桑寄生顽拗性种子生物学特性及脱水敏感性机理研究
发布时间：2018-07-18 08:31
【摘要】：桑寄生是地位重要但不易繁育的特殊药材,其种子为典型的顽拗性种子,失活快,脱水敏感性强。而种子作为其唯一的繁殖材料,严重制约其繁育。目前顽拗性种子保存研究多,但成功实例较少,其根本原因是对脱水敏感性引起的死亡机理知之甚少,而种子脱水敏感性是引起顽拗性种子死亡的关键科学问题。本研究从桑寄生种子生物学特性、脱水敏感性、低温保存等方面进行系统的研究,实现桑寄生种子繁育与保存的目标;围绕桑寄生种子脱水过程中细胞的形态结构及抗氧化系统进行综合研究,筛选脱水敏感性关键时期的种子进行高通量测序,挖掘与脱水敏感性相关的关键基因,从而揭示桑寄生顽拗性种子脱水敏感性的分子机理,不仅为研制桑寄生种子的繁育与保存关键技术提供理论依据,也为研究其它顽拗性种子提供新的知识与思路。其主要研究结果如下:(1)影响检测桑寄生种子生活力的因素的主次为染色时间TTC浓度染色温度,最佳鉴定方法为沿种子长轴的两侧纵向切开成两半,在30℃下,将种子至于浓度为0.8%TTC的溶液中黑暗染色8小时。(2)种子最佳发芽床是纸床;不同温度处理种子发芽率之间差异显著,最佳发芽温度是变温处理(25/30℃);光照对种子的萌发无影响;种子不用消毒,用灭菌水处理效果最好;不同批次的种子质量有差异,春季采收的种子发芽率最高。(3)桑寄生种子是典型的顽拗性种子,对低温极其敏感,长期保存困难,自然状态下保存7天后就失去了活力。其最佳的保存方法是:将用无菌水处理过的新鲜种子浸泡到2mg/L的ABA溶液中30 min,取出放置于铺有2层滤纸的培养皿内,然后用封口袋包装好置于4℃的冰箱,120天后种子仍有活力。(4)根据桑寄生种子的萌发特性,将桑寄生种子的质量分为三级,要求纯度和净度都为100%。一级发芽率不低于75.0%,水分不低于50.0%,百粒重不低于60.0 g;二级发芽率不低于70.0%,水分不低于40%,百粒重不低于50.0 g;三级发芽率不低于60.0%,水分小于40.0%,百粒重低于50.0 g。(5)桑寄生最佳的繁殖方法为采集新鲜、饱满、无病虫害的果实去掉果皮,粘于2年生桑树上部直径大于1.0cm的枝条之上,早晚洒水,使桑林间空气湿度保持在80%以上。(6)桑寄生种子对脱水非常敏感,其活力和发芽率与含水量显著相关。新鲜种子的发芽率为86%(活力为99%),脱水16h后发芽率迅速下降到39%(活力为66%,含水量为35.17%),36h后含水量会下降至24.93%,种子活力变为15%,而发芽率仅为6%,40h种子含水量为23.47%时,种子基本丧失了活力,只有9%,而发芽率为0。(7)桑寄生种子脱水过程中细胞显微和超微结构变化很大。显微观察显示脱水过程中细胞间隙逐步增大,细胞质和细胞壁逐渐分离,从而导致细胞进一步降解;超微结构显示脱水过程中各种细胞器受到不同程度的损伤,线粒体结构破裂,核膜变得模糊不清,核仁逐步降解,淀粉粒和脂肪体进一步分解劣变;脱水过程中生理指标变化较大,随着脱水处理时间的增加,内源激素ABA、ZR、GA、IAA含量总体上呈现先上升再下降的变化趋势,种子内的SOD活性呈总体下降趋势,超氧阴离子自由基含量、可溶性蛋白和MDA含量则随种子含水量下降而增加。(8)对不同部位桑寄生总RNA提取方法研究表明,CTAB-LiC1法提取效果最好,TriZol试剂盒法和改良TriZol法均不能有效的去除桑寄生中的多糖和蛋白质等杂质。(9)利用高通量测序技术,对未脱水的桑寄生种子(CK),脱水16小时(Tac-16)和脱水36小时(Tac-36)进行了转录组分析,一共得到386,542,846条高质量序列。利用Trinity软件,组装得到164,546转录本(对应114,971基因)。通过与NR,UniProt,GO,KEGG pathway和COG数据库进行比对,并对组装得到的转录本进行了详细的注释。在CK,Tac16和Tac-36样本中,分别检测到60,695,56,027和66,389转录本(1FPKM)。通过与CK进行比较,在Tac-16中发现了 2,102个上调和1,344个下调的转录本,而在Tac-36中发现了 1,649个上调和2,135个下调的转录本。这些差异表达的转录本包含一些与已知的脱水过程相关的基因,比如RD22,热休克蛋白(HSP),转录因子(MYB,WRKY和乙烯反应性转录因子)。研究结果显示HSP和核糖体蛋白(ribosomal proteins)属于桑寄生顽拗性种子早期响应脱水过程的重要基因。原始数据已提交到NCBISRA数据库(SRA309567)。本研究系统地对桑寄生种子的生物学特性进行研究,也是第一次利用转录组技术研究桑寄生顽拗性种子脱水敏感性的机理,所得结果对于桑寄生种子的繁育、保存及脱水过程中的基因调控具有重要的指导意义,并拓宽了我们对于顽拗性种子脱水及萌发过程中基因变化的认识。
[Abstract]:The seed is the typical recalcitrant seed, which is the typical recalcitrant seed, and the seed is the only reproduction material, and the seed is the only reproduction material, which seriously restricts its breeding. At present, the recalcitrant seeds have been preserved more and more, but the basic reason is the death mechanism caused by the dehydration sensitivity. Little is known, and seed dehydration sensitivity is the key scientific problem that causes recalcitrant seed death. This study systematically studies the biological characteristics, dehydration sensitivity and cryopreservation of the seeds of the parasitic parasitic parasitic seeds to realize the breeding and preservation of the seeds of the parasitic seeds, and the morphology and structure of the cells in the process of the dehydration of the seeds of the parasitic parasitic parasitic seeds. A comprehensive study of the antioxidant system was carried out to screen the seeds of the critical period of dehydration sensitivity, and the key genes related to the sensitivity of dehydration were identified. The molecular mechanism of the dehydration sensitivity of the recalcitrant seeds of the parasitic parasitic recalcitrant seeds was revealed, which not only provided a theoretical basis for the development of the key techniques for the propagation and preservation of the seeds of the parasitic parasitic parasitic seeds, but also the research for the key techniques for the development and preservation of the seeds of the parasitic parasitic parasitic seeds. Other recalcitrant seeds provide new knowledge and ideas. The main results are as follows: (1) the main factors affecting the viability of the seed of the parasitic seeds are the dyeing temperature of the dyeing time TTC concentration, and the best method is to cut into two halves along the two sides of the long axis of the seed, and the black seed in a solution with a concentration of 0.8%TTC at 30. 