金柑FcSOC1同源基因克隆及其遗传转化

发布时间：2018-03-10 09:35

本文选题：金柑　切入点：FcSOC1　出处：《广西大学》2016年硕士论文　论文类型：学位论文

【摘要】：MADS-box家族基因在植物生长发育中起着重要的作用,尤其在控制植物花器官的形成发育方面起着重要的作用。SOC1基因作为编码MADS-box家族的重要一员,能够集成多种温度、激素、年龄相关的开花信号,是重要的开花调控关键基因。近年来,科研人员对植物SOC1基因的研究不断深入,在水稻、葡萄、苹果、草莓、牡丹、豌豆、玉米、白桦、拟南芥等多种植物中相继克隆SOC1基因,不断完善其参与的功能及机理。但对于金柑的SOC1基因的研究未见报道,为了深入研究金柑SOC1基因的功能,本研究基于金柑转录组测序数据de novo组装分析的基础上,获得SOCl同源基因片段序列信息。通过同源基因克隆方法,克隆出SOC1同源基因cDNA全长,分析了融安金柑SOC1的生物学特点,探讨融安金柑SOC1在生殖器官和营养器官的表达特性,并对其进行转基因研究,初步获得如下结果：(1)通过生物信息学分析表明FcSOCl同源基因的cDNA序列为660bp和636bp,编码220和212个氨基酸,分别命名为FcSOC1a和FcSOC1b。FcSOC1a理论蛋白质的分子量为：25204.7 Da,等电点：9.36；原子个数为3561；分子式为：C1085H1796N332O339S9;脂肪族指数(Aliphatic index)大致为76.73；不稳定指数(Instability index)大致为53.23；蛋白质疏水性(Grand average of hydropathicity, GRAVY)为：-0.831。FcSOC1b理论蛋白质的分子量为24829.4Da、等电点8.9、原子个数为3488、分子式C1067H17S5N319O333S14、不稳定指数53.73；脂肪族指数为77.31、蛋白质疏水性为-0.823。FcSOC1a和FcSOC1b序列的N端为均为M(甲硫氨酸),两者表达蛋白在活体哺乳动物的红细胞的半衰期均为30小时左右,在活体酵母中半衰期均在20小时以上,在活体大肠杆菌均在10以上。在基因的同源性比较分析中,结果显示其与同属于芸香科的克里曼丁桔SOCl相似性达98%,和甜橙SOC1的相似性达到90%以上。FcSOC1a和FcSOC1b与葡萄的相似性也达到75%和65%。这对同源基因与麻风树、荷花、醉蝶花、拟南芥同源相似性达到50%以上。系统进化树分析表明,FcSOC1a与克里曼丁桔聚在一起,FcSOC1b与葡萄聚在一起。FcSOC1a和FcSOC1b这对同源基因相距较远。序列对比和进化树分析表明,FcSOC1a和FcSOC1b编码的蛋白中含有一个保守的MADS盒结构以及包含半保守的k结构域。(2)通过实时荧光定量PCR分析探讨融安金柑SOC1基因的表达模式发现：FcSOC1a和FcSOC1b在生殖器官和营养器官中均有表达,并且都在花器官中高水平表达。在营养器官中,FcSOC1同源基因在嫩叶、嫩茎、老叶、老茎中高表达,在幼树茎和叶中几乎没有表达。在生殖器官各个部位中,FcSOC1a和FcSOC1b均在花蕾中表达,在1.0 mm(花1)、花蕾2.0-3.0 mm(花2)、花蕾7.0 mm(花6)、花蕾8.0-9.0 mm(花7)、花芽、花蕾7.0 mm时的花瓣(花瓣6)、初花期时的花药(花药8)均有表达。不同之处在,FcSOC1a在花初开放时的子房(子房8)、花蕾8.0～9.0 mm时花托(花托7)表达明显。FcSOC1b在花蕾7.0 mm时(子房6)中表达明显,在花托中表达不明显。在日周期材料中,FcSOCl在融安金桔花、茎、叶中均有表达,基因的表达量在白天(6～12am,12～18pm)阶段要大于夜晚(18～24pm,0～6am)的表达量,并且白天的表达相对于晚上稳定。与茎、叶等样品中基因的表达量相比,在花这一器官中基因的表达量相对比较高,同时表达的不稳定性也相对较高。这对同源基因在金柑生殖器官和营养器官表达几乎相同,但也存在非常明显的不同之处,可以推断这对同源基因之间出现了功能分化。(3)成功构建融安金柑FcSOC1a和FcSOC1b基因的真核表达载体Cam35S-gfp,利用农杆菌借导采用花序浸染法转化拟南芥进,已成功得到T0代种子,其基因功能需进一步研究。
[Abstract]:The MADS-box gene family in plant growth and development plays an important role in the formation and development, especially in the control of floral organ plays an important role as an important member of the.SOC1 gene encoding the MADS-box family, can integrate a variety of temperature, hormone, flowering signal associated with the age of flowering is key genes important in recent years. Researchers, research on plant SOC1 genes in rice, deepening, grape, apple, strawberry, peony, pea, corn, birch, Arabidopsis and other plants have been cloned SOC1 gene, and constantly improve its function and mechanism involved. But for the SOC1 gene of Kumquat has not been reported. In order to study the kumquat the function of SOC1 gene, this study analyzes the basic composition based on kumquat transcriptome sequencing data on novo De, SOCl homologous gene fragment sequences by homologous cloning, cloning SOC1 gene full-length cDNA, analyzed the biological characteristics of SOC1 to investigate the expression of Rongan kumquat kumquat, characteristics of SOC1 in Rong'an reproductive organs and vegetative organs, and transgenic research on the preliminary results obtained are as follows: (1) by bioinformatics analysis showed that FcSOCl gene sequence of 660bp source cDNA and 636bp, encoding 220 and 212 amino acids, named FcSOC1a and FcSOC1b.FcSOC1a theory of protein molecular weight is 25204.7 Da, isoelectric point of 9.36; the atomic number is 3561; the molecular formula: C1085H1796N332O339S9 (Aliphatic