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Fusion of Protoplasts from Wild Type of Bangia atropurpurea and Green Type of Porphyra tenera

野生型紫菜原生质体与绿色紫菜原生质体的融合

基本信息

批准号：
04660203
负责人：
ARAKI Toshiyoshi
金额：
$ 1.28万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660203/
关键词：
Bangia atropurpurea Protoplast beta-1, 4-Mannanase beta-1, 3-Xylanase Porphyranase ポルフィラナーゼ

项目摘要

Three kinds of enzymes, beta-1, 4-mannanase, beta-1, 3-xylanase, and agarase, required for isolation of protoplats from a red alga, Bangia atropurpurea, were prepared from bacterial culture fluids of Vibrio sp. MA-138, Alcaligenes sp. XY-234, and Vibrio sp. P0-303, isolated from sea environment. The optimal pHs of all the crude enzymes were around 7.5. The suitable condition for protoplast isolation was examined. The treatment of the fronds with papain solution (20 mM MES buffer, pH 7.5, containing 2 % papain and 0.5 M mannitol) have contributed to isolatating protoplasts. When razorcut fragments of the fronds (about 200 mg in weight) dipped in the cell wall-digestive enzyme solution (1 unit each of 1, 4-beta-mannanase, 1, 3-beta-xylanase, and agarase, and 0.5 M mannitol in 20 mM MES buffer, pH 7.5) were incubated at 17 ﾟC for 90 min with gentle agitation, 7.1 x 10^6 of protoplasts were released from them.For carrying out cell fusion polyethylen glycol (PEG) was added into the suspension of protoplasts from the wild type of B.atropurpurea and the green varient of Porphyra tenera. Homo- and hetero-plasmic adhesion of protoplasts were observed. The PEG-treated protoplasts were cultured in artificial light of about 48 muEm^<-2>s^<-1> on a 9 h light/15 h dark cycle at 17 ﾟC.They grew into calli and plantrets of brown or green color after 6-week culture. A pair of protoplast which parts of their menbrans adhesed also developed into plantlets.

从紫云弧菌(Vibrio sp.)的细菌培养液中制备了分离赤潮藻原生质体所需的3种酶：β-1，4-甘露聚糖酶、β-1，3-木聚糖酶和琼脂酶。Ma-138，产碱菌属。XY-234和弧菌属(Vibrio sp.)P0-303，与海洋环境分离。所有粗酶的最适pH值均在7.5左右。对原生质体分离的适宜条件进行了考察。用木瓜蛋白酶溶液(20 mM MES缓冲液，pH 7.5，含2%木瓜酶和0.5M甘露醇)处理有利于分离原生质体。用细胞壁消化酶溶液(1，4-β-甘露聚糖酶、1，3-β-木聚糖酶和琼脂酶各1个单位，0.5M甘露醇在20 mM MES缓冲液中，pH 7.5)在17ﾟC温和搅拌90min，释放出7.1×10~(-6)的原生质体，以进行细胞融合。观察到原生质体的同质和异质粘连。经聚乙二醇化处理的原生质体在约48微米的人工光照下培养，光照周期为9h/15h，暗周期为17ﾟC，培养6周后长成棕色或绿色的愈伤组织和植物体。它们膜的一部分粘着的一对原生质体也发育成了植株。