8 hours of dark dyeing. (2) the best seed germination bed of seed is paper bed; the difference of germination rate between different temperature treatment seeds is significant, the best germination temperature is temperature change treatment (25/30 C); light has no effect on seed germination; seeds do not have to be sterilized, the best effect is treated with sterilization water; the seed quality of different batch seeds is different, and the germination rate of seed harvest in spring is different. (3) (3) the seed of the parasitic parasitic seed is a typical recalcitrant seed, extremely sensitive to low temperature, difficult to preserve for a long time, and loses vitality in the natural state for 7 days. The best preservation method is to soak the fresh seeds treated with aseptic water to 30 min in the ABA solution, and put out in a Petri dish with 2 layers of filter paper, and then put it in a Petri dish. Then, the seeds are placed in a Petri dish with 2 layers of filter paper. The seeds were packed in a pocket at 4 degrees centigrade, and the seeds were still alive after 120 days. (4) according to the germination characteristics of the parasitic seeds, the quality of the parasitic seeds was divided into three grades. The purity and cleanliness of the seeds were not less than 75%, the moisture was not less than 50%, the weight of 100 grains was not less than 60 g, and the two germination rate was not less than 70%, and the moisture was not low. In 40%, the weight of 100 grain is not less than 50 g, the three grade germination rate is not less than 60%, the water is less than 40%, the weight of 100 grain weight is less than 50 g. (5), the best breeding method for mulberry parasitic is to collect fresh, full, fruit without pests and pests, and stick to the branches above the 1.0cm in the upper diameter of the mulberry trees for 2 years, and keep the air humidity between the mulberry and the forest at 80%. (6) (6) the seed of the parasitic parasitic seed is very sensitive to dehydration. The vigor and germination rate are significantly related to the water content. The germination rate of fresh seeds is 86% (vitality 99%). The germination rate of the fresh seeds is 86% (66%, 35.17%), the water content will fall to 24.93%, the seed vigor becomes 15%, and the germination rate is only 6%, 40H seed. When the water content was 23.47%, the vigor of the seeds was lost, only 9%, and the germination rate was 0. (7) of the Sang Jisheng seed. The microscopic and ultrastructure of the cells changed greatly during the dehydration process of the seeds. The microscopic observation showed that the cell gap gradually increased during the dehydration process and the cytoplasm and cell wall gradually separated, which resulted in the further degradation of the cells, and the ultrastructure showed dehydration. During the process, the organelles were damaged in varying degrees, the structure of mitochondria broke, the nuclear membrane became blurred, the nucleolus was degraded gradually, the starch granules and the fat body were further decomposed, and the physiological indexes of the dehydration process changed greatly. As the dehydration time increased, the content of endogenous hormones ABA, ZR, GA, and IAA decreased first and then declined. The SOD activity in the seeds decreased in general, the content of superoxide anion radical and the content of soluble protein and MDA increased with the decrease of seed water content. (8) the study on the extraction method of total RNA in different parts of the parasitic parasitic fungi showed that the extraction effect of CTAB-LiC1 method was the best, TriZol Kit Method and the modified TriZol method were not effective. (9) a transcriptional analysis was carried out on the non dehydrated parasitic seeds (CK), dehydration 16 hours (Tac-16) and 36 hours of dehydration (Tac-36) by high throughput sequencing technology. A total of 386542846 high quality sequences were obtained. The 164546 transcript (corresponding 114971 gene) was assembled by Trinity software. By comparing with the NR, UniProt, GO, KEGG pathway and COG databases, and detailed annotations for the transcripts obtained, 60695,56027 and 66389 transcripts (1FPKM) were detected in CK, Tac16 and Tac-36 samples. By comparing with CK, 2102 up-regulated and 1344 down-regulated transcripts were found. 