,GC含量為40.68%。將組裝得到的Unigene進行七大功能數(shù)據(jù)庫注釋,NR注釋結(jié)果顯示所得Unige.nes與麻瘋樹Unigenes相似性最高。GO功能注釋可知有75135個Unigenes參與生物過程功能,有47900個Unigenes執(zhí)行細胞組成功能,有43235個Unigenes執(zhí)行分子功能。KEGG注釋結(jié)果發(fā)現(xiàn)這些Unigenes參與許多生物學通路,主要包括膜轉(zhuǎn)運代謝、碳水化合物運輸與代謝以及能量代謝通路,由此推測這些通路的相關(guān)基因可能參與橡膠樹生殖發(fā)育過程,并在其中起重要調(diào)控作用。2、根據(jù)轉(zhuǎn)錄組測序結(jié)果,從中獲得了 75個候選的橡膠樹MADS-box基因序列,經(jīng)過去冗余、檢查CDS編碼區(qū)以及查找典型的MADS-box結(jié)構(gòu)域,總共鑒定出26個參與橡膠樹花器官發(fā)育的MADS-box基因,其中6個基因是前人已經(jīng)報道過的。隨后將新發(fā)現(xiàn)的20個橡膠樹MADS-box基因提交NCBI數(shù)據(jù)庫,并將其命名為HbSEP、HbAGL9.1、HbAGL9.2、HbCMB1、HbCMB1-L、HbAGL6、HbAGL8、HbAP1、HbAGL12、HbAG、HbAGL11、HbAGL15、HbSVP1、HbSVP2、HbTT16、HbDEFL、HbPMADS2、HbAGL30、HbAGL61 以及 HbAGL65。3、對獲得的26個橡膠樹MADS-box基因進行序列特征分析,結(jié)果表明這些基因的ORF長度分布范圍在609bp(HbAGL12)-1116bp(HbAGL30)之間,編碼的氨基酸范圍在202-371之間,相應的分子量分布范圍在23.09-41.86kDa之間,推測蛋白等電點在6.06(HbSVP2)-9.34(HbAGL11)之間。對獲得的26個HbMADS-box基因進行序列比對分析,結(jié)果表明這些基因編碼蛋白的序列一致性在13.67%-98.57%之間,平均一致性為39.99%,其中HbMADS4和HbSVP1蛋白序列的一致性最大,達到98.5%,HbAGL30與HbMADS3蛋白序列的一致性最小,僅為25%。4、對所得的26個HbMADS-box基因編碼蛋白的保守區(qū)域進行分析,結(jié)果表明HbMADS-box基因都含有1個長度約為50個氨基酸殘基的、高度保守的MADS-box結(jié)構(gòu)域,1個半保守的具有明顯堿性螺旋環(huán)的K結(jié)構(gòu)域,1個較保守的Ⅰ結(jié)構(gòu)域,以及1個非常不保守的C結(jié)構(gòu)域,表明以上獲得的HbMADS-box基因?qū)儆诘湫偷腗ADS-box基因家族。進一步對26個HbMADS-box基因編碼蛋白的保守區(qū)域進行分析,結(jié)果表明這26個HbMADS-box基因在MADS-box結(jié)構(gòu)域的3(R)、17(R)、20(T)、23-24(KR)、27(G)、30-31(KK)、34(E)、38-39(LC)、48(F)以及 52(G)等 13 個位點高度保守。5、對所得的26個HbMADS-box基因進行結(jié)構(gòu)特征分析,結(jié)果表明HbMADS-box基因通常含有多個內(nèi)含子(HbAGL61除外),且大部分HbMADS-box基因含有5-7個內(nèi)含子,最多含有10個內(nèi)含子。進一步對26個HbMADS-box基因進行系統(tǒng)進化樹分析,結(jié)果表明這些基因分為3個進化支,主要包括.SEP、AGL6、FUL/AP1、AGL12、AG、SOC1、AGL15、AGL17、SVP、BS以 AP3/PI等11個亞家族。6、對所得的26個HbMADS-box基因進行時空特異性表達模式分析,結(jié)果表明HbSEP、HbAGL9.1、HbAGL9.2、HbCMB1、HbCMB1-L、HbAGL6、HbAG、HbDEFL以及HbPADS2在莖尖和花器官中顯著高調(diào)表達;HbAGL15、HbTT16、HbMADS2以及HbAG主要在果實中顯著高調(diào)表達,表明這些HbMADS-box基因可能參與了橡膠樹花器官和生殖發(fā)育過程。7、挑選在橡膠樹花器官發(fā)育中顯著高調(diào)表達的HbAG基因進行亞細胞定位與轉(zhuǎn)基因功能驗證,結(jié)果表明HbAG定位于核內(nèi),符合轉(zhuǎn)錄因子特征。轉(zhuǎn)基因結(jié)果顯示HbAG的過表達可導致植株矮小,使得營養(yǎng)生長期縮短,從而促進植株提前開花。進一步對轉(zhuǎn)基因擬南芥中花發(fā)育相關(guān)內(nèi)源基因的表達情況進行檢測,結(jié)果顯示在轉(zhuǎn)基因擬南芥植株中HbAG的過表達可以促進AtFT、AtAP1、AtLFY和AtFUL基因的表達,以及抑制AtSVP基因的表達,推測HbAG可能通過與這些基因的互作來影響橡膠樹的生殖發(fā)育進程。
[Abstract]:Brazil rubber tree commonly known as the rubber tree, is an important economic crop in tropical areas of China, the natural rubber production is an important strategic material. As beneficial to the people's livelihood domestic natural rubber consumption increasing year by year, affected by environmental conditions planting area limit, to expand the scale of planting the premise, increase the intensity of breeding of rubber the tree yielding new varieties, to further improve the yield per unit area is between the effective relief of domestic natural rubber production and consumption of one of the effective way of contradictions. However, the rubber tree is a perennial tree, sexual generation cycle is long, in the breeding of new varieties of rubber tree in flower fruit, fruit problem is low, resulting in many hybrid combinations cannot succeed with the group, the rubber tree breeding efficiency is limited. Therefore, in-depth study of rubber tree flower development and reproductive development mechanism, to change the rubber tree flowers Less real "phenomenon, improve the fruit setting rate, can provide a theoretical basis and technical means for the rubber tree elite hybrid configuration. In plant floral organs and reproductive development, MADS-box gene as an important transcription