龙须菜紫外诱变育种及抗逆速生优良品系的选育
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摘要
龙须菜（Gracilariopsis lemaneiformis）是我国重要的产琼胶红藻，目前已经成为我国继海带和紫菜之后的第三大栽培海藻。野生型龙须菜自然分布在我国的山东半岛的潮间带，其最适的生长温度是12-23°C。随着龙须菜养殖业的不断发展，其耐高温性、生长速率及琼胶含量仍亟待提高，以扩大其栽培地域并延长栽培时间，进一步提高产品产量和质量。因此有必要对其进行遗传改良，选育出更具应用价值的新品种。
本研究首次以龙须菜四分孢子和果孢子做为材料进行紫外线辐射诱变，以耐高温和生长速率作为主要性状筛选抗逆速生突变体，就高温胁迫对突变株与981和野生型藻体的生理和分子特性等方面的影响进行了比较分析。本文旨在探索适于龙须菜的遗传育种方法，建立一套耐高温品系的筛选方法，为选育龙须菜的高产抗逆品系，并进一步将其应用于生产打下基础。
诱变育种是龙须菜新品种选育的重要手段之一。本实验选择放散刚附着在平皿中的四分孢子（单倍体）和果孢子（二倍体）作为紫外诱变的材料，确定了其紫外诱变条件分别为20W紫外灯，照射距离为30cm，照射时间为45s和48s。建立了龙须菜四分孢子和果孢子的紫外诱变技术方法。实验中观察到了单倍体四分孢子和二倍体果孢子的紫外诱变效果的差异，四分孢子紫外诱变后，观察到细胞出现了色素凝集现象，而诱变后的果孢子没有；四分孢子紫外诱变后，经高温胁迫筛选到了6株色素突变体，而在诱变后的果孢子中没有获得色素突变体。研究结果显示，刚放散附着的四分孢子和果孢子均为单个细胞，是紫外诱变的良好材料。
本实验成功建立起了一套龙须菜耐高温藻株的筛选方法。针对龙须菜在不同发育阶段耐高温性可能会不同，设置了在其不同发育阶段都进行高温胁迫筛选。配子体的耐高温筛选分为三个阶段，分别在四分孢子发育到盘状体期（诱变2周后）、直立体期（诱变6周后）和成熟藻体（海上挂养采回）时，在培养箱中经35°C高温胁迫72h，经过三个时期筛选后最终存活的藻体，被初步确定为耐高温配子体。四分孢子体的耐高温筛选分为两个阶段，在果孢子发育到5cm长幼苗期（诱变8周后）时，采用41°C水浴高温胁迫30min，将存活的藻体经海上挂养至成熟藻体时（海上挂养采回），再经过在培养箱中35°C高温胁迫72h，经过两个时期筛选最终存活的藻体，被初步确定为耐高温四分孢子体。
通过高温筛选，初步获得了具有耐高温、生长快速等优良性状的雌配子体和四分孢子体突变株各两株，比较了突变株与981和野生型藻体在高温胁迫下的生理响应，包括它们在海上和实验室内的日平均生长速率（SGR），在32°C热胁迫下藻体内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、脯氨酸含量和热激蛋白基因（hsp70）表达等方面的变化，测定了藻体的琼胶含量。
研究结果显示，两株雌配子体在实验室内和海上的日平均生长速率与981差异不显著，但明显高于野生型，四分孢子体突变株的日平均生长速率均明显高于981和野生型，推测四分孢子体可能因为二倍体的原因，具有明显的生长优势；在热胁迫后，配子体和四分孢子突变体MDA含量和增加的幅度均明显低于野生型，SOD活性高于野生型，脯氨酸含量的累积高于野生型，hsp70基因的表达水平也显著高于野生型，显示了明显的抗逆性。这些结果进一步说明了该四株突变藻体比野生型具有更强的生长优势和耐高温性。基于这些分析结果将有助于龙须菜耐高温品系的选育。
研究发现，不同耐热性的藻体在整个热胁迫过程中各种生理和分子指标的变化趋势是一致的。热胁迫下，藻体的MDA含量一直处于升高的趋势，由于MDA是膜脂过氧化的产物，说明随着时间的延长，热胁迫导致细胞膜受到损害的程度增加。所有的藻株SOD活性在第一天内呈上升趋势，随着胁迫时间的延长活性降低。热胁迫后较短的时间内SOD活性升高，有利于保护细胞免受氧化胁迫的伤害。推测随着胁迫时间的延长，SOD酶的活性受到抑制，使细胞受损害程度增加，导致胁迫过程中MDA含量的持续升高。脯氨酸在非酶促抗氧化系统中起着重要的作用，在热胁迫后脯氨酸含量呈现先上升再下降的趋势，显示了在胁迫初期能够有效地提高藻体的抗逆性。藻体经4h高温胁迫后，hsp70基因的表达水平显著增高，说明hsp70基因快速响应，大量表达缓解高温胁迫带来的伤害，产生更强的耐热性。通过高温胁迫后藻体各项生理和分子指标的比较分析，为进一步揭示龙须菜耐高温机理提供参考，为其耐高温品系的快速选育提供依据。
Gracilariopsis lemaneiformis is a macroscopic red alga well known as the majoragarophyte in China. It becomes the third largest cultivated seaweed followingLaminaria and Porphyra in China. Gp. lemaneiformis distribute naturally along thecoast of Shandong Peninsula in China. The suitable temperature for growth of thewild population is between12-23°C. With the development of Gp. lemaneiformis, it isurgently needed to improve it's heat tolerance, growth rate and agar content in orderto extend the cultivation period, enlarge the cultivation area and increase theproductivity and quality. Therefore it is essential to improve it genetically and toselect more worthy strains.
