CN114424803A - 一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用 - Google Patents
一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用,属于单环刺螠育种技术领域。本发明所述诱导剂包括FILa6成熟肽、双蒸水和海水,并具体限定了FILa6成熟肽的浓度为5~30μM。本发明含有FILa6成熟肽的诱导剂能显著提高单环刺螠幼虫附着变态率。实验结果表明,本发明诱导剂在诱导24h时,附着变态率为53.3%,诱导48h时,附着变态率高达93.3%,显著高于对照。
Description
技术领域
本发明属于单环刺螠育种技术领域,尤其涉及一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用。
背景技术
螠虫门(Echiura)动物是一类海洋底栖无脊椎动物。单环刺螠(Urechisunicinctus)俗称海肠,是一种分布于中国、韩国、日本和俄罗斯沿岸潮间带泥沙底质U形洞穴内的无管螠目(Xenopneusta)螠虫动物(李诺等,1995;陈宗涛,2005),也是一种新兴的海洋经济无脊椎动物,近年来的商品价格倍增,已接近甚至超过鲜海参的价格。目前,市场上的单环刺螠主要来自沿海采捕,但由于其捕捞的野蛮性和破坏性,自然种群数量受到严重威胁,因此,发展其人工育苗和养殖产业迫在眉睫。
作为沿岸生活的螠虫动物代表,单环刺螠生活史中具有典型的幼虫沉降附着和变态过程。其浮游生活的担轮幼虫历经体节幼虫,然后发育成为底栖穴居生活的蠕虫状幼虫,启动其底栖穴居生活。前期研究发现,单环刺螠由浮游生活的体节幼虫发育至底栖生活的蠕虫状幼虫持续时间长、死亡率高,是其苗种发育过程中的重要环节。因此,开展单环刺螠人工繁育,缩短单环刺螠幼虫附着、变态时间迫在眉睫。
目前,诱导单环刺螠变态主要通过投喂饵料的方式,如专利号为CN104663587A的一种缩短单环刺螠幼虫浮游期的养殖方法,通过投喂开口饵料等鞭金藻3011,达到减少单环刺螠幼虫浮游期变态附着时间的效果,总体变态成活率为50%以上;胡丽萍等研究了饵料品种、配比及投喂方式显著影响单环刺螠幼虫的变态率,混合饵料的变态率最高为21.72%(胡丽萍等.饵料种类,配比及投喂方式对单环刺螠幼虫生长和变态的影响[J].大连海洋大学学报)。然而,采用投喂饵料的方法,单环刺螠幼虫的变态率较低。所以,亟需发明一种提高单环刺螠幼虫变态效率的物质和方法。
发明内容
有鉴于此,本发明的目的在于提供一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用,通过添加由成熟肽FILa6制备的诱导剂,能够显著提高单环刺螠幼虫的变态率。
为了实现上述发明目的,本发明提供了以下技术方案:
一种单环刺螠幼虫附着变态的诱导剂,所述诱导剂包括FILa6成熟肽、双蒸水和海水;所述诱导剂中FILa6成熟肽的浓度为5~30μM。
优选的,所述FILa6成熟肽的氨基酸序列如SEQ ID No.1所示。
优选的,所述海水的盐度为25~30。
优选的,所述双蒸水与海水的体积比为7.36:3000。
本发明还提供了上述诱导剂的制备方法,包括以下步骤:将所述FILa6成熟肽和双蒸水混合,得到储存液,再与海水混合得到所述诱导剂。
优选的,所述储存液中FILa6成熟肽的浓度为2.5~15mg/mL。
本发明还提供了上述诱导剂在诱导单环刺螠幼虫附着变态中的应用。
优选的,所述诱导的方法包括,将单环刺螠幼虫加入至所述诱导剂中诱导24~48h。
优选的,所述诱导的温度为19~20℃。
相对于现有技术,本发明具有如下有益效果:
本发明含有FILa6成熟肽的诱导剂能显著提高单环刺螠幼虫附着变态率。实验结果表明,本发明诱导剂在诱导24h时,附着变态率为53.3%,诱导48h时,附着变态率高达93.3%,显著高于对照。
附图说明
