芒属植物组织培养及南荻胚性愈伤组织形成基因研究

发布时间：2018-01-13 08:20

  本文关键词：芒属植物组织培养及南荻胚性愈伤组织形成基因研究　出处：《武汉大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 芒属 南荻 组织培养 胚性愈伤 植株再生 基因克隆 荧光定量PCR

【摘要】：随着煤炭、石油、天然气等传统能源的过度开发利用以及引起的环境污染,科学家们开始致力于寻找可替代的清洁新型能源。芒草为C4植物,具有光合效率高、二氧化碳排放量低以及纤维素含量丰富等特点,从而被选为最具潜力的生物质能源作物。中国是芒草分布的中心,其芒草多样性高、生态型丰富。本研究选取其中最具生物质能源应用价值的五个物种：五节芒、芒、荻、南荻以及奇岗作为研究对象,从芒属植物组织培养体系、南荻转化体系、南荻胚性愈伤组织形成基因三个方面进行研究。本研究主要结果如下：1.利用五节芒、芒、荻、南荻以及奇岗的未成熟幼穗为外植体,探讨了最佳的外植体诱导培养基及其pH值；分化培养基以及生根培养基的激素配比。成功建立了五种芒属植物基于胚性愈伤组织的快繁体系。发现五种芒属植物最佳胚性愈伤组织诱导培养基为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值为5.5时诱导效率最高。然而,胚性愈伤组织在附加6-BA和NAA两种激素的分化培养基培养四周后,五节芒、芒和荻的分化率均达到100%,南荻也达到了99%,只有奇岗的分化率较低为57.4%。五种植物在MS基本培养基上生根率均达到100%。2.利用南荻成熟种子为外植体,通过外植体消毒、诱导、分化、生根、驯化以及移栽建立了完整的体胚组织培养再生体系。种子的最佳诱导培养基为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。诱导出的黄色致密愈伤组织以及白色致密愈伤组织能分化出芽,其中白色致密愈伤组织在KT 5.0 mg/L时分化率最高,为45.6%。另外,重点研究了南荻不同基因型、不同幼穗发育期对胚性愈伤组织诱导的影响。结果表明37个取样点中,只有9个点的幼穗能够诱导出胚性愈伤,且1093这个基因型的B发育时期(即幼穗长度在50-100 mm)未成熟幼穗能诱导出胚性愈伤组织其诱导率为95%,其胚性愈伤组织占有比例为74.8%,且这两种基因型的胚性愈伤组织在附加KT 5.0 mg/L的优化分化培养基上其分化效果最好,再生频率为91.5%。3.农杆菌介导的南荻转基因体系初步研究。建立了完善的预培养、侵染培养、共培养和筛选培养体系,得到了阳性愈伤组织,并对阳性愈伤组织进行了初步的PCR检测,初步证明外源基因已整合到南荻愈伤组织的基因组中。发现预培养采用AS浓度100mM预培养五天阳性愈伤组织得率较高,农杆菌侵染时OD值在0.7左右共培养2天或者3天均可,筛选培养基附加Basta 30mg/L、头孢750mg/L时筛选效果最佳整个过程其基本培养基为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.克隆得到了五个南荻胚性愈伤组织发生相关基因MIARF-GEP、MIKHCP、 MlSERK1、MISERK2和MlTypA, Genbank登录号为KU640196-KU640200。通过多重序列比对分析,发现五个基因保守性很高。进化分析表明,南荻的五个基因各自与禾本科植物中玉米或者高粱的同源基因亲缘关系最近。利用qRT-PCR技术,发现五个基因在南荻胚性愈伤组织以及非胚性愈伤组织培养的四周内表达量存在显著差异,在胚性愈伤组织内的表达量均高于非胚性愈伤组织,说明五个基因与南荻胚性愈伤组织形成密切相关。除此以外,SSR标记统计结果的卡方分析表明,MISERK1基因的基因型与南荻胚性愈伤组织的诱导显著相关。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S578
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本文编号：1418211