花椰菜核型分析及单条染色体的显微分离
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摘要
花椰菜是重要的蔬菜作物，对其染色体进行核型分析及显微分离，可为花椰菜DNA文库和遗传图谱的构建以及重要基因的分离提供重要的指导作用。本文以“福州60天”花椰菜品种为材料，对以下内容进行了研究。
1．对花椰菜染色体制片技术进行优化，结果表明：
(1)酸解以60℃1N HCl解离10min左右效果较好，常温下用1N HCl解离12～16min也得到了较好的效果。
(2)根尖长度以0.5cm左右时取材较好。
(3)预处理以8-羟基喹啉、饱和对二氯苯、放线菌酮、α-溴萘处理的染色体形态比较好、染色体易于辨别，尤其以8-羟基喹啉处理的效果最好。经8-羟基喹啉处理后染色体具有分散、缢痕显示清晰、随体染色体更易于辨认等优点。
(4)经1～1.5h酶解后所制得的片子，染色体容易充分展开，着色较深，清晰度好，背景没有细胞壁和细胞质的干扰，适合于染色体显微分离。
2．分析了“福州60天”花椰菜的核型，结果表明：花椰菜体细胞染色体数2n=18，染色体绝对长度为2.05～3.90μm，第7对染色体为随体染色体，其臂比值为2.01；最长染色体与最短染色体相对长度变化范围为7.97％～15.17％，大小之间相差7.20％；最长与最短染色体的相对长度比为1.90，平均臂比为1.62；第1、7两对染色体臂比介于1.71～3.00，为近中部着丝点染色体(sm)，其余7对染色体臂比介于1.01～1.70，为中部着丝点染色体(m)；核型公式为2n=18=14m+4sm(2SAT)，染色体属2A型，属对称性核型。
3．在核型分析的基础上，对花椰菜具随体的单条染色体进行了识别，并成功地将具随体的第7号染色体和其它染色体中的任一条染色体进行了显微分离。为花椰菜染色体的微切割、微分离和微克隆奠定了基础。
Cauliflower (Brassica oleracea .vsx.botrytis L) is one of the most important vegetables. It is very practically meaningful to study karyotype and to microdissect single chromosome of cauliflower for establishing its DNA library and genetic map as well as cloning its key genes. In this paper, the cauliflower variety, "Fuzhou 60 day", was used as experimental material for following studies.
1.The experimental conditions of karyotype analysis and miscrodisscetion were optimized. The results were shown as follows:
(1)Very good chromosome samples were obtained by macerating 60癈 IN HC1 for 10 minutes, and similar effect could also be obtained by macerating IN HC1 for 12-16 minutes at room temperature.
(2)When the root length was about 0.5cm was the best time to pretreat.
(3)Good shape of chromosomes could be obtained and the chromosomes were easy to be identified by pretreating with 8-Hydroxyquinoline, a-Bromonaphthalene, Cycloheximide and p-Dichlorobenzene. And pretreatment with 8-Hydroxyquinoline could reach the best result, namely, chromosomes were fairly well-dispersed, the primary and secondary constrictions were distinct and the chromosomes with satellite were easy to be identified.
(4)For microdissection, the chromosome sample should further be macerated with a solution containing 2% cellulase and 2% pectinase at 37癈 for 1~1.5 hour, which could refrain from the background interference of cytoplasm and cell wall.
2.The analysis of karyotype of "Fuzhou 60 day" showed that: the somatic chromosome numbers were 2n=18, the chromosome with satellite was located in chromosome 7 with a arm ratio 2.01. The length of the chromosomes was between 2.05 and 3.9Q\im, the relative length was between 7.97% and 15.17%. Chromosome 1 and 7 were sub-metacentric chromosomes with arm ratio between 1.71 and 3.00, and the rest were metacenrric chromosomes with arm ratio between
1.01 and 1.70. The length ratio of the longest and shortest chromosome was less 2.0, the average arm ratio was 1.62, which indicated that the chromosome type of "Fuzhou 60 day" belonged to 2A type, a symmetrical karyotype.