1649 up-regulated and 2135 down-regulated transcripts were found in ac-36. These differentially expressed transcripts included genes associated with known dehydration processes, such as RD22, heat shock protein (HSP), transcription factors (MYB, WRKY, and ethylene reactive transcription factors). The results showed that HSP and ribosomal protein (ribosomal proteins) belonged to mulberry. An important gene for the early response to dehydration process in the parasitic recalcitrant seeds. The original data have been submitted to the NCBISRA database (SRA309567). This study systematically studies the biological characteristics of the seeds of the parasite, and is the first mechanism to study the dehydration sensitivity of the recalcitrant seeds of the parasitic parasite by using the transcriptional technique. The results are for the parasitism of the parasite. Gene regulation in the process of seed breeding, preservation and dehydration has important guiding significance, and broadens our understanding of gene changes during dewatering and germination of recalcitrant seeds.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S567.19
本文编号：2131338
[Abstract]:The seed is the typical recalcitrant seed, which is the typical recalcitrant seed, and the seed is the only reproduction material, and the seed is the only reproduction material, which seriously restricts its breeding. At present, the recalcitrant seeds have been preserved more and more, but the basic reason is the death mechanism caused by the dehydration sensitivity. Little is known, and seed dehydration sensitivity is the key scientific problem that causes recalcitrant seed death. This study systematically studies the biological characteristics, dehydration sensitivity and cryopreservation of the seeds of the parasitic parasitic parasitic seeds to realize the breeding and preservation of the seeds of the parasitic seeds, and the morphology and structure of the cells in the process of the dehydration of the seeds of the parasitic parasitic parasitic seeds. A comprehensive study of the antioxidant system was carried out to screen the seeds of the critical period of dehydration sensitivity, and the key genes related to the sensitivity of dehydration were identified. The molecular mechanism of the dehydration sensitivity of the recalcitrant seeds of the parasitic parasitic recalcitrant seeds was revealed, which not only provided a theoretical basis for the development of the key techniques for the propagation and preservation of the seeds of the parasitic parasitic parasitic seeds, but also the research for the key techniques for the development and preservation of the seeds of the parasitic parasitic parasitic seeds. Other recalcitrant seeds provide new knowledge and ideas. The main results are as follows: (1) the main factors affecting the viability of the seed of the parasitic seeds are the dyeing temperature of the dyeing time TTC concentration, and the best method is to cut into two halves along the two sides of the long axis of the seed, and the black seed in a solution with a concentration of 0.8%TTC at 30. 8 hours of dark dyeing. (2) the best seed germination bed of seed is paper bed; the difference of germination rate between different temperature treatment seeds is significant, the best germination temperature is temperature change treatment (25/30 C); light has no effect on seed germination; seeds do not have to be sterilized, the best effect is treated with sterilization water; the seed quality of different batch seeds is different, and the germination rate of seed harvest in spring is different. (3) (3) the seed of the parasitic parasitic seed is a typical recalcitrant seed, extremely sensitive to low temperature, difficult to preserve for a long time, and loses vitality in the natural state for 7 days. The best preservation method is to soak the fresh seeds treated with aseptic water to 30 min in the ABA solution, and put out in a Petri dish with 2 layers of filter paper, and then put it in a Petri dish. Then, the seeds are placed in a Petri dish with 2 layers of filter paper. The seeds were packed in a pocket at 4 degrees centigrade, and the seeds were still alive after 120 days. (4) according to the germination characteristics of the parasitic seeds, the quality of the parasitic seeds was divided into three grades. The purity and cleanliness of the seeds were not