factor gene family, which plays a critical role in the growth and signal transduction in plant in the previous research. It has been confirmed that MADS-box gene in the regulation of flowering time, decided the development of floral organs, affecting fruit maturation, regulation of vegetative organs, and plays an important role in response to external signals. However, so far, of a kind of rubber trees MADS-box gene, expression characteristics and regulation function is still unknown. Based on the different development stages of rubber tree flower organs analyzed transcriptome sequencing, identified a number of participating rubber trees of floral organ development MADS-box gene, further to those involved in rubber Bioinformatics analysis of MADS-box gene tree of floral organ development, phylogenetic analysis, pattern analysis, expression and subcellular localization, transgenic functional verification, the results obtained are as follows: 1, in the early development of inflorescence of rubber tree using IluminaHiseq platform, stamen and pistil of transcriptome sequencing, were measured 16.23 Gb assembly data. And to get redundant 109411 Unigene, the average length of 1044 BP, N50 1805 BP, GC content of 40.68%. will be assembled by Unigene seven function database notes, NR notes the result of Unige.nes and leprosy tree Unigenes the highest similarity.GO functional annotation of 75135 Unigenes in the process of biological function, 47900 Unigenes cell function, there are 43235 Unigenes.KEGG molecular function annotation results found that these Unigenes are involved in many biological pathways, including membrane transport generation Thanks, carbohydrate transport and metabolism and energy metabolism pathway, which indicated that these genes may be involved in the pathway of rubber tree reproductive development process, and play an important role in.2, according to the transcriptome sequencing results, the rubber tree MADS-box gene sequences obtained from the 75 candidate, after going to check the CDS encoding region and redundancy. Find the typical MADS-box domain, identify a total of 26 in the rubber tree of floral organ development MADS-box gene, of which 6 genes are the predecessors have reported. Then the newly discovered 20 rubber tree MADS-box gene submitted to NCBI database, and named HbSEP, HbAGL9.1, HbAGL9.2, HbCMB1, HbCMB1-L, HbAGL6 HbAGL8, HbAP1, HbAGL12, HbAG, HbAGL11, HbAGL15, HbSVP1, HbSVP2, HbTT16, HbDEFL, HbPMADS2, HbAGL30, HbAGL61, and HbAGL65.3, to obtain the 26 rubber tree MADS-box gene sequence. Analysis results show that these genes, the ORF length distribution in the range of 609bp (HbAGL12) -1116bp (HbAGL30), encoding the amino acid in the range of 202-371, corresponding to the distribution of molecular weight in the range of 23.09-41.86kDa, speculated that the isoelectric point of the protein in 6.06 (HbSVP2) -9.34 (HbAGL11). The sequences of the 26 the HbMADS-box gene, the results show that these genes encoding protein sequence similarity between 13.67%-98.57%, the average consistency was 39.99%, the consistency of HbMADS4 and HbSVP1 protein sequence is the largest, reached 98.5%, HbAGL30 and HbMADS3 protein sequence consistency minimum, only 25%.4, analyze the conserved region of HbMADS-box gene encoding protein of 26 The results showed that, HbMADS-box gene contains 1 length of about 50 amino acid residues, highly conserved MADS-box domain and 1.5 conserved has obvious alkali 鎬ц灪鏃嬬幆鐨凨緇撴瀯鍩，

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