海带EST-SSR标记开发及TPS基因的克隆和比较遗传学研究
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摘要
海带是重要大型经济海藻,也是我国人工栽培历史最长和产量最高的海藻,我国海带养殖面积、养殖产量均居世界首位。海带广泛应用于食品保健、海洋药物、化工原料、农业肥料等方面,具有较高的经济价值,是我国海水养殖的支柱产业之一。另外海带生物量巨大,是近海潮下带的重要初级生产力之一,对调节优化养殖环境具有重要的生态作用,其综合利用和可持续发展越来越受重视。
海带生活史中典型的异型世代交替现象以及孤雌生殖和无配生殖现象,也使之成为藻类学和遗传学研究的良好模式生物。自海带引入我国后,经过几十年的发展,其遗传育种、育苗及栽培技术取得了很多进展。与海带栽培技术研究相比,目前有关海带遗传多样性分析、经济性状遗传等应用基础研究还相对落后。
由于海带中多糖和酚类物质含量较高、染色体数目众多且基因组较大,海带的分子遗传学、基因研究及分子标记工作进展较为缓慢。目前,已公布的可直接应用于遗传性状研究的分子标记数量有限。在开发利用海带资源的过程中,种质问题也是是困扰其生产的一个重大问题,本研究利用海带EST数据,进行EST-SSR筛选、引物设计和验证应用,进行种质遗传多样性分析。从4099条海带属EST中(其中971条来自我们构建的两个cDNA文库,3128条从NCBI网站下载)筛选微卫星位点,设计合成63对引物,优化反应体系后,应用于12个海带属材料分析,最终开发出23对EST-SSR引物,用这23对EST-SSR引物对12个海带属材料进行了遗传多样性分析,并能较好的将不同来源的样品区分开来。这23对EST-SSR引物具有很好的实用性,可以用于海带的分子生物学研究,特别是功能基因的研究。
海藻糖是一种广泛存在于自然界中的非还原性双糖,研究表明它能提高植物抵抗温度、pH等变化的能力,是一种前景广阔的食品药品稳定剂、添加剂和植物抗逆保护剂。分离有关海藻糖合成酶的基因,应用生物工程的手段引入植物,可以提高植物抗干旱、抗盐碱的能力。本研究用同源克隆的方法,通过优化PCR反应体系,从海带、裙带菜、亨氏马尾藻等海藻中克隆了海藻糖合成酶基因TPS,其保守域长度为2727bp,并用生物信息学方法对已得到的海藻TPS基因的cDNA序列进行了比较研究,发现它们之间的同源性都高于94.4%,海带的TPS蛋白与其它各种海藻TPS蛋白的氨基酸序列同源性则都高于96.6%。将这些海藻TPS基因的DNA序列与已报道的其它几种生物(大肠杆菌、酿酒酵母、拟南芥、水稻)的TPS基因的序列(EcOtsAB, ScTPS2、AtTPS5、OsTPS)进行了比对,发现十种海藻TPS基因的序列是相对保守的,这说明他们的功能应该是相似的；其它几种生物的TPS基因中,与海藻同源性最高的是水稻和拟南芥的TPS基因,它们之间的同源性达到40%以上,同源性次之的是酵母的TPS基因,它们之间的同源性只有27%左右；最低的是与大肠杆菌之间的同源性,它们的同源性仅仅只有20%左右。这与它们的进化的等级是相同的。大肠杆菌为原核生物,在进化树的最低层,稍早于酵母的位置,所以与酵母的同源性最高,它们都在进化树的低层。而藻类和其他的高等植物在进化树的高层,所以它们之间同一个基因会出现这么大差别。由于海洋生存环境的原因,海带TPS基因(LjTPS)和其他海藻TPS基因可能是农作物抗旱耐盐遗传改良的优良基因源。将海藻TPS基因转入农作物有望在一定程度上提高作物的抗干旱、耐盐碱的能力,在农作物改良中具有良好的应用前景。
Laminaria is one of the most important seaweeds, which is the earliest cultivatedand and high-yielding algae in china. Its farming area and yield-production rank first of seaweed in the world. The main producing area of Laminaria is concentrated in northwest Pacific and Atlantic, especially in China and Japan.