In the present study, tetraspores and carpospores were firstly used as the materialfor Ultraviolet (UV) mutagenesis. Stress tolerance mutants were screened by hightemperature tolerance and growth rate mainly. The influence of high temperature onphysiological and molecular character of the mutants, cultivar981and wild strainswas analyzed. The aim of our research was to set up an appropriate means for geneticbreeding, to develop a suitable screening procedure for high temperature tolerantstains and to lay the foundation of breeding and application for stress tolerance andfast growth strains of Gp. lemaneiformis.
Mutagenesis breeding is one of the most important methods for the selection ofnew strains of Gp. lemaneiformis. Newly attached tetraspores and carpospores weremutated by UV irradiation. The UV mutagenesis condition was set as:20W UV light,30cm distance，45s and48s irradiation respectively. The method of tetraspores andcarpospores UV mutagenesis was set up. The differences between haploid tetrasporesand diploid carpospores after UV mutagenesis were discovered. After UVmutagenesis, the pigment aggregation was discovered in tetraspores cell while it wasnot found in carpospores. Also, six pigment mutants were isolated after heat-stresswhile there was no in carpospores. It was indicated that the newly released tetrasporesand carpospores which were single cells were the nice materials for UV mutagenesis.
A set of method for screening for high temperature tolerance mutants was set upsuccessfully. Mutants at different development stages were screened under heat shockin order to obtain mutants with constant heat-resistance. The screening ofthormo-tolerate gametophytes was divided into three phases. Heat shock of35°C inincubator for72h was imposed to tetraspores when they developed into gametophyticdiscs (2weeks after mutagenesis), germlings of gametophyte (6weeks aftermutagenesis) and mature algae (after cultivation in the sea) respectively. The survivalsafter the three screening were preliminary considered as thermo-tolerate strains. Thethormo-tolerate screening method of tetrasporophyte was optimized to two phases.When carpospores developed into5cm germlings of tetrasporophyte (8weeks aftermutagenesis), heat shock of41°C water bath for30min was carried on. When carpospores developed into mature algae (after cultivation in the sea), heat shock of35°C for72h in incubator was carried on. The survivals after the two screening phaseswere preliminary considered as thermo-tolerate strains.
