桑寄生頑拗性種子生物學(xué)特性及脫水敏感性機理研究
發(fā)布時間：2018-07-18 08:31
【摘要】：桑寄生是地位重要但不易繁育的特殊藥材,其種子為典型的頑拗性種子,失活快,脫水敏感性強。而種子作為其唯一的繁殖材料,嚴重制約其繁育。目前頑拗性種子保存研究多,但成功實例較少,其根本原因是對脫水敏感性引起的死亡機理知之甚少,而種子脫水敏感性是引起頑拗性種子死亡的關(guān)鍵科學(xué)問題。本研究從桑寄生種子生物學(xué)特性、脫水敏感性、低溫保存等方面進行系統(tǒng)的研究,實現(xiàn)桑寄生種子繁育與保存的目標;圍繞桑寄生種子脫水過程中細胞的形態(tài)結(jié)構(gòu)及抗氧化系統(tǒng)進行綜合研究,篩選脫水敏感性關(guān)鍵時期的種子進行高通量測序,挖掘與脫水敏感性相關(guān)的關(guān)鍵基因,從而揭示桑寄生頑拗性種子脫水敏感性的分子機理,不僅為研制桑寄生種子的繁育與保存關(guān)鍵技術(shù)提供理論依據(jù),也為研究其它頑拗性種子提供新的知識與思路。其主要研究結(jié)果如下:(1)影響檢測桑寄生種子生活力的因素的主次為染色時間TTC濃度染色溫度,最佳鑒定方法為沿種子長軸的兩側(cè)縱向切開成兩半,在30℃下,將種子至于濃度為0.8%TTC的溶液中黑暗染色8小時。(2)種子最佳發(fā)芽床是紙床;不同溫度處理種子發(fā)芽率之間差異顯著,最佳發(fā)芽溫度是變溫處理(25/30℃);光照對種子的萌發(fā)無影響;種子不用消毒,用滅菌水處理效果最好;不同批次的種子質(zhì)量有差異,春季采收的種子發(fā)芽率最高。(3)桑寄生種子是典型的頑拗性種子,對低溫極其敏感,長期保存困難,自然狀態(tài)下保存7天后就失去了活力。其最佳的保存方法是:將用無菌水處理過的新鮮種子浸泡到2mg/L的ABA溶液中30 min,取出放置于鋪有2層濾紙的培養(yǎng)皿內(nèi),然后用封口袋包裝好置于4℃的冰箱,120天后種子仍有活力。(4)根據(jù)桑寄生種子的萌發(fā)特性,將桑寄生種子的質(zhì)量分為三級,要求純度和凈度都為100%。一級發(fā)芽率不低于75.0%,水分不低于50.0%,百粒重不低于60.0 g;二級發(fā)芽率不低于70.0%,水分不低于40%,百粒重不低于50.0 g;三級發(fā)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為采集新鮮、飽滿、無病蟲害的果實去掉果皮,粘于2年生桑樹上部直徑大于1.0cm的枝條之上,早晚灑水,使桑林間空氣濕度保持在80%以上。(6)桑寄生種子對脫水非常敏感,其活力和發(fā)芽率與含水量顯著相關(guān)。新鮮種子的發(fā)芽率為86%(活力為99%),脫水16h后發(fā)芽率迅速下降到39%(活力為66%,含水量為35.17%),36h后含水量會下降至24.93%,種子活力變?yōu)?5%,而發(fā)芽率僅為6%,40h種子含水量為23.47%時,種子基本喪失了活力,只有9%,而發(fā)芽率為0。(7)桑寄生種子脫水過程中細胞顯微和超微結(jié)構(gòu)變化很大。顯微觀察顯示脫水過程中細胞間隙逐步增大,細胞質(zhì)和細胞壁逐漸分離,從而導(dǎo)致細胞進一步降解;超微結(jié)構(gòu)顯示脫水過程中各種細胞器受到不同程度的損傷,線粒體結(jié)構(gòu)破裂,核膜變得模糊不清,核仁逐步降解,淀粉粒和脂肪體進一步分解劣變;脫水過程中生理指標變化較大,隨著脫水處理時間的增加,內(nèi)源激素ABA、ZR、GA、IAA含量總體上呈現(xiàn)先上升再下降的變化趨勢,種子內(nèi)的SOD活性呈總體下降趨勢,超氧陰離子自由基含量、可溶性蛋白和MDA含量則隨種子含水量下降而增加。(8)對不同部位桑寄生總RNA提取方法研究表明,CTAB-LiC1法提取效果最好,TriZol試劑盒法和改良TriZol法均不能有效的去除桑寄生中的多糖和蛋白質(zhì)等雜質(zhì)。(9)利用高通量測序技術(shù),對未脫水的桑寄生種子(CK),脫水16小時(Tac-16)和脫水36小時(Tac-36)進行了轉(zhuǎn)錄組分析,一共得到386,542,846條高質(zhì)量序列。利用Trinity軟件,組裝得到164,546轉(zhuǎn)錄本(對應(yīng)114,971基因)。通過與NR,UniProt,GO,KEGG pathway和COG數(shù)據(jù)庫進行比對,并對組裝得到的轉(zhuǎn)錄本進行了詳細的注釋。在CK,Tac16和Tac-36樣本中,分別檢測到60,695,56,027和66,389轉(zhuǎn)錄本(1FPKM)。通過與CK進行比較,在Tac-16中發(fā)現(xiàn)了 2,102個上調(diào)和1,344個下調(diào)的轉(zhuǎn)錄本,而在Tac-36中發(fā)現(xiàn)了 1,649個上調(diào)和2,135個下調(diào)的轉(zhuǎn)錄本。這些差異表達的轉(zhuǎn)錄本包含一些與已知的脫水過程相關(guān)的基因,比如RD22,熱休克蛋白(HSP),轉(zhuǎn)錄因子(MYB,WRKY和乙烯反應(yīng)性轉(zhuǎn)錄因子)。研究結(jié)果顯示HSP和核糖體蛋白(ribosomal proteins)屬于桑寄生頑拗性種子早期響應(yīng)脫水過程的重要基因。原始數(shù)據(jù)已提交到NCBISRA數(shù)據(jù)庫(SRA309567)。本研究系統(tǒng)地對桑寄生種子的生物學(xué)特性進行研究,也是第一次利用轉(zhuǎn)錄組技術(shù)研究桑寄生頑拗性種子脫水敏感性的機理,所得結(jié)果對于桑寄生種子的繁育、保存及脫水過程中的基因調(diào)控具有重要的指導(dǎo)意義,并拓寬了我們對于頑拗性種子脫水及萌發(fā)過程中基因變化的認識。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S567.19
本文編號：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S567.19
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