index); aliphatic index is approximately 76.73; the instability index (Instability index) is approximately 53.23; protein hydrophobicity (Grand average of hydropathicity, GRAVY): theoretical molecular weight of -0.831.FcSOC1b protein is 24829.4Da, isoelectric point of 8.9, the atomic number is 3488, the molecular formula C1067H17S5N319O333S 14, the instability index 53.73; aliphatic index was 77.31, protein hydrophobicity is -0.823.FcSOC1a and FcSOC1b sequences of N were M (end for methionine), both protein expression in mammalian red blood cells in vivo half-life is 30 hours or so, in vivo yeast half2life in more than 20 hours in vivo E. coli were above 10. The gene homology analysis, the results showed that the same belongs to Rutaceae crimmen Ding SOCl similarity of 98% orange, orange similarity and SOC1 reached more than 90% similarity with.FcSOC1a and FcSOC1b grapes has reached 75% 65%. and the homologous gene and jatropha, lotus, spiderflower, Arabidopsis homology reached more than 50%. Phylogenetic tree analysis showed that FcSOC1a and Jieju crimmen Ding together, FcSOC1b and.FcSOC1a and FcSOC1b grapes together in the distance far homologous gene sequence. Comparison and phylogenetic analysis showed that a conserved MADS box structure of FcSOC1a and FcSOC1b encoding the protein containing K domain containing and semi conservative. (2) analyzed by real-time quantitative PCR to investigate the expression pattern of SOC1 gene discovery: Rongan kumquat FcSOC1a and FcSOC1b in the reproductive organs and vegetative organs and tissues. In the high level expression in floral organs. In vegetative organs, FcSOC1 homologous genes in leaves, stems, old leaves, high expression in older stems, young stems and leaves almost no expression. In each part of reproductive organs, FcSOC1a and FcSOC1b were expressed in flower buds, flowers 1 (at 1 mm 2.0-3.0 (mm), bud flower bud 2), 7 mm (flower 6), 8.0-9.0 mm (7) bud, flower bud, flower petals (7 mm, 6 petals) during the initial period of the anther (anther 8) were expressed. The difference is, the ovary FcSOC1a open in flowers at the beginning (8), 8. buds of ovary 0 ~ 9 mm (receptacle 7) receptacle expression was.FcSOC1b in bud 7 mm (ovary 6) was obviously in the receptacle, expression is not obvious. In the daily cycle of material, FcSOCl in Rongan kumquat flowers, stems and leaves were expressed, the expression in the daytime (6 ~ 12am, 12 ~ 18pm) stage than at night (18 ~ 24pm, 0 ~ 6am) expression, and the expression of relative stability. During the day and night stems, compared gene expression levels in samples of leaves, the expression of this gene in flower organs is relatively high, while the expression of instability is relatively high. The expression of homologous genes in reproductive and vegetative organs were almost the same, but there are also differences obviously, it can be inferred the function and differentiation of homologous genes. (3) the successful construction of the eukaryotic expression of FcSOC1b gene FcSOC1a and Rongan kumquat carrier Cam35S-gfp, by using Agrobacterium tumefaciens The transformation of Arabidopsis into the Arabidopsis by inflorescence staining method has been successfully obtained from the T0 generation, and its gene function needs further study.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S666.1

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