3.The chromosome with satellite was identified and successfully isolated with glass needle by means of micromanipulation. One of the rest chromosomes was also microdissected. These provided a possible chance for microcloning of single chromosome in Brassica oleracea vsx.botrytis L.
1．对花椰菜染色体制片技术进行优化，结果表明：
(1)酸解以60℃1N HCl解离10min左右效果较好，常温下用1N HCl解离12～16min也得到了较好的效果。
(2)根尖长度以0.5cm左右时取材较好。
(3)预处理以8-羟基喹啉、饱和对二氯苯、放线菌酮、α-溴萘处理的染色体形态比较好、染色体易于辨别，尤其以8-羟基喹啉处理的效果最好。经8-羟基喹啉处理后染色体具有分散、缢痕显示清晰、随体染色体更易于辨认等优点。
(4)经1～1.5h酶解后所制得的片子，染色体容易充分展开，着色较深，清晰度好，背景没有细胞壁和细胞质的干扰，适合于染色体显微分离。
2．分析了“福州60天”花椰菜的核型，结果表明：花椰菜体细胞染色体数2n=18，染色体绝对长度为2.05～3.90μm，第7对染色体为随体染色体，其臂比值为2.01；最长染色体与最短染色体相对长度变化范围为7.97％～15.17％，大小之间相差7.20％；最长与最短染色体的相对长度比为1.90，平均臂比为1.62；第1、7两对染色体臂比介于1.71～3.00，为近中部着丝点染色体(sm)，其余7对染色体臂比介于1.01～1.70，为中部着丝点染色体(m)；核型公式为2n=18=14m+4sm(2SAT)，染色体属2A型，属对称性核型。
3．在核型分析的基础上，对花椰菜具随体的单条染色体进行了识别，并成功地将具随体的第7号染色体和其它染色体中的任一条染色体进行了显微分离。为花椰菜染色体的微切割、微分离和微克隆奠定了基础。
Cauliflower (Brassica oleracea .vsx.botrytis L) is one of the most important vegetables. It is very practically meaningful to study karyotype and to microdissect single chromosome of cauliflower for establishing its DNA library and genetic map as well as cloning its key genes. In this paper, the cauliflower variety, "Fuzhou 60 day", was used as experimental material for following studies.
1.The experimental conditions of karyotype analysis and miscrodisscetion were optimized. The results were shown as follows:
(1)Very good chromosome samples were obtained by macerating 60癈 IN HC1 for 10 minutes, and similar effect could also be obtained by macerating IN HC1 for 12-16 minutes at room temperature.
(2)When the root length was about 0.5cm was the best time to pretreat.
(3)Good shape of chromosomes could be obtained and the chromosomes were easy to be identified by pretreating with 8-Hydroxyquinoline, a-Bromonaphthalene, Cycloheximide and p-Dichlorobenzene. And pretreatment with 8-Hydroxyquinoline could reach the best result, namely, chromosomes were fairly well-dispersed, the primary and secondary constrictions were distinct and the chromosomes with satellite were easy to be identified.
(4)For microdissection, the chromosome sample should further be macerated with a solution containing 2% cellulase and 2% pectinase at 37癈 for 1~1.5 hour, which could refrain from the background interference of cytoplasm and cell wall.
2.The analysis of karyotype of "Fuzhou 60 day" showed that: the somatic chromosome numbers were 2n=18, the chromosome with satellite was located in chromosome 7 with a arm ratio 2.01. The length of the chromosomes was between 2.05 and 3.9Q\im, the relative length was between 7.97% and 15.17%. Chromosome 1 and 7 were sub-metacentric chromosomes with arm ratio between 1.71 and 3.00, and the rest were metacenrric chromosomes with arm ratio between
1.01 and 1.70. The length ratio of the longest and shortest chromosome was less 2.0, the average arm ratio was 1.62, which indicated that the chromosome type of "Fuzhou 60 day" belonged to 2A type, a symmetrical karyotype.
3.The chromosome with satellite was identified and successfully isolated with glass needle by means of micromanipulation. One of the rest chromosomes was also microdissected. These provided a possible chance for microcloning of single chromosome in Brassica oleracea vsx.botrytis L.
引文
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