Two strains of female gametophytes and tetrasporophytes each withthermo-tolerance and high growth were isolated through high temperature screeningand analyzed with cultivar981and wild strains under heat stress. The specific growthrate (SGR) were compared and malondialdehyde (MDA) contents, SuperoxideDismutase (SOD) activities, proline contents and the expressions of heat shockproteins70(hsp70) under heat stress were also compared among the mutants, cultivar981and the wild strains. The agar contents were measured.
Results demonstrated that the SGR of two female gametophytes growed and inlab and sea were similar with that of the cultivar981and higher significantly than thewild strains. While the SGR of two tetrasporophytes were higher than cultivar981and wild strains. It was inferred that tetrasporophytes may have the growth superioritybecause of diploid. The contents of MDA of gametophytes and tetrasporophytesmutants were lower than the wild, and SOD activities and hsp70expression werehigher than wild, which indicated their stress tolerance. All these may indicated thatthe mutants were more tolerant to high temperature and growed faster than the wild.The analysis process on the basis of these criteria may help to develop heat-toleranceGp. lemaneiformis cultivars in the future.
In our study, the physiological and molecular responses of different strains to heatstress were coordinate. Research revealed, MDA contents that are the ultimate productof membrane peroxidation increased during the heat stress, which indicated the cellmembrane was harmed increasingly. SOD activity in all strains increased within thefirst day and decreased thereafter gradually within the heat stress period. Increasedactivity of SOD during short-term heat stress may provide protection from oxidativestress. We deduced that with prolonged periods of heat stress, the activities of SODwere inhibited and the degree of cell damage increased, which lead to MDA contentsaccumulated significantly. Proline plays an important role in the nonenzymaticantioxidant system and it's contents increased firstly and then decreased under theheat stress. It showed proline can improve the stress tolerance of the strains at thebeginning. The expression of hsp70increased significantly after4h's heat shock. Theexcessive expression of hsp70rapidly can relieve the harm of heat stress and producehigher heat tolerance. The physiological and molecular indicators provided data forunderstanding the heat stress mechanisms of Gp. lemaneiformis, and for breeding forhigh temperature resistant strains.
本研究首次以龙须菜四分孢子和果孢子做为材料进行紫外线辐射诱变，以耐高温和生长速率作为主要性状筛选抗逆速生突变体，就高温胁迫对突变株与981和野生型藻体的生理和分子特性等方面的影响进行了比较分析。本文旨在探索适于龙须菜的遗传育种方法，建立一套耐高温品系的筛选方法，为选育龙须菜的高产抗逆品系，并进一步将其应用于生产打下基础。