图1为实验例1中本发明诱导剂和对照在24h、48h诱导单环刺螠幼虫的附着变态率。
具体实施方式
本发明提供了一种单环刺螠幼虫附着变态的诱导剂,所述诱导剂包括FILa6成熟肽、双蒸水和海水;所述诱导剂中FILa6成熟肽的浓度为5~30μM。
在本发明中,为了确定诱导剂中FILa6成熟肽的浓度,将含有浓度分别为5、10、20、30μM FILa6成熟肽的诱导剂用于诱导单环刺螠幼虫,其他条件相同。将配置好的上述诱导剂分别添加到24孔细胞培养板中。选取单环刺螠感受态幼虫10只置于上述24孔细胞培养板中,每组设置3个平行。将24孔细胞培养板漂浮于19.5℃恒温海水中,24h和48h后观察统计各组幼虫的附着变态率。若幼虫触底5s以上并出现探索底质行为视为附着,幼虫形态变化为蠕虫状,纤毛褪去,体节消失则视为变态。附着变态率=(附着变态幼虫数/总幼虫数)×100%,附着变态提高率=实验组附着变态率均值-对照组幼虫附着变态均值。具体结果见表1。
表1不同浓度诱导剂诱导单环刺螠幼虫附着变态率(%)
由表1结果可知,本发明所述诱导剂中FILa6成熟肽的浓度优选为10μM。
在本发明中,所述FILa6成熟肽的氨基酸序列为SMPQYDDFIL,如SEQ ID No.1所示。
本发明对所述FILa6成熟肽的合成没有特殊限定,采用本领域常规合成方法即可。作为一种实施方式,将FILa6成熟肽序列送至上海生工生物有限公司,对成熟肽C端进行酰胺化处理,合成具有活性的FILa6成熟肽,FILa6成熟肽的合成纯度大于98%。
在本发明中,所述海水的盐度优选为25~30,更优选为29。
在本发明中,所述双蒸水与海水的体积比优选为7.36:3000;
本发明还提供了上述诱导剂的制备方法,包括以下步骤:将所述FILa6成熟肽和双蒸水混合,得到储存液,再与海水混合得到所述诱导剂。
在本发明中,所述储存液中FILa6成熟肽的浓度优选为2.5~15mg/mL,更优选为5mg/mL。
本发明还提供了上述诱导剂在诱导单环刺螠幼虫附着变态中的应用。
在本发明中,所述诱导的方法优选包括,将单环刺螠幼虫加入至所述诱导剂中诱导24~48h。
在本发明中,所述诱导的温度优选为19~20℃,更优选为19.5℃。
本发明对未提及的原料来源没有特殊限定,采用本领域市售产品即可。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
FILa6成熟肽的合成
将FILa6成熟肽序列(SMPQYDDFIL)送至上海生工生物有限公司,对成熟肽C端进行酰胺化处理,合成具有活性的FILa6成熟肽,FILa6成熟肽的合成纯度大于98%。
实施例2
将实施例1得到的FILa6成熟肽和7.36μL双蒸水混合,得到浓度为5mg/mL的储存液,再与3mL盐度为29的海水混合,得到FILa6成熟肽浓度为10μM的诱导剂。
实施例3
将实施例1得到的FILa6成熟肽和7.36μL双蒸水混合,得到浓度为2.5mg/mL的储存液,再与3mL盐度为25的海水混合,得到FILa6成熟肽浓度为5μM的诱导剂。
实施例4
将实施例1得到的FILa6成熟肽和7.36μL双蒸水混合,得到浓度为15mg/mL的储存液,再与3mL盐度为30的海水混合,得到FILa6成熟肽浓度为30μM的诱导剂。
实验例1
分组:实施例2的诱导剂和对照组(含有7.36μL双蒸水和3mL盐度为29的海水)。
将实施例2的诱导剂和对照组分别添加到24孔细胞培养板中。每组设置3个平行,选取单环刺螠感受态幼虫10只置于上述24孔细胞培养板中。将24孔细胞培养板漂浮于19.5℃恒温海水中,24h和48h后观察统计各组幼虫的附着变态率。若幼虫触底5s以上并出现探索底质行为视为附着,幼虫形态变化为蠕虫状,纤毛褪去,体节消失则视为变态。附着变态率=(附着变态幼虫数/总幼虫数)×100%,附着变态提高率=实验组附着变态率均值-对照组幼虫附着变态均值。具体结果见表2。
表2本发明诱导剂和对照诱导单环刺螠幼虫附着变态率(%)
