石蒜属植物小鳞茎繁殖及基于转录组学的基因发掘研究
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摘要
石蒜属(Lycoris)植物是我国原产的重要球根花卉资源,分布范围广,适应性和抗逆性强,又具有很高的观赏价值和药用价值,是切花、盆栽、园林地被植物的常用材料。然而,由于石蒜鳞茎的生长周期长、自然繁殖系数低等限制因素,目前难以达到规模化生产和产业化开发的要求,大量挖掘野生资源的现状,导致资源保护堪忧。如能缩短鳞茎繁育周期,扩大繁殖系数,并改良观赏性状,将对石蒜属植物的资源保护、开发利用具有重要实践意义。
本文在对换锦花等石蒜属重要野生资源调查的基础上,通过筛选、优化鳞片扦插繁殖技术和组培小鳞茎诱导技术,以有效缩短鳞茎发育周期,提高繁殖系数；尝试利用化学诱变和辐照诱变手段探讨换锦花的倍性育种。同时,利用目前先进的高通量测序技术,从RNA水平建立了换锦花的转录组数据库,获得大规模基因信息,对其基因功能进行预测,并在此基础上比较幼年期鳞茎与成熟鳞茎的差异表达基因及其功能,以发掘与鳞茎发育、代谢途径相关的基因,为石蒜属植物的分子育种奠定重要理论基础。主要研究结果如下：
1.实地调查了浙江舟山群岛、南麂列岛和台湾地区马祖列岛,均发现稀少但保存完好的换锦花群落,其野生生境和伴生植物群落存在差异,证实换锦花具有较阔的生态幅,并发现可能存在生态型变异。
2.通过对红花石蒜、忽地笑、换锦花等鳞茎进行切分扦插试验,认为四月是江南地区适宜的扦插季节,其鳞茎块的成活率和繁殖系数高；母鳞茎越大,则繁殖力越强。试验发现,四分鳞茎块的气培法是一种稳定、有效的扦插方式,可发生大量整齐而健壮的腋芽,为周年批量培育小鳞茎提供可能。
3.针对组培过程中鳞茎块易污染、褐化的普遍问题,比较了不同消毒时间、外植体选择和预处理等方法,提出0.1%升汞的适宜消毒时间,双鳞片诱导、外植体基盘朝上接种和利用幼嫩鳞茎降低污染率和褐化率的有效方法,尤其使用20 mg/L抗坏血酸预处理可有效防止褐化发生。
4.创造性地提出利用扦插获得的一年生小鳞茎为外植体进行悬浮培养,其污染率低且出芽大量、迅速；利用内生菌含量极低的未成熟胚为外植体,成功地通过胚挽救手段形成胚根和不定芽。试验筛选出石蒜属植物适宜的初代培养和继代培养培养基,即10 mg/L BA和0.5 mg/L NAA组合的不定芽诱导率最高,8 mg/LBA和1-2 mg/L NAA组合的增殖率较高。
5.对换锦花鳞茎进行秋水仙素处理,确定了半致死处理时间为48 h,并经根尖染色体计数、叶表皮气孔保卫细胞大小统计、流式细胞仪DNA相对含量检测,确认获得了二倍体与四倍体嵌合体。利用60co Y射线辐照换锦花鳞茎,多数材料死亡,部分可能有变异产生。
6.利用新一代高通量测序技术对换锦花鳞茎进行了大规模转录组测序,共获得98150个基因,其中45.9%的序列经蛋白质数据库比对获得注释。对功能基因进行G0、COG及KEGG分类研究,得知脂氧合酶、α葡聚糖H-型块茎磷酸化酶(GHTP)、a-海藻糖磷酸合酶等在换锦花鳞茎中表达量最高,与代谢相关的途径、植物-病原互作、剪接、植物激素的生物合成途径等是换锦花最活跃的生物代谢途径。
7.利用数字基因表达谱技术(DGE)对换锦花休眠期的多年生成熟鳞茎和一年生幼年期小鳞茎进行测序,由表达量分析及注释结果可知换锦花中有较多基因编码的蛋白质功能未知,并有很多新的基因片段。与换锦花转录组数据库比对,两个发育阶段共存在4389个表达差异显著的基因,其中上调表达基因数多于下调表达基因数,说明大部分基因在换锦花鳞茎发育过程中表达受到抑制。在差异表达基因中,病程相关蛋白上调表达最显著,假想的钙反应转录共激活因子下调表达最显著。利用KEGG数据库以及转录组参考数据库找到7条在差异表达中显著富集的Pathway。这7条途径中,光合作用途径和α-亚麻酸途径全部上调,说明这两个途径在鳞茎发育早期非常活跃。
The plants of Lycoris genus mainly originated and distributed in the east of China. They grow in very tough conditions and can easily fit in wicked environment in landscaping. These years they became popular in the market of cutting flowers, potting flowers, and landscape. But they were also restricted by the long life cyle and low proliferation, and can not meet the needs of the industrial development. Since most of the wild resources had been seriously destroyed till now, the most emergent thing today is to shorten the breeding periods, increase the proliferation,and improve the ornamental treats of Lycoris plants.
Basing on survey of the wild resources of Lycoris sprengeri, we compared different culture skills like scales cutting and tissue culture, in the hope of shortening the developing period and increasing the proliferation. On the other hand, we use both chemical and radiation mutation breeding methods to investigate the ploidy of L. sprengeri. At the same time, the next generation of high-through sequenceing technique was used to build up the transcriptome library of L. sprengeri on the RNA level. Massive gene information was obtained, and the gene functions were predicted. According to this, the differentially expressed genes and their functions were compared between the juvenile bulbs and mature bulbs. Next, we will search for the genes related to bulb developing and metabolism pathways, which will definitely support the following molecular breeding of Lycoris. The majoy results were as follows:
1. Among the three islands named Zhoushan island, Nanji island, and Mazu island, well preserved Lycoris sprengri were found. The habitata and plant community of I. sprengri showed differently. It meant that the L. sprengri was able to adapt to a wide ecological amplitude, and some mutant traits on the plants caused by the environment were discovered.
2. Scale cutting were applied to L. radiate, L. aurea, and L. sprengeri. April was considered to be the best season to cut, when the survival rate and proliferation were highest. And the bigger the mother bulbs were, the stronger the reproductive capacity would be. Also, we found that the air culture of the four-excision scales generated many buds, and was a stable and effective method.
3. In order to overcome the problems of polluting and browning which widely existed in the tissue culture, we compared different time of mercurous chloride treatment, the type of explants and many ways of pretreatment. As a result, using the twin-scales as explants was better than the four-excision scales, and laying the basal part of explant out of medium could also prevent the diffusing of pollution.20 mg/L VC was effectively stopping the browning.
4. We used the bulblets from scale cutting as explants in tissue culure for the first time, and combined with liquid culture. Few pollution took place and massive buds came out immediately. By the way, the best medium component for buds inducing was 10 mg/L BA+0.5 mg/L NAA; and the combination of 8 mg/L BA+1-2 mg/L NAA was best for reproductivity.
5. Bulbs of L. sprengeri were treated with colchicine, and 48 h of cochicine treating were fixed as the half-death time. According to the chromosome calculation of root tip, number and size of guard cells, as well as DNA content detecting by flow cytometer, some mosaic were produced. While the radiation of L. sprengeri bulbs by 60Coy killed many bulbs, and also produced several potencial mutants.
6. Illumina's new generation of high-throughput sequencing technology was used to establish a transcriptome data library from the bulbs of L. sprengeri. A total of 98150 unigenes with an average length of 90 nt were generated,41.6% of these unigenes were annotated using BLAST. According to GO, COG and KEGG classification, lipoxygenase showed the highest expression level, and metabolic pathways were the most active pathways in L. sprengeri..
