巴西橡胶树MADS-box基因家族的克

发布时间：2018-03-10 23:29

  本文选题：巴西橡胶树　切入点：MADS-box基因　出处：《海南大学》2017年硕士论文　论文类型：学位论文

【摘要】：巴西橡胶树俗称橡胶树,是我国热带地区重要的经济作物,其生产的天然橡胶是关系到国计民生的重要战略物资。随着国内天然橡胶消费量的逐年增加,受植胶区环境条件的限制,在难以通过扩大种植规模的前提下,加大橡胶树高产新品种选育力度,进一步提高单位面积产量是有效缓解国内天然橡胶生产量与消耗量之间矛盾的有效途径之一。然而,橡胶树属多年生乔木,有性世代周期长,在橡胶树新品种选育中存在花多果少,座果率偏低等问题,导致许多杂交组合不能成功组配,使得橡胶树选育种效率受到很大限制。因此,深入研究橡胶树花发育及生殖发育机理,旨在改变橡胶树"繁花少实"的现象,提高其坐果率,可为橡胶树优良杂交组合的配置提供理论基础与技术手段。在植物花器官及生殖发育中,MADS-box基因作为一类重要的转录因子基因家族,它们在植物的生长发育和信号转导中起着关键性的调控作用。如前人研究已证实MADS-box基因在调控开花时间、决定花器官发育、影响果实成熟、调节营养器官发育、以及对外界信号的响应中起着重要作用。然而,到目前为止,关于橡胶树中MADS-box基因的种类、表达特征及调控功能仍然未知。本研究通过对不同发育时期的橡胶树花器官进行转录组测序分析,鉴定出多个参与橡胶树花器官发育的MADS-box基因,进一步对这些参与橡胶树花器官发育的MADS-box基因进行生物信息学分析、进化树分析、时空表达模式分析、亚细胞定位、以及转基因功能验证,获得如下结果:1、利用IluminaHiseq平台对橡胶树早期发育的花序、雄蕊及雌蕊进行转录组测序,共测得16.23 Gb数据。组装并去冗余后得到109411个Unigene,平均长度1044 bp,N50为1805 bp,GC含量为40.68%。将组装得到的Unigene进行七大功能数据库注释,NR注释结果显示所得Unige.nes与麻疯树Unigenes相似性最高。GO功能注释可知有75135个Unigenes参与生物过程功能,有47900个Unigenes执行细胞组成功能,有43235个Unigenes执行分子功能。KEGG注释结果发现这些Unigenes参与许多生物学通路,主要包括膜转运代谢、碳水化合物运输与代谢以及能量代谢通路,由此推测这些通路的相关基因可能参与橡胶树生殖发育过程,并在其中起重要调控作用。2、根据转录组测序结果,从中获得了 75个候选的橡胶树MADS-box基因序列,经过去冗余、检查CDS编码区以及查找典型的MADS-box结构域,总共鉴定出26个参与橡胶树花器官发育的MADS-box基因,其中6个基因是前人已经报道过的。随后将新发现的20个橡胶树MADS-box基因提交NCBI数据库,并将其命名为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、对获得的26个橡胶树MADS-box基因进行序列特征分析,结果表明这些基因的ORF长度分布范围在609bp(HbAGL12)-1116bp(HbAGL30)之间,编码的氨基酸范围在202-371之间,相应的分子量分布范围在23.09-41.86kDa之间,推测蛋白等电点在6.06(HbSVP2)-9.34(HbAGL11)之间。对获得的26个HbMADS-box基因进行序列比对分析,结果表明这些基因编码蛋白的序列一致性在13.67%-98.57%之间,平均一致性为39.99%,其中HbMADS4和HbSVP1蛋白序列的一致性最大,达到98.5%,HbAGL30与HbMADS3蛋白序列的一致性最小,仅为25%。4、对所得的26个HbMADS-box基因编码蛋白的保守区域进行分析,结果表明HbMADS-box基因都含有1个长度约为50个氨基酸残基的、高度保守的MADS-box结构域,1个半保守的具有明显碱性螺旋环的K结构域,1个较保守的Ⅰ结构域,以及1个非常不保守的C结构域,表明以上获得的HbMADS-box基因属于典型的MADS-box基因家族。进一步对26个HbMADS-box基因编码蛋白的保守区域进行分析,结果表明这26个HbMADS-box基因在MADS-box结构域的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 个位点高度保守。5、对所得的26个HbMADS-box基因进行结构特征分析,结果表明HbMADS-box基因通常含有多个内含子(HbAGL61除外),且大部分HbMADS-box基因含有5-7个内含子,最多含有10个内含子。进一步对26个HbMADS-box基因进行系统进化树分析,结果表明这些基因分为3个进化支,主要包括.SEP、AGL6、FUL/AP1、AGL12、AG、SOC1、AGL15、AGL17、SVP、BS以 AP3/PI等11个亚家族。6、对所得的26个HbMADS-box基因进行时空特异性表达模式分析,结果表明HbSEP、HbAGL9.1、HbAGL9.2、HbCMB1、HbCMB1-L、HbAGL6、HbAG、HbDEFL以及HbPADS2在茎尖和花器官中显著高调表达;HbAGL15、HbTT16、HbMADS2以及HbAG主要在果实中显著高调表达,表明这些HbMADS-box基因可能参与了橡胶树花器官和生殖发育过程。7、挑选在橡胶树花器官发育中显著高调表达的HbAG基因进行亚细胞定位与转基因功能验证,结果表明HbAG定位于核内,符合转录因子特征。转基因结果显示HbAG的过表达可导致植株矮小,使得营养生长期缩短,从而促进植株提前开花。进一步对转基因拟南芥中花发育相关内源基因的表达情况进行检测,结果显示在转基因拟南芥植株中HbAG的过表达可以促进AtFT、AtAP1、AtLFY和AtFUL基因的表达,以及抑制AtSVP基因的表达,推测HbAG可能通过与这些基因的互作来影响橡胶树的生殖发育进程。
[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灪鏃嬬幆鐨凨缁撴瀯鍩，

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