less than 75%, the moisture was not less than 50%, the weight of 100 grains was not less than 60 g, and the two germination rate was not less than 70%, and the moisture was not low. In 40%, the weight of 100 grain is not less than 50 g, the three grade germination rate is not less than 60%, the water is less than 40%, the weight of 100 grain weight is less than 50 g. (5), the best breeding method for mulberry parasitic is to collect fresh, full, fruit without pests and pests, and stick to the branches above the 1.0cm in the upper diameter of the mulberry trees for 2 years, and keep the air humidity between the mulberry and the forest at 80%. (6) (6) the seed of the parasitic parasitic seed is very sensitive to dehydration. The vigor and germination rate are significantly related to the water content. The germination rate of fresh seeds is 86% (vitality 99%). The germination rate of the fresh seeds is 86% (66%, 35.17%), the water content will fall to 24.93%, the seed vigor becomes 15%, and the germination rate is only 6%, 40H seed. When the water content was 23.47%, the vigor of the seeds was lost, only 9%, and the germination rate was 0. (7) of the Sang Jisheng seed. The microscopic and ultrastructure of the cells changed greatly during the dehydration process of the seeds. The microscopic observation showed that the cell gap gradually increased during the dehydration process and the cytoplasm and cell wall gradually separated, which resulted in the further degradation of the cells, and the ultrastructure showed dehydration. During the process, the organelles were damaged in varying degrees, the structure of mitochondria broke, the nuclear membrane became blurred, the nucleolus was degraded gradually, the starch granules and the fat body were further decomposed, and the physiological indexes of the dehydration process changed greatly. As the dehydration time increased, the content of endogenous hormones ABA, ZR, GA, and IAA decreased first and then declined. The SOD activity in the seeds decreased in general, the content of superoxide anion radical and the content of soluble protein and MDA increased with the decrease of seed water content. (8) the study on the extraction method of total RNA in different parts of the parasitic parasitic fungi showed that the extraction effect of CTAB-LiC1 method was the best, TriZol Kit Method and the modified TriZol method were not effective. (9) a transcriptional analysis was carried out on the non dehydrated parasitic seeds (CK), dehydration 16 hours (Tac-16) and 36 hours of dehydration (Tac-36) by high throughput sequencing technology. A total of 386542846 high quality sequences were obtained. The 164546 transcript (corresponding 114971 gene) was assembled by Trinity software. By comparing with the NR, UniProt, GO, KEGG pathway and COG databases, and detailed annotations for the transcripts obtained, 60695,56027 and 66389 transcripts (1FPKM) were detected in CK, Tac16 and Tac-36 samples. By comparing with CK, 2102 up-regulated and 1344 down-regulated transcripts were found. 1649 up-regulated and 2135 down-regulated transcripts were found in ac-36. These differentially expressed transcripts included genes associated with known dehydration processes, such as RD22, heat shock protein (HSP), transcription factors (MYB, WRKY, and ethylene reactive transcription factors). The results showed that HSP and ribosomal protein (ribosomal proteins) belonged to mulberry. An important gene for the early response to dehydration process in the parasitic recalcitrant seeds. The original data have been submitted to the NCBISRA database (SRA309567). This study systematically studies the biological characteristics of the seeds of the parasite, and is the first mechanism to study the dehydration sensitivity of the recalcitrant seeds of the parasitic parasite by using the transcriptional technique. The results are for the parasitism of the parasite. Gene regulation in the process of seed breeding, preservation and dehydration has important guiding significance, and broadens our understanding of gene changes during dewatering and germination of recalcitrant seeds.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S567.19
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