诱变育种是龙须菜新品种选育的重要手段之一。本实验选择放散刚附着在平皿中的四分孢子（单倍体）和果孢子（二倍体）作为紫外诱变的材料，确定了其紫外诱变条件分别为20W紫外灯，照射距离为30cm，照射时间为45s和48s。建立了龙须菜四分孢子和果孢子的紫外诱变技术方法。实验中观察到了单倍体四分孢子和二倍体果孢子的紫外诱变效果的差异，四分孢子紫外诱变后，观察到细胞出现了色素凝集现象，而诱变后的果孢子没有；四分孢子紫外诱变后，经高温胁迫筛选到了6株色素突变体，而在诱变后的果孢子中没有获得色素突变体。研究结果显示，刚放散附着的四分孢子和果孢子均为单个细胞，是紫外诱变的良好材料。
本实验成功建立起了一套龙须菜耐高温藻株的筛选方法。针对龙须菜在不同发育阶段耐高温性可能会不同，设置了在其不同发育阶段都进行高温胁迫筛选。配子体的耐高温筛选分为三个阶段，分别在四分孢子发育到盘状体期（诱变2周后）、直立体期（诱变6周后）和成熟藻体（海上挂养采回）时，在培养箱中经35°C高温胁迫72h，经过三个时期筛选后最终存活的藻体，被初步确定为耐高温配子体。四分孢子体的耐高温筛选分为两个阶段，在果孢子发育到5cm长幼苗期（诱变8周后）时，采用41°C水浴高温胁迫30min，将存活的藻体经海上挂养至成熟藻体时（海上挂养采回），再经过在培养箱中35°C高温胁迫72h，经过两个时期筛选最终存活的藻体，被初步确定为耐高温四分孢子体。
通过高温筛选，初步获得了具有耐高温、生长快速等优良性状的雌配子体和四分孢子体突变株各两株，比较了突变株与981和野生型藻体在高温胁迫下的生理响应，包括它们在海上和实验室内的日平均生长速率（SGR），在32°C热胁迫下藻体内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、脯氨酸含量和热激蛋白基因（hsp70）表达等方面的变化，测定了藻体的琼胶含量。
研究结果显示，两株雌配子体在实验室内和海上的日平均生长速率与981差异不显著，但明显高于野生型，四分孢子体突变株的日平均生长速率均明显高于981和野生型，推测四分孢子体可能因为二倍体的原因，具有明显的生长优势；在热胁迫后，配子体和四分孢子突变体MDA含量和增加的幅度均明显低于野生型，SOD活性高于野生型，脯氨酸含量的累积高于野生型，hsp70基因的表达水平也显著高于野生型，显示了明显的抗逆性。这些结果进一步说明了该四株突变藻体比野生型具有更强的生长优势和耐高温性。基于这些分析结果将有助于龙须菜耐高温品系的选育。
研究发现，不同耐热性的藻体在整个热胁迫过程中各种生理和分子指标的变化趋势是一致的。热胁迫下，藻体的MDA含量一直处于升高的趋势，由于MDA是膜脂过氧化的产物，说明随着时间的延长，热胁迫导致细胞膜受到损害的程度增加。所有的藻株SOD活性在第一天内呈上升趋势，随着胁迫时间的延长活性降低。热胁迫后较短的时间内SOD活性升高，有利于保护细胞免受氧化胁迫的伤害。推测随着胁迫时间的延长，SOD酶的活性受到抑制，使细胞受损害程度增加，导致胁迫过程中MDA含量的持续升高。脯氨酸在非酶促抗氧化系统中起着重要的作用，在热胁迫后脯氨酸含量呈现先上升再下降的趋势，显示了在胁迫初期能够有效地提高藻体的抗逆性。藻体经4h高温胁迫后，hsp70基因的表达水平显著增高，说明hsp70基因快速响应，大量表达缓解高温胁迫带来的伤害，产生更强的耐热性。通过高温胁迫后藻体各项生理和分子指标的比较分析，为进一步揭示龙须菜耐高温机理提供参考，为其耐高温品系的快速选育提供依据。
Gracilariopsis lemaneiformis is a macroscopic red alga well known as the majoragarophyte in China. It becomes the third largest cultivated seaweed followingLaminaria and Porphyra in China. Gp. lemaneiformis distribute naturally along thecoast of Shandong Peninsula in China. The suitable temperature for growth of thewild population is between12-23°C. With the development of Gp. lemaneiformis, it isurgently needed to improve it's heat tolerance, growth rate and agar content in orderto extend the cultivation period, enlarge the cultivation area and increase theproductivity and quality. Therefore it is essential to improve it genetically and toselect more worthy strains.
In the present study, tetraspores and carpospores were firstly used as the materialfor Ultraviolet (UV) mutagenesis. Stress tolerance mutants were screened by hightemperature tolerance and growth rate mainly. The influence of high temperature onphysiological and molecular character of the mutants, cultivar981and wild strainswas analyzed. The aim of our research was to set up an appropriate means for geneticbreeding, to develop a suitable screening procedure for high temperature tolerantstains and to lay the foundation of breeding and application for stress tolerance andfast growth strains of Gp. lemaneiformis.
