發(fā)布時(shí)間：2018-01-13 08:20

  本文關(guān)鍵詞：芒屬植物組織培養(yǎng)及南荻胚性愈傷組織形成基因研究　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 芒屬 南荻 組織培養(yǎng) 胚性愈傷 植株再生 基因克隆 熒光定量PCR

【摘要】：隨著煤炭、石油、天然氣等傳統(tǒng)能源的過度開發(fā)利用以及引起的環(huán)境污染,科學(xué)家們開始致力于尋找可替代的清潔新型能源。芒草為C4植物,具有光合效率高、二氧化碳排放量低以及纖維素含量豐富等特點(diǎn),從而被選為最具潛力的生物質(zhì)能源作物。中國是芒草分布的中心,其芒草多樣性高、生態(tài)型豐富。本研究選取其中最具生物質(zhì)能源應(yīng)用價(jià)值的五個(gè)物種：五節(jié)芒、芒、荻、南荻以及奇崗作為研究對(duì)象,從芒屬植物組織培養(yǎng)體系、南荻轉(zhuǎn)化體系、南荻胚性愈傷組織形成基因三個(gè)方面進(jìn)行研究。本研究主要結(jié)果如下：1.利用五節(jié)芒、芒、荻、南荻以及奇崗的未成熟幼穗為外植體,探討了最佳的外植體誘導(dǎo)培養(yǎng)基及其pH值；分化培養(yǎng)基以及生根培養(yǎng)基的激素配比。成功建立了五種芒屬植物基于胚性愈傷組織的快繁體系。發(fā)現(xiàn)五種芒屬植物最佳胚性愈傷組織誘導(dǎo)培養(yǎng)基為MS附加4.0 mg/L 2,4-D、750 mg/L MgCl2·6H2O、2.88 g/L脯氨酸、3 g/L Phytagel、30 g/L蔗糖,且其pH值為5.5時(shí)誘導(dǎo)效率最高。然而,胚性愈傷組織在附加6-BA和NAA兩種激素的分化培養(yǎng)基培養(yǎng)四周后,五節(jié)芒、芒和荻的分化率均達(dá)到100%,南荻也達(dá)到了99%,只有奇崗的分化率較低為57.4%。五種植物在MS基本培養(yǎng)基上生根率均達(dá)到100%。2.利用南荻成熟種子為外植體,通過外植體消毒、誘導(dǎo)、分化、生根、馴化以及移栽建立了完整的體胚組織培養(yǎng)再生體系。種子的最佳誘導(dǎo)培養(yǎng)基為M524附加5.0 mg/L 2,4-D、0.5 mg/L NAA、750 mg/L MgCl2·6H2O、 2.88 g/L脯氨酸、2 g/LPVP、3 g/L Phytagel、30 g/L蔗糖。誘導(dǎo)出的黃色致密愈傷組織以及白色致密愈傷組織能分化出芽,其中白色致密愈傷組織在KT 5.0 mg/L時(shí)分化率最高,為45.6%。另外,重點(diǎn)研究了南荻不同基因型、不同幼穗發(fā)育期對(duì)胚性愈傷組織誘導(dǎo)的影響。結(jié)果表明37個(gè)取樣點(diǎn)中,只有9個(gè)點(diǎn)的幼穗能夠誘導(dǎo)出胚性愈傷,且1093這個(gè)基因型的B發(fā)育時(shí)期(即幼穗長度在50-100 mm)未成熟幼穗能誘導(dǎo)出胚性愈傷組織其誘導(dǎo)率為95%,其胚性愈傷組織占有比例為74.8%,且這兩種基因型的胚性愈傷組織在附加KT 5.0 mg/L的優(yōu)化分化培養(yǎng)基上其分化效果最好,再生頻率為91.5%。3.農(nóng)桿菌介導(dǎo)的南荻轉(zhuǎn)基因體系初步研究。建立了完善的預(yù)培養(yǎng)、侵染培養(yǎng)、共培養(yǎng)和篩選培養(yǎng)體系,得到了陽性愈傷組織,并對(duì)陽性愈傷組織進(jìn)行了初步的PCR檢測(cè),初步證明外源基因已整合到南荻愈傷組織的基因組中。發(fā)現(xiàn)預(yù)培養(yǎng)采用AS濃度100mM預(yù)培養(yǎng)五天陽性愈傷組織得率較高,農(nóng)桿菌侵染時(shí)OD值在0.7左右共培養(yǎng)2天或者3天均可,篩選培養(yǎng)基附加Basta 30mg/L、頭孢750mg/L時(shí)篩選效果最佳整個(gè)過程其基本培養(yǎng)基為MS附加4.0 mg/L 2,4-D、750 mg/L MgCl2·6H2O、2.88 g/L甫氨酸、3g/L Phytagel、30 g/L蔗糖。4.克隆得到了五個(gè)南荻胚性愈傷組織發(fā)生相關(guān)基因MIARF-GEP、MIKHCP、 MlSERK1、MISERK2和MlTypA, Genbank登錄號(hào)為KU640196-KU640200。通過多重序列比對(duì)分析,發(fā)現(xiàn)五個(gè)基因保守性很高。進(jìn)化分析表明,南荻的五個(gè)基因各自與禾本科植物中玉米或者高粱的同源基因親緣關(guān)系最近。利用qRT-PCR技術(shù),發(fā)現(xiàn)五個(gè)基因在南荻胚性愈傷組織以及非胚性愈傷組織培養(yǎng)的四周內(nèi)表達(dá)量存在顯著差異,在胚性愈傷組織內(nèi)的表達(dá)量均高于非胚性愈傷組織,說明五個(gè)基因與南荻胚性愈傷組織形成密切相關(guān)。除此以外,SSR標(biāo)記統(tǒng)計(jì)結(jié)果的卡方分析表明,MISERK1基因的基因型與南荻胚性愈傷組織的誘導(dǎo)顯著相關(guān)。
[Abstract]:With the coal, oil, over exploitation of natural gas and other traditional energy and environmental pollution caused by clean energy, scientists began to find alternative. Miscanthus C4 plants with high photosynthetic efficiency, low carbon dioxide emissions and cellulose content rich features, which was selected as the most potential the biomass energy crops. Chinese is grass distribution center, the grass high diversity, ecological rich. This study selected five species among the most biomass energy application value: miscanthusfloridulus, awn, Di, Triarrhena and Qi Gang as the research object, which belongs to the plant tissue culture system from Mount, Triarrhena transformation system, Triarrhena embryogenic callus formation three gene were studied. The main results of this study are as follows: 1. using Miscanthus, awn, reed, immature Triarrhena and Qi Gang spike as explants, the optimum explants were discussed In vitro induction culture medium and the hormone ratio pH value; the differentiation medium and rooting medium. We successfully established five Miscanthus micropropagation of embryogenic callus