7. Bulbs of different developing stage were sampled and sequenced by DGE. The expression level and annotated results indicated that much of the protein function was unknown. Compared with the transcriptome data base,4389 differentially expressed gene were found, and most of them were up-regulated.
本文在对换锦花等石蒜属重要野生资源调查的基础上,通过筛选、优化鳞片扦插繁殖技术和组培小鳞茎诱导技术,以有效缩短鳞茎发育周期,提高繁殖系数；尝试利用化学诱变和辐照诱变手段探讨换锦花的倍性育种。同时,利用目前先进的高通量测序技术,从RNA水平建立了换锦花的转录组数据库,获得大规模基因信息,对其基因功能进行预测,并在此基础上比较幼年期鳞茎与成熟鳞茎的差异表达基因及其功能,以发掘与鳞茎发育、代谢途径相关的基因,为石蒜属植物的分子育种奠定重要理论基础。主要研究结果如下：
1.实地调查了浙江舟山群岛、南麂列岛和台湾地区马祖列岛,均发现稀少但保存完好的换锦花群落,其野生生境和伴生植物群落存在差异,证实换锦花具有较阔的生态幅,并发现可能存在生态型变异。
2.通过对红花石蒜、忽地笑、换锦花等鳞茎进行切分扦插试验,认为四月是江南地区适宜的扦插季节,其鳞茎块的成活率和繁殖系数高；母鳞茎越大,则繁殖力越强。试验发现,四分鳞茎块的气培法是一种稳定、有效的扦插方式,可发生大量整齐而健壮的腋芽,为周年批量培育小鳞茎提供可能。
3.针对组培过程中鳞茎块易污染、褐化的普遍问题,比较了不同消毒时间、外植体选择和预处理等方法,提出0.1%升汞的适宜消毒时间,双鳞片诱导、外植体基盘朝上接种和利用幼嫩鳞茎降低污染率和褐化率的有效方法,尤其使用20 mg/L抗坏血酸预处理可有效防止褐化发生。
4.创造性地提出利用扦插获得的一年生小鳞茎为外植体进行悬浮培养,其污染率低且出芽大量、迅速；利用内生菌含量极低的未成熟胚为外植体,成功地通过胚挽救手段形成胚根和不定芽。试验筛选出石蒜属植物适宜的初代培养和继代培养培养基,即10 mg/L BA和0.5 mg/L NAA组合的不定芽诱导率最高,8 mg/LBA和1-2 mg/L NAA组合的增殖率较高。
5.对换锦花鳞茎进行秋水仙素处理,确定了半致死处理时间为48 h,并经根尖染色体计数、叶表皮气孔保卫细胞大小统计、流式细胞仪DNA相对含量检测,确认获得了二倍体与四倍体嵌合体。利用60co Y射线辐照换锦花鳞茎,多数材料死亡,部分可能有变异产生。
6.利用新一代高通量测序技术对换锦花鳞茎进行了大规模转录组测序,共获得98150个基因,其中45.9%的序列经蛋白质数据库比对获得注释。对功能基因进行G0、COG及KEGG分类研究,得知脂氧合酶、α葡聚糖H-型块茎磷酸化酶(GHTP)、a-海藻糖磷酸合酶等在换锦花鳞茎中表达量最高,与代谢相关的途径、植物-病原互作、剪接、植物激素的生物合成途径等是换锦花最活跃的生物代谢途径。
7.利用数字基因表达谱技术(DGE)对换锦花休眠期的多年生成熟鳞茎和一年生幼年期小鳞茎进行测序,由表达量分析及注释结果可知换锦花中有较多基因编码的蛋白质功能未知,并有很多新的基因片段。与换锦花转录组数据库比对,两个发育阶段共存在4389个表达差异显著的基因,其中上调表达基因数多于下调表达基因数,说明大部分基因在换锦花鳞茎发育过程中表达受到抑制。在差异表达基因中,病程相关蛋白上调表达最显著,假想的钙反应转录共激活因子下调表达最显著。利用KEGG数据库以及转录组参考数据库找到7条在差异表达中显著富集的Pathway。这7条途径中,光合作用途径和α-亚麻酸途径全部上调,说明这两个途径在鳞茎发育早期非常活跃。
The plants of Lycoris genus mainly originated and distributed in the east of China. They grow in very tough conditions and can easily fit in wicked environment in landscaping. These years they became popular in the market of cutting flowers, potting flowers, and landscape. But they were also restricted by the long life cyle and low proliferation, and can not meet the needs of the industrial development. Since most of the wild resources had been seriously destroyed till now, the most emergent thing today is to shorten the breeding periods, increase the proliferation,and improve the ornamental treats of Lycoris plants.