由图1和表2可知,本发明实施例2的诱导剂诱导24h后,单环刺螠幼虫的附着变态率为53.3%,对照组为6.7%,本发明实施例2比对照组高46.6%;本发明实施例2的诱导剂诱导48h后,单环刺螠幼虫的附着变态率为93.3%,对照组为56.7%,本发明实施例2的诱导率比对照组高36.6%。可见,本发明诱导剂能显著提高诱导单环刺螠幼虫的附着变态率。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国海洋大学三亚海洋研究院
<120> 一种单环刺螠幼虫附着变态的诱导剂及其制备方法和应用
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Claims (9)
1.一种单环刺螠幼虫附着变态的诱导剂,其特征在于,所述诱导剂包括FILa6成熟肽、双蒸水和海水;所述诱导剂中FILa6成熟肽的浓度为5~30μM。
2.根据权利要求1所述的诱导剂,其特征在于,所述FILa6成熟肽的氨基酸序列如SEQID No.1所示。
3.根据权利要求1所述的诱导剂,其特征在于,所述海水的盐度为25~30。
4.根据权利要求1所述的诱导剂,其特征在于,所述双蒸水与海水的体积比为7.36:3000。
5.权利要求1~4任意一项所述诱导剂的制备方法,其特征在于,包括以下步骤:将所述FILa6成熟肽和双蒸水混合,得到储存液,再与海水混合得到所述诱导剂。
6.根据权利要求5所述的制备方法,其特征在于,所述储存液中FILa6成熟肽的浓度为2.5~15mg/mL。
7.权利要求1~4任意一项所述诱导剂或权利要求5或6所述制备方法制备得到的诱导剂在诱导单环刺螠幼虫附着变态中的应用。
8.根据权利要求7所述的应用,其特征在于,所述诱导的方法包括,将单环刺螠幼虫加入至所述诱导剂中诱导24~48h。
9.根据权利要求8所述的应用,其特征在于,所述诱导的温度为19~20℃。
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Citations (9)
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CN112753627A (zh) * | 2019-11-06 | 2021-05-07 | 大连市现代农业生产发展服务中心(大连市农业科学研究院) | 一种单环刺螠苗种培育方法 |
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CN101530064A (zh) * | 2008-03-15 | 2009-09-16 | 盘锦光合水产有限公司 | 单环刺螠繁育养成方法 |
CN101878862A (zh) * | 2010-06-08 | 2010-11-10 | 大连水产学院 | 海参多肽饲料添加剂 |
CN104206829A (zh) * | 2014-08-13 | 2014-12-17 | 青岛胶南市金龙饲料有限公司 | 一种单环刺螠饲料及其制备方法 |
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CN112753627A (zh) * | 2019-11-06 | 2021-05-07 | 大连市现代农业生产发展服务中心(大连市农业科学研究院) | 一种单环刺螠苗种培育方法 |
AU2020102887A4 (en) * | 2020-10-20 | 2020-12-17 | Dalian modern agricultural production development service center | Industrial three-dimensional ecological cultivation method of stichopus japonicus and urechis unicinctus |
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