坛紫菜转鲎素基因研究
摘要
本研究对坛紫菜转基因育种涉及的下述三个方面进行了系统研究:坛紫菜转基因受体系统;GUS基因在坛紫菜叶状体体细胞内的瞬时表达;鲎素基因转化坛紫菜叶状体体细胞。研究结果具体如下。
(1)坛紫菜转基因受体系统研究。本研究比较了源自银口凹螺、朝鲜花冠小月螺及杂色鲍的海藻解壁酶中琼胶酶、木聚糖酶、纤维素酶的比活力,研究结果表明,银口凹螺和朝鲜花冠小月螺较之杂色鲍更适合作为应用于紫菜等红藻的海藻解壁酶的酶源生物;本研究结果表明,细胞密度、光照强度、培养液比重对坛紫菜叶状体体细胞的发育分化具有显著影响;本研究结果表明,体细胞对氯霉素很敏感,表明氯霉素可作为筛选坛紫菜转基因植株的有效选择压力。
(2)GUS基因在坛紫菜叶状体体细胞内的瞬时表达研究。以电穿孔法将质粒pBI221导入坛紫菜叶状体体细胞内,组织化学检测结果表明,电击培养72 h、6 d的转化组体细胞内GUS基因进行了瞬时表达,表达效率分别为1.1×10-6、1.3×10-6;荧光分光光度检测表明,电击培养2 d-5 d的转化组体细胞的GUS比活力都显著大于对照组的,表明GUS基因进行了瞬时表达。上述结果表明,本研究建立的坛紫菜叶状体体细胞外源基因转化体系是有效的。
(3)鲎素基因转化坛紫菜叶状体体细胞研究。本研究构建了鲎素I基因同源重组表达载体,利用电穿孔法将其导入坛紫菜叶状体体细胞。通过PCR、PCR-Southern杂交、Southern杂交等分子生物学检测方法对导入后的植株进行检测,结果表明鲎素I基因已成功整合到基因组内。本研究首次将鲎素I基因成功转入并整合到坛紫菜体细胞基因组内,Northern点杂交检测结果进一步表明鲎素I基因在RNA水平得以表达。本研究推进了紫菜转基因抗病育种的进展,并为紫菜转基因生物反应器的研究工作提供了可资借鉴的基础资料。此外,本研究结果还表明,以坛紫菜18S rDNA的460bp、1200bp的序列分别作为外源基因3’端和5’端同源重组序列,同时以家蚕的MARs序列作为增强表达序列,有利于鲎素I基因在坛紫菜叶状体体细胞内的转化、整合、表达。本研究构建的鲎素基因同源重组表达载体可用于相关大型海藻的转基因研究中。
The three contents involved in Porphyra haitanensis transgenic breeding were studied systemically: P. haitanensis transgenic receptor system; GUS gene transient expression within the P. haitanensis thallus somatic cells; The research of tachyplesin I transfer into P. haitanensis cells. Results of the study as below.
(1)Study on P. haitanensis transgenic receptor system. Firstly, special activities of three enzymes of cell wall lytic enzymes for seaweed from three species of Gastropoda were compared, the result indicates that Chlorostoma argyrostoma and Lunella coronata coreensis are more suitable as sources of cell wall lytic enzymes for seaweed for red algae as P. haitanensis than Haliotis diversicolor; Secondly, result shows that cell density, illumination intensity and specific gravity have a significant effect on the development and differentiation of P. haitanensis thallus somatic cells; Thirdly, result shows that P. haitanensis thallus somatic cells are sensitive to chloromycetin, this indicates that chloromycetin is a effective seltive pressure for transgenic P. haitanensis.
(2)Study on gus transient expression within P. haitanensis thallus somatic cells. Plasmid pBI221 was transformed into thallus somatic cells with the method of electroporation, result of histochemical assay shows that gus expressed transiently within the cells cultured for 72 h and 6 d, expression efficiency were 1.1×10-6 and 1.3×10-6 respectively; Result of quantitative enzyme assay shows that special activities of GUS extracted from transformed cells cultured for 2 d-5 d were all higher than those of controls significantly, this indicates that gus expressed transiently. The results above indicate that exogenous gene transformation system of P. haitanensis thallus somatic cells is effective.