Mutagenesis breeding is one of the most important methods for the selection ofnew strains of Gp. lemaneiformis. Newly attached tetraspores and carpospores weremutated by UV irradiation. The UV mutagenesis condition was set as:20W UV light,30cm distance，45s and48s irradiation respectively. The method of tetraspores andcarpospores UV mutagenesis was set up. The differences between haploid tetrasporesand diploid carpospores after UV mutagenesis were discovered. After UVmutagenesis, the pigment aggregation was discovered in tetraspores cell while it wasnot found in carpospores. Also, six pigment mutants were isolated after heat-stresswhile there was no in carpospores. It was indicated that the newly released tetrasporesand carpospores which were single cells were the nice materials for UV mutagenesis.
A set of method for screening for high temperature tolerance mutants was set upsuccessfully. Mutants at different development stages were screened under heat shockin order to obtain mutants with constant heat-resistance. The screening ofthormo-tolerate gametophytes was divided into three phases. Heat shock of35°C inincubator for72h was imposed to tetraspores when they developed into gametophyticdiscs (2weeks after mutagenesis), germlings of gametophyte (6weeks aftermutagenesis) and mature algae (after cultivation in the sea) respectively. The survivalsafter the three screening were preliminary considered as thermo-tolerate strains. Thethormo-tolerate screening method of tetrasporophyte was optimized to two phases.When carpospores developed into5cm germlings of tetrasporophyte (8weeks aftermutagenesis), heat shock of41°C water bath for30min was carried on. When carpospores developed into mature algae (after cultivation in the sea), heat shock of35°C for72h in incubator was carried on. The survivals after the two screening phaseswere preliminary considered as thermo-tolerate strains.
Two strains of female gametophytes and tetrasporophytes each withthermo-tolerance and high growth were isolated through high temperature screeningand analyzed with cultivar981and wild strains under heat stress. The specific growthrate (SGR) were compared and malondialdehyde (MDA) contents, SuperoxideDismutase (SOD) activities, proline contents and the expressions of heat shockproteins70(hsp70) under heat stress were also compared among the mutants, cultivar981and the wild strains. The agar contents were measured.
Results demonstrated that the SGR of two female gametophytes growed and inlab and sea were similar with that of the cultivar981and higher significantly than thewild strains. While the SGR of two tetrasporophytes were higher than cultivar981and wild strains. It was inferred that tetrasporophytes may have the growth superioritybecause of diploid. The contents of MDA of gametophytes and tetrasporophytesmutants were lower than the wild, and SOD activities and hsp70expression werehigher than wild, which indicated their stress tolerance. All these may indicated thatthe mutants were more tolerant to high temperature and growed faster than the wild.The analysis process on the basis of these criteria may help to develop heat-toleranceGp. lemaneiformis cultivars in the future.
In our study, the physiological and molecular responses of different strains to heatstress were coordinate. Research revealed, MDA contents that are the ultimate productof membrane peroxidation increased during the heat stress, which indicated the cellmembrane was harmed increasingly. SOD activity in all strains increased within thefirst day and decreased thereafter gradually within the heat stress period. Increasedactivity of SOD during short-term heat stress may provide protection from oxidativestress. We deduced that with prolonged periods of heat stress, the activities of SODwere inhibited and the degree of cell damage increased, which lead to MDA contentsaccumulated significantly. Proline plays an important role in the nonenzymaticantioxidant system and it's contents increased firstly and then decreased under theheat stress. It showed proline can improve the stress tolerance of the strains at thebeginning. The expression of hsp70increased significantly after4h's heat shock. Theexcessive expression of hsp70rapidly can relieve the harm of heat stress and producehigher heat tolerance. The physiological and molecular indicators provided data forunderstanding the heat stress mechanisms of Gp. lemaneiformis, and for breeding forhigh temperature resistant strains.
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