蜡梅开花过程基因表达的ESTs分析与凝集素基因功能的初步鉴定
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摘要
蜡梅(Chimonanthus praecox (L．) Link)是蜡梅科(Calycanthaceae)蜡梅属(Chimonanthus Lindl．)植物，为原产我国的落叶丛生灌木。蜡梅花在12月至翌年早春2月孕蕾开花、花香宜人以及其特殊的抗逆性状令人关注。目前人们对蜡梅开花与花香形成的分子机理研究缺乏，没有从蜡梅中克隆到抗逆基因的报道，对蜡梅开花过程中的基因表达情况不清楚。本论文从构建蜡梅花cDNA文库入手，利用EST技术分析开花过程中的基因表达情况，在此基础上，克隆蜡梅凝集素基因，利用转基因手段初步分析其抗虫性。主要取得以下结果：
1．蜡梅花cDNA文库构建
用SMART cDNA文库构建试剂盒成功构建了蕾期、露瓣期、初开期、盛开期蜡梅花混合cDNA文库。原始文库滴度达到1.4×10~6pfu／ml，扩增文库滴度约为10~(10)pfu／ml，重组率达96％，插入片段在0.5 kb以上，平均约1.1kb。通过PCR检测，从扩增文库中检测到了蜡梅PI、AP3和LTP基因的特异片段，说明所构建的文库覆盖度高、代表性强。LTP基因具有内含子，而通过PCR从文库中扩增出的LTP基因不含内含子，说明所构建的cDNA文库无基因组DNA污染。这是第一次正式报道蜡梅cDNA文库的构建。
2．蜡梅花EST序列测定与拼接
从蜡梅花cDNA文库首先挑取噬菌斑克隆，然后转变为质粒，PCR筛选阳性克隆进行正向序列测定获得了896个原始序列，经SeqMan、BLAST分析后得到蜡梅花的有效EST序列867条，有效率为96.76％。全部片段长为584201bp，平均读长为673.8bp。进而转化成GenBank提交格式提交GenBank数据库，登录号从DW222667到DW223533。
利用DNASTAR软件包的SeqMan Ⅱ程序对867条有效EST序列进行序列拼接后得到479个不同的独立基因(contigs，含singlet)，冗余序列占全部序列的44.8％。
根据不同独立基因(contigs，含singlet)的EST数量统计，蜡梅开花过程低丰度表达
Calycanthaceae are a family of 10 species in three genera, Calycanthus L. with three species, Chimonanthus Lindley with six, and Idiospermum Blake with a single species. Wintersweet (Chimonanthus praecox Link) is an important species originating from China, where it is cultivated with a long history owing to its important characteristics, such as blooming in deep winter, having strong fragrance and stress resistance et al.. However, the genic expression situations of wintersweet are not yet reported. In this dissertation, we report the construction of the cDNA library from wintersweet flower, sequence and functional analysis of the expressed sequence tags (ESTs), cloning and functional identification of a lectin gene (Cplectin )from the library. The main results are as follows:
1. Construction of wintersweet flower cDNA library
A cDNA library was constructed with SMART cDNA Library Construction Kit using the RNA from the wintersweet flowers of sprout period, flower-bud period, display-petal period, initiating bloom period, bloom period and wither period. The primary library had a high titer of 1.4×10~6, in which 96% clones were recombinant and the length of insert cDNAs was over 0.5 kb. The amplified library had a titer of 10~(10). The positive signals of PI, AP3 and LTP were detected by PCR in the amplified library, although their expression abundances were different. The LTP gene from the amplified library had no intron, which showed no genomic DNA in the library. These meant that a high quality cDNA library of wintersweet flower was obtained.
2. Sequencing and assembling of expressed sequence tags (ESTs)
1．蜡梅花cDNA文库构建
用SMART cDNA文库构建试剂盒成功构建了蕾期、露瓣期、初开期、盛开期蜡梅花混合cDNA文库。原始文库滴度达到1.4×10~6pfu／ml，扩增文库滴度约为10~(10)pfu／ml，重组率达96％，插入片段在0.5 kb以上，平均约1.1kb。通过PCR检测，从扩增文库中检测到了蜡梅PI、AP3和LTP基因的特异片段，说明所构建的文库覆盖度高、代表性强。LTP基因具有内含子，而通过PCR从文库中扩增出的LTP基因不含内含子，说明所构建的cDNA文库无基因组DNA污染。这是第一次正式报道蜡梅cDNA文库的构建。
2．蜡梅花EST序列测定与拼接
从蜡梅花cDNA文库首先挑取噬菌斑克隆，然后转变为质粒，PCR筛选阳性克隆进行正向序列测定获得了896个原始序列，经SeqMan、BLAST分析后得到蜡梅花的有效EST序列867条，有效率为96.76％。全部片段长为584201bp，平均读长为673.8bp。进而转化成GenBank提交格式提交GenBank数据库，登录号从DW222667到DW223533。
利用DNASTAR软件包的SeqMan Ⅱ程序对867条有效EST序列进行序列拼接后得到479个不同的独立基因(contigs，含singlet)，冗余序列占全部序列的44.8％。
根据不同独立基因(contigs，含singlet)的EST数量统计，蜡梅开花过程低丰度表达
Calycanthaceae are a family of 10 species in three genera, Calycanthus L. with three species, Chimonanthus Lindley with six, and Idiospermum Blake with a single species. Wintersweet (Chimonanthus praecox Link) is an important species originating from China, where it is cultivated with a long history owing to its important characteristics, such as blooming in deep winter, having strong fragrance and stress resistance et al.. However, the genic expression situations of wintersweet are not yet reported. In this dissertation, we report the construction of the cDNA library from wintersweet flower, sequence and functional analysis of the expressed sequence tags (ESTs), cloning and functional identification of a lectin gene (Cplectin )from the library. The main results are as follows:
1. Construction of wintersweet flower cDNA library
A cDNA library was constructed with SMART cDNA Library Construction Kit using the RNA from the wintersweet flowers of sprout period, flower-bud period, display-petal period, initiating bloom period, bloom period and wither period. The primary library had a high titer of 1.4×10~6, in which 96% clones were recombinant and the length of insert cDNAs was over 0.5 kb. The amplified library had a titer of 10~(10). The positive signals of PI, AP3 and LTP were detected by PCR in the amplified library, although their expression abundances were different. The LTP gene from the amplified library had no intron, which showed no genomic DNA in the library. These meant that a high quality cDNA library of wintersweet flower was obtained.
2. Sequencing and assembling of expressed sequence tags (ESTs)
引文
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