lines based on five kinds of Miscanthus. Found the best embryogenic callus induction medium was MS with 4 mg/L 2,4-D, 750 mg/ L MgCl2 6H2O 2.88 g/L, 3 g/L Phytagel, proline, 30 g/L sucrose, and its pH value was 5.5 by the highest efficiency. However, the embryogenic callus in differentiation with 6-BA and NAA two kinds of hormones were cultured for four weeks, Miscanthus, awns and sacchariflorus differentiation rate reached 100%, Triarrhena reached 99% the gang, only odd low differentiation rates for five species of 57.4%. in MS medium on rooting rate of 100%.2. using Triarrhena mature seeds as explants, the sterilization of explants, induction, differentiation, rooting and transplanting, domestication established a complete set of somatic embryo tissue culture regeneration Seed system. The best medium is M524 add 5 mg/L 2,4-D mg/L, 0.5 NAA, 750 mg/L MgCl2, 2.88 6H2O, 2 g/LPVP, 3 g/L proline, g/L Phytagel, 30 g/L sucrose. The induced yellow compact calli and white compact callus differentiation bud, the white compact callus in KT 5 mg/L when the highest differentiation rate is 45.6%., in addition, focuses on the research of Triarrhena of different genotypes, different panicle development stage to the induction of embryogenic callus. The results showed that 37 sampling points, only 9 points in the spike could induce embryogenic callus, and the genotype of B 1093 developmental stages (i.e. spike length in 50-100 mm) immature spike can induce embryogenic callus induction rate was 95%, the proportion of embryogenic callus was 74.8%, and the two genotypes of embryogenic callus and differentiation in 5 additional KT mg/L culture Based on the best differentiation effect, regeneration frequency is the preliminary study of Triarrhena 91.5%.3. transgenic system mediated by Agrobacterium. The establishment of a sound pre culture, infection and screening culture culture, co culture system, obtained the positive callus, and callus were detected positive on initial PCR, proved that exogenous gene has been integrated to Triarrhena callus genome. It was found that pre culture with AS concentration of 100mM pre cultured for five days the calli were higher, Agrobacterium infection when the OD value of around 0.7 in 2 days or 3 days coculture can screening medium supplemented with Basta 30mg/L, of 750mg/L screening the best medium for the whole process MS added 4 mg/L 2,4-D, 750 mg/L, 2.88 MgCl2 6H2O, g/L 3g/L Phytagel, Fu ammonia acid, 30 g/L sucrose.4. cloned five Triarrhena embryogenic callus associated genes MIARF-GEP, MIKHCP, MlSERK 1, MISERK2 and MlTypA, Genbank accession number KU640196-KU640200. through the analysis of multiple sequence alignment, found five genes was highly conservative. Phylogenetic analysis showed that five genes Triarrhena each with corn or sorghum grasses in recent homologous gene phylogeny. Using qRT-PCR technology, found five gene expression differences in Triarrhena embryogenic callus and non embryogenic callus culture around the inner expression in embryogenic calli were higher than those in the non embryogenic callus, five genes and Triarrhena embryogenic callus form Chengmiqie. In addition, the chi square statistics SSR marker analysis showed that induction of MISERK1 gene type and Triarrhena embryogenic callus was significantly correlated.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S578
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本文編號(hào)：1418211