本文選題：金柑　切入點(diǎn)：FcSOC1　出處：《廣西大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：MADS-box家族基因在植物生長發(fā)育中起著重要的作用,尤其在控制植物花器官的形成發(fā)育方面起著重要的作用。SOC1基因作為編碼MADS-box家族的重要一員,能夠集成多種溫度、激素、年齡相關(guān)的開花信號(hào),是重要的開花調(diào)控關(guān)鍵基因。近年來,科研人員對(duì)植物SOC1基因的研究不斷深入,在水稻、葡萄、蘋果、草莓、牡丹、豌豆、玉米、白樺、擬南芥等多種植物中相繼克隆SOC1基因,不斷完善其參與的功能及機(jī)理。但對(duì)于金柑的SOC1基因的研究未見報(bào)道,為了深入研究金柑SOC1基因的功能,本研究基于金柑轉(zhuǎn)錄組測(cè)序數(shù)據(jù)de novo組裝分析的基礎(chǔ)上,獲得SOCl同源基因片段序列信息。通過同源基因克隆方法,克隆出SOC1同源基因cDNA全長,分析了融安金柑SOC1的生物學(xué)特點(diǎn),探討融安金柑SOC1在生殖器官和營養(yǎng)器官的表達(dá)特性,并對(duì)其進(jìn)行轉(zhuǎn)基因研究,初步獲得如下結(jié)果：(1)通過生物信息學(xué)分析表明FcSOCl同源基因的cDNA序列為660bp和636bp,編碼220和212個(gè)氨基酸,分別命名為FcSOC1a和FcSOC1b。FcSOC1a理論蛋白質(zhì)的分子量為：25204.7 Da,等電點(diǎn)：9.36；原子個(gè)數(shù)為3561；分子式為：C1085H1796N332O339S9;脂肪族指數(shù)(Aliphatic index)大致為76.73；不穩(wěn)定指數(shù)(Instability index)大致為53.23；蛋白質(zhì)疏水性(Grand average of hydropathicity, GRAVY)為：-0.831。FcSOC1b理論蛋白質(zhì)的分子量為24829.4Da、等電點(diǎn)8.9、原子個(gè)數(shù)為3488、分子式C1067H17S5N319O333S14、不穩(wěn)定指數(shù)53.73；脂肪族指數(shù)為77.31、蛋白質(zhì)疏水性為-0.823。FcSOC1a和FcSOC1b序列的N端為均為M(甲硫氨酸),兩者表達(dá)蛋白在活體哺乳動(dòng)物的紅細(xì)胞的半衰期均為30小時(shí)左右,在活體酵母中半衰期均在20小時(shí)以上,在活體大腸桿菌均在10以上。在基因的同源性比較分析中,結(jié)果顯示其與同屬于蕓香科的克里曼丁桔SOCl相似性達(dá)98%,和甜橙SOC1的相似性達(dá)到90%以上。FcSOC1a和FcSOC1b與葡萄的相似性也達(dá)到75%和65%。這對(duì)同源基因與麻風(fēng)樹、荷花、醉蝶花、擬南芥同源相似性達(dá)到50%以上。系統(tǒng)進(jìn)化樹分析表明,FcSOC1a與克里曼丁桔聚在一起,FcSOC1b與葡萄聚在一起。FcSOC1a和FcSOC1b這對(duì)同源基因相距較遠(yuǎn)。序列對(duì)比和進(jìn)化樹分析表明,FcSOC1a和FcSOC1b編碼的蛋白中含有一個(gè)保守的MADS盒結(jié)構(gòu)以及包含半保守的k結(jié)構(gòu)域。(2)通過實(shí)時(shí)熒光定量PCR分析探討融安金柑SOC1基因的表達(dá)模式發(fā)現(xiàn)：FcSOC1a和FcSOC1b在生殖器官和營養(yǎng)器官中均有表達(dá),并且都在花器官中高水平表達(dá)。在營養(yǎng)器官中,FcSOC1同源基因在嫩葉、嫩莖、老葉、老莖中高表達(dá),在幼樹莖和葉中幾乎沒有表達(dá)。在生殖器官各個(gè)部位中,FcSOC1a和FcSOC1b均在花蕾中表達(dá),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時(shí)的花瓣(花瓣6)、初花期時(shí)的花藥(花藥8)均有表達(dá)。不同之處在,FcSOC1a在花初開放時(shí)的子房(子房8)、花蕾8.0～9.0 mm時(shí)花托(花托7)表達(dá)明顯。FcSOC1b在花蕾7.0 mm時(shí)(子房6)中表達(dá)明顯,在花托中表達(dá)不明顯。在日周期材料中,FcSOCl在融安金桔花、莖、葉中均有表達(dá),基因的表達(dá)量在白天(6～12am,12～18pm)階段要大于夜晚(18～24pm,0～6am)的表達(dá)量,并且白天的表達(dá)相對(duì)于晚上穩(wěn)定。與莖、葉等樣品中基因的表達(dá)量相比,在花這一器官中基因的表達(dá)量相對(duì)比較高,同時(shí)表達(dá)的不穩(wěn)定性也相對(duì)較高。這對(duì)同源基因在金柑生殖器官和營養(yǎng)器官表達(dá)幾乎相同,但也存在非常明顯的不同之處,可以推斷這對(duì)同源基因之間出現(xiàn)了功能分化。(3)成功構(gòu)建融安金柑FcSOC1a和FcSOC1b基因的真核表達(dá)載體Cam35S-gfp,利用農(nóng)桿菌借導(dǎo)采用花序浸染法轉(zhuǎn)化擬南芥進(jìn),已成功得到T0代種子,其基因功能需進(jìn)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.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S666.1

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