Laminaria can be widely used as food, marine drugs, chemical materials and agricultural fertilizers, so it plays an important role in our aquaculture industries. In addition, with huge biomass, Laminaria contributes a large proportion of the primary productivity of the subtidal area, and improves the culture environment and prevent red tide correspondingly, therefore utilization and sustainable development research of Laminaria should be given more and more attention. Heterogenesis, parthenogenesis and apogamy in the life history of Laminaria make it a favourable model organism for genetics and algology, both academically and practically. After introduced to our country, the genetic breeding, propagation and cultivation techniques of Laminaria have made great progress. Compared with cultivation of seaweed, the researches in molecular biological levele are delayed relatively. Rich in polysaccharides and phenolic compounds, numerous chromosomes, and a huge genome, makes the progress in researches for Laminaria genes and new molecular markers more slowly. Nowadays, there are not enough recearches for molecular markers for Laminaria. Through the sequence analysis of 4099 ESTs from Laminaria (971 ESTs were generated from our two constructed cDNA libraries and 3128 ESTs were downloaded from NCBI databases) with SSRIT software (www.gramene.org/db/searches/ssrtool), we found 254 SSRs in 201 ESTs, from which 63 SSR primer-pairs were designed and then tested in optimized SSR reaction conditions using 12 Laminara DNA samples as templates. Results showed that 23 SSR primer-pairs gave good amplification patterns on most (more than 80%) of the testing samples and can be used as SSR markers. The developed 23 SSR markers were used in genetic diversity analysis of 12 selected seaweed species, which belong to red, brown and green algae respectively; the obtained result is basically similar to that from traditional classifications. The developed EST-SSR markers showed a good practicability. They will be useful in molecular biological research in Laminaria, especially for functional gene research.
Trehalose is a non-reducing disaccharide sugar, composed of two glucose units, and can be found in many organisms. It could serve as a protective agent against heat, and saline-alkali stresses. And this character makes trehalose synthase gene as the target gene for plant gene transformation to improve its drought-salinity tolerance and adverse resistance. In this study, the trehalose-6-phosphate synthase gene (TPS) was isolated by homologous cloning method from Laminaria japonica and other 9 seaweed species. The obtained ORF (open reading frame) contained 2727bp. Sequences of the obtained TPS genes from seaweeds were deposited in GenBank. All of the ten seaweed TPS genes encoded peptides of 908 amino acids and the sequences were highly conservative both in nucleotide composition (>94.4%) and in amino acid composition (>96.6%). Homology alignment for seaweed TPS and the TPS proteins from bacteria, yeast and higher plants indicated that, unlike the TPS genes from some other plants, there was no intron existed in all of the ten isolated seaweed TPS genes. And the most closely related sequences to seaweed TPS gene were those from higher plants (OsTPS and AtTPS5, about 40% similarity), whereas the most distant sequence was the one from bacteria (EcOtsAB, about 20% similarity). Based on the living environment, LyTPS and other seaweed TPS genes are considered as good candidate gene resources for genetic improvement of the drought- and salt-resitance in crops. If the seaweed TPS genes are transformed into crops, the transgenic plants are expected to increase salt/ drought resistance. That will be useful in crop improvement and with bright application furture.
海带生活史中典型的异型世代交替现象以及孤雌生殖和无配生殖现象,也使之成为藻类学和遗传学研究的良好模式生物。自海带引入我国后,经过几十年的发展,其遗传育种、育苗及栽培技术取得了很多进展。与海带栽培技术研究相比,目前有关海带遗传多样性分析、经济性状遗传等应用基础研究还相对落后。
由于海带中多糖和酚类物质含量较高、染色体数目众多且基因组较大,海带的分子遗传学、基因研究及分子标记工作进展较为缓慢。目前,已公布的可直接应用于遗传性状研究的分子标记数量有限。在开发利用海带资源的过程中,种质问题也是是困扰其生产的一个重大问题,本研究利用海带EST数据,进行EST-SSR筛选、引物设计和验证应用,进行种质遗传多样性分析。从4099条海带属EST中(其中971条来自我们构建的两个cDNA文库,3128条从NCBI网站下载)筛选微卫星位点,设计合成63对引物,优化反应体系后,应用于12个海带属材料分析,最终开发出23对EST-SSR引物,用这23对EST-SSR引物对12个海带属材料进行了遗传多样性分析,并能较好的将不同来源的样品区分开来。这23对EST-SSR引物具有很好的实用性,可以用于海带的分子生物学研究,特别是功能基因的研究。