Basing on survey of the wild resources of Lycoris sprengeri, we compared different culture skills like scales cutting and tissue culture, in the hope of shortening the developing period and increasing the proliferation. On the other hand, we use both chemical and radiation mutation breeding methods to investigate the ploidy of L. sprengeri. At the same time, the next generation of high-through sequenceing technique was used to build up the transcriptome library of L. sprengeri on the RNA level. Massive gene information was obtained, and the gene functions were predicted. According to this, the differentially expressed genes and their functions were compared between the juvenile bulbs and mature bulbs. Next, we will search for the genes related to bulb developing and metabolism pathways, which will definitely support the following molecular breeding of Lycoris. The majoy results were as follows:
1. Among the three islands named Zhoushan island, Nanji island, and Mazu island, well preserved Lycoris sprengri were found. The habitata and plant community of I. sprengri showed differently. It meant that the L. sprengri was able to adapt to a wide ecological amplitude, and some mutant traits on the plants caused by the environment were discovered.
2. Scale cutting were applied to L. radiate, L. aurea, and L. sprengeri. April was considered to be the best season to cut, when the survival rate and proliferation were highest. And the bigger the mother bulbs were, the stronger the reproductive capacity would be. Also, we found that the air culture of the four-excision scales generated many buds, and was a stable and effective method.
3. In order to overcome the problems of polluting and browning which widely existed in the tissue culture, we compared different time of mercurous chloride treatment, the type of explants and many ways of pretreatment. As a result, using the twin-scales as explants was better than the four-excision scales, and laying the basal part of explant out of medium could also prevent the diffusing of pollution.20 mg/L VC was effectively stopping the browning.
4. We used the bulblets from scale cutting as explants in tissue culure for the first time, and combined with liquid culture. Few pollution took place and massive buds came out immediately. By the way, the best medium component for buds inducing was 10 mg/L BA+0.5 mg/L NAA; and the combination of 8 mg/L BA+1-2 mg/L NAA was best for reproductivity.
5. Bulbs of L. sprengeri were treated with colchicine, and 48 h of cochicine treating were fixed as the half-death time. According to the chromosome calculation of root tip, number and size of guard cells, as well as DNA content detecting by flow cytometer, some mosaic were produced. While the radiation of L. sprengeri bulbs by 60Coy killed many bulbs, and also produced several potencial mutants.
6. Illumina's new generation of high-throughput sequencing technology was used to establish a transcriptome data library from the bulbs of L. sprengeri. A total of 98150 unigenes with an average length of 90 nt were generated,41.6% of these unigenes were annotated using BLAST. According to GO, COG and KEGG classification, lipoxygenase showed the highest expression level, and metabolic pathways were the most active pathways in L. sprengeri..
7. Bulbs of different developing stage were sampled and sequenced by DGE. The expression level and annotated results indicated that much of the protein function was unknown. Compared with the transcriptome data base,4389 differentially expressed gene were found, and most of them were up-regulated.
引文
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