(1)坛紫菜转基因受体系统研究。本研究比较了源自银口凹螺、朝鲜花冠小月螺及杂色鲍的海藻解壁酶中琼胶酶、木聚糖酶、纤维素酶的比活力,研究结果表明,银口凹螺和朝鲜花冠小月螺较之杂色鲍更适合作为应用于紫菜等红藻的海藻解壁酶的酶源生物;本研究结果表明,细胞密度、光照强度、培养液比重对坛紫菜叶状体体细胞的发育分化具有显著影响;本研究结果表明,体细胞对氯霉素很敏感,表明氯霉素可作为筛选坛紫菜转基因植株的有效选择压力。
(2)GUS基因在坛紫菜叶状体体细胞内的瞬时表达研究。以电穿孔法将质粒pBI221导入坛紫菜叶状体体细胞内,组织化学检测结果表明,电击培养72 h、6 d的转化组体细胞内GUS基因进行了瞬时表达,表达效率分别为1.1×10-6、1.3×10-6;荧光分光光度检测表明,电击培养2 d-5 d的转化组体细胞的GUS比活力都显著大于对照组的,表明GUS基因进行了瞬时表达。上述结果表明,本研究建立的坛紫菜叶状体体细胞外源基因转化体系是有效的。
(3)鲎素基因转化坛紫菜叶状体体细胞研究。本研究构建了鲎素I基因同源重组表达载体,利用电穿孔法将其导入坛紫菜叶状体体细胞。通过PCR、PCR-Southern杂交、Southern杂交等分子生物学检测方法对导入后的植株进行检测,结果表明鲎素I基因已成功整合到基因组内。本研究首次将鲎素I基因成功转入并整合到坛紫菜体细胞基因组内,Northern点杂交检测结果进一步表明鲎素I基因在RNA水平得以表达。本研究推进了紫菜转基因抗病育种的进展,并为紫菜转基因生物反应器的研究工作提供了可资借鉴的基础资料。此外,本研究结果还表明,以坛紫菜18S rDNA的460bp、1200bp的序列分别作为外源基因3’端和5’端同源重组序列,同时以家蚕的MARs序列作为增强表达序列,有利于鲎素I基因在坛紫菜叶状体体细胞内的转化、整合、表达。本研究构建的鲎素基因同源重组表达载体可用于相关大型海藻的转基因研究中。
The three contents involved in Porphyra haitanensis transgenic breeding were studied systemically: P. haitanensis transgenic receptor system; GUS gene transient expression within the P. haitanensis thallus somatic cells; The research of tachyplesin I transfer into P. haitanensis cells. Results of the study as below.
(1)Study on P. haitanensis transgenic receptor system. Firstly, special activities of three enzymes of cell wall lytic enzymes for seaweed from three species of Gastropoda were compared, the result indicates that Chlorostoma argyrostoma and Lunella coronata coreensis are more suitable as sources of cell wall lytic enzymes for seaweed for red algae as P. haitanensis than Haliotis diversicolor; Secondly, result shows that cell density, illumination intensity and specific gravity have a significant effect on the development and differentiation of P. haitanensis thallus somatic cells; Thirdly, result shows that P. haitanensis thallus somatic cells are sensitive to chloromycetin, this indicates that chloromycetin is a effective seltive pressure for transgenic P. haitanensis.
(2)Study on gus transient expression within P. haitanensis thallus somatic cells. Plasmid pBI221 was transformed into thallus somatic cells with the method of electroporation, result of histochemical assay shows that gus expressed transiently within the cells cultured for 72 h and 6 d, expression efficiency were 1.1×10-6 and 1.3×10-6 respectively; Result of quantitative enzyme assay shows that special activities of GUS extracted from transformed cells cultured for 2 d-5 d were all higher than those of controls significantly, this indicates that gus expressed transiently. The results above indicate that exogenous gene transformation system of P. haitanensis thallus somatic cells is effective.
引文
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