海藻糖是一种广泛存在于自然界中的非还原性双糖,研究表明它能提高植物抵抗温度、pH等变化的能力,是一种前景广阔的食品药品稳定剂、添加剂和植物抗逆保护剂。分离有关海藻糖合成酶的基因,应用生物工程的手段引入植物,可以提高植物抗干旱、抗盐碱的能力。本研究用同源克隆的方法,通过优化PCR反应体系,从海带、裙带菜、亨氏马尾藻等海藻中克隆了海藻糖合成酶基因TPS,其保守域长度为2727bp,并用生物信息学方法对已得到的海藻TPS基因的cDNA序列进行了比较研究,发现它们之间的同源性都高于94.4%,海带的TPS蛋白与其它各种海藻TPS蛋白的氨基酸序列同源性则都高于96.6%。将这些海藻TPS基因的DNA序列与已报道的其它几种生物(大肠杆菌、酿酒酵母、拟南芥、水稻)的TPS基因的序列(EcOtsAB, ScTPS2、AtTPS5、OsTPS)进行了比对,发现十种海藻TPS基因的序列是相对保守的,这说明他们的功能应该是相似的；其它几种生物的TPS基因中,与海藻同源性最高的是水稻和拟南芥的TPS基因,它们之间的同源性达到40%以上,同源性次之的是酵母的TPS基因,它们之间的同源性只有27%左右；最低的是与大肠杆菌之间的同源性,它们的同源性仅仅只有20%左右。这与它们的进化的等级是相同的。大肠杆菌为原核生物,在进化树的最低层,稍早于酵母的位置,所以与酵母的同源性最高,它们都在进化树的低层。而藻类和其他的高等植物在进化树的高层,所以它们之间同一个基因会出现这么大差别。由于海洋生存环境的原因,海带TPS基因(LjTPS)和其他海藻TPS基因可能是农作物抗旱耐盐遗传改良的优良基因源。将海藻TPS基因转入农作物有望在一定程度上提高作物的抗干旱、耐盐碱的能力,在农作物改良中具有良好的应用前景。
Laminaria is one of the most important seaweeds, which is the earliest cultivatedand and high-yielding algae in china. Its farming area and yield-production rank first of seaweed in the world. The main producing area of Laminaria is concentrated in northwest Pacific and Atlantic, especially in China and Japan.
Laminaria can be widely used as food, marine drugs, chemical materials and agricultural fertilizers, so it plays an important role in our aquaculture industries. In addition, with huge biomass, Laminaria contributes a large proportion of the primary productivity of the subtidal area, and improves the culture environment and prevent red tide correspondingly, therefore utilization and sustainable development research of Laminaria should be given more and more attention. Heterogenesis, parthenogenesis and apogamy in the life history of Laminaria make it a favourable model organism for genetics and algology, both academically and practically. After introduced to our country, the genetic breeding, propagation and cultivation techniques of Laminaria have made great progress. Compared with cultivation of seaweed, the researches in molecular biological levele are delayed relatively. Rich in polysaccharides and phenolic compounds, numerous chromosomes, and a huge genome, makes the progress in researches for Laminaria genes and new molecular markers more slowly. Nowadays, there are not enough recearches for molecular markers for Laminaria. Through the sequence analysis of 4099 ESTs from Laminaria (971 ESTs were generated from our two constructed cDNA libraries and 3128 ESTs were downloaded from NCBI databases) with SSRIT software (www.gramene.org/db/searches/ssrtool), we found 254 SSRs in 201 ESTs, from which 63 SSR primer-pairs were designed and then tested in optimized SSR reaction conditions using 12 Laminara DNA samples as templates. Results showed that 23 SSR primer-pairs gave good amplification patterns on most (more than 80%) of the testing samples and can be used as SSR markers. The developed 23 SSR markers were used in genetic diversity analysis of 12 selected seaweed species, which belong to red, brown and green algae respectively; the obtained result is basically similar to that from traditional classifications. The developed EST-SSR markers showed a good practicability. They will be useful in molecular biological research in Laminaria, especially for functional gene research.
Trehalose is a non-reducing disaccharide sugar, composed of two glucose units, and can be found in many organisms. It could serve as a protective agent against heat, and saline-alkali stresses. And this character makes trehalose synthase gene as the target gene for plant gene transformation to improve its drought-salinity tolerance and adverse resistance. In this study, the trehalose-6-phosphate synthase gene (TPS) was isolated by homologous cloning method from Laminaria japonica and other 9 seaweed species. The obtained ORF (open reading frame) contained 2727bp. Sequences of the obtained TPS genes from seaweeds were deposited in GenBank. All of the ten seaweed TPS genes encoded peptides of 908 amino acids and the sequences were highly conservative both in nucleotide composition (>94.4%) and in amino acid composition (>96.6%). Homology alignment for seaweed TPS and the TPS proteins from bacteria, yeast and higher plants indicated that, unlike the TPS genes from some other plants, there was no intron existed in all of the ten isolated seaweed TPS genes. And the most closely related sequences to seaweed TPS gene were those from higher plants (OsTPS and AtTPS5, about 40% similarity), whereas the most distant sequence was the one from bacteria (EcOtsAB, about 20% similarity). Based on the living environment, LyTPS and other seaweed TPS genes are considered as good candidate gene resources for genetic improvement of the drought- and salt-resitance in crops. If the seaweed TPS genes are transformed into crops, the transgenic plants are expected to increase salt/ drought resistance. That will be useful in crop improvement and with bright application furture.
引文
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