百合种球生产关键技术与休眠解除中碳水化合物代谢机理研究

发布时间：2018-02-19 23:25

本文关键词： 百合种球生产休眠解除碳水化合物代谢淀粉酶基因　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：百合是世界范围内重要的观赏植物,也是世界范围内重要的鲜切花种类之一。2014年中国仅百合切花产量就超20亿枝,即使扣除其中多茬球的使用外,我国每年从荷兰等国进口百合种球超过4亿粒,占百合种球总需求的80%以上,使得我国百合产业对外依存度很高。本研究立足于北京周边地区的自然环境资源和实际生产条件,开展了以实现本土化的百合种球规模化生产的关键技术研发与实践应用,包括：规模化籽球脱毒快繁、鳞片扦插繁殖、商品球种植、栽培管理以及种球贮藏等百合规模化生产的关键技术。此外,鉴于种球冷藏期间休眠解除的重要性,重点探讨了种球休眠解除可能的生理和分子机制。获得以下主要结果：籽球繁殖和商品球生产技术方面：1)建立了通过茎尖离体培养、隔离种植获得百合脱毒种球后,从百合种球鳞片扦插、籽球扩繁到商品球生产的北京地区规模化百合脱毒种球繁育生产技术规程。2)优化了脱毒球鳞片扦插扩繁条件。选择蛭石作为扦插基质扦插效果好,基质与鳞片的体积比为3：1时鳞片球鲜重和直径均最优,脱毒鳞片扦插的环境温度应控制在20℃-25℃ 16 w+17℃ 2w+5℃ 9w的变温条件下,脱毒鳞片球形成的数量多,质量优,外层鳞片形成的鳞片球显著多于内层鳞片。3)明确了冷库控温精度对鳞茎冷藏效果影响较大,冷藏温度为-1.5±0.2℃的条件下,采用阶梯式温度调控法,可有效避免鳞茎芽冷害。发现随着种球规格的增大,鳞茎芽的活动明显放缓,冷害发生率降低。对冷库的高精度控制可以有效延长不同规格的百合种球贮藏期,而在冷库精度低于士1℃的冷库中,贮藏的时间越长冷害和青霉病发生的可能性越大。种球休眠解除机制解析方面：1)通过转录组测序,进一步明确淀粉和蔗糖代谢途径在差异表达基因中显著富集。以休眠解除前的'Siberia'品种百合种球作为对照组,选取4℃储藏8w的已解除休眠的种球作为试验组,使用Illumina HiSeq 2000系统对cDNA文库进行双末端测序。分别获得50648075和51254807个reads。将这些序列组装获得79,354个unigene,平均长度为968bp。共得到5,834条差异明显的基因,其中2,619条基因表达明显上调,3,215条基因表达明显下调。生化途径(KEGG)富集分析发现百合鳞茎休眠解除与激素信号转导,能量代谢相关。2)在百合休眠解除转录组中筛选到两个在休眠解除过程中表达上调的淀粉酶基因,其表达模式与碳水化合物含量和淀粉酶的活性变化相吻合。基于生理指标和基因表达的转录组分析,发现淀粉酶基因可能参与了百合种球的休眠解除。通过RACE获得了α-淀粉酶(LiAMY)和β-淀粉酶(LiBAM)基因。qRT-PCR分析表明,与对照相比,LiAMY和LiBAM基因在贮藏于4℃百合鳞茎中,在前6w的转录逐渐积累,并在第8w大幅增加,达到10.6倍的表达高峰；之后淀粉酶基因的表达略有下降,但在12w仍保持7.8倍的表达量。这一表达特性与碳水化合物含量和淀粉酶的活性相一致,表明该基因转录水平的增加至少部分程度上促进了水解淀粉和可溶性糖的积累,可能参与了百合种球的休眠解除。
[Abstract]:Lily is an important ornamental plant in the world, only China lily flower yield also in the world one of the important types of fresh cut.2014 over 2 billion branches, even excluding the use one more crop ball, China's annual imports from Holland more than 400 million grains of lily bulbs of lily bulbs, accounted for more than the total demand 80%, make our lily industry a high degree of dependence on foreign. This research is based on the surrounding areas of Beijing, the natural environment resources and the actual production conditions, to carry out the research on the key technology of Lily ball scale production and practice, in order to achieve localization include: large-scale seed ball virus-free propagation, cutting propagation of scale the ball, commercial cultivation, the key technology of Lily Scale Cultivation and bulb storage. In addition, in view of the importance of the ball during dormancy refrigerated, focusing on the bulb dormancy physiology can be And the molecular mechanism. The main results were as follows: seed breeding and production technology of ball ball goods: 1) was established by stem tip culture, plant quarantine to obtain virus-free bulb lily, from lily bulb scale cutting, the scale of Beijing lily seed ball propagation to commodity production of virus-free ball ball the breeding production techniques of.2) optimization of virus-free ball scale cutting propagation conditions. Using vermiculite as cuttage cutting effect is good, and the matrix scales at the volume ratio of 3:1 bulb fresh weight and diameter were optimal, detoxification scale cutting environment temperature should be controlled in a temperature condition of 20 DEG -25 DEG w+17 to 16 DEG 2w+5 DEG 9W, the number of virus-free bulb formation, excellent quality, outer scales formed bulb inner scales significantly more than.3) the cold storage temperature control accuracy of bulb storage effect influence, cold temperature is -1.5 - 0.2 DEG C A Next, using the ladder type temperature control method, which can effectively avoid the damage. With the increase of bulb bud ball specifications, bulb bud activity slowed down significantly, reduce the incidence of chilling injury. Lily bulbs during storage of cold storage and high precision control can effectively extend the different specifications, and in the cold storage, 1 degree accuracy is lower than the base the possibility of the longer storage, chilling injury and Penicillium disease more. Bulb dormancy mechanisms analysis: 1) by transcriptome sequencing, to further clarify the starch and sucrose metabolism in the differentially expressed genes were significantly enriched in dormancy. Prior to the termination of the'Siberia'varieties of lily bulbs were selected as the control group. 4 C storage 8W has lifted the bulbs dormancy as experimental group, using Illumina HiSeq 2000 system were sequenced. The cDNA library are respectively 50648075 and 51254807 reads. will receive 79354 of these sequence assembly UniGene, the average length of 968bp. gene were obtained 5834 obvious differences, of which 2619 genes were up-regulated and 3215 genes were downregulated. Biochemical pathways (KEGG) enrichment analysis showed that the lily bulbs dormancy and hormone signal transduction, energy metabolism related.2) in the lily dormancy transcriptome screened two in the dormancy genes up-regulated expression of amylase in the process of changes in activity and its expression pattern of carbohydrate and amylase coincide. Transcriptome analysis based on gene expression and physiological indexes, found that amylase gene may be involved in the dormancy of lily bulbs. Obtained by RACE alpha amylase (LiAMY) and beta amylase (LiBAM) gene.QRT-PCR analysis showed that compared with the control, LiAMY and LiBAM genes in storage at the temperature of 4 bulbs, accumulated in the transcription of 6W before, and a substantial increase in the 8w, The expression reached a peak of 10.6 times; after the expression of amylase gene expression declined slightly, but remained 7.8 times the amount in 12W. This is consistent with the expression characteristics and carbohydrate content and amylase activity, indicated that the increase of the level of gene transcription at least in part to promote the hydrolysis of starch and soluble sugar accumulation, dormancy may in the lily bulbs.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.29

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