Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize...Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.展开更多
文摘红藻真江蓠(Gracilaria vermiculophylla)是西北太平洋地区特有种,但在过去100年间它借助海运(太平洋牡蛎养殖)快速入侵到北美、欧洲和地中海等沿海栖息地,对当地的生物多样性、海洋环境和生态系统等造成重大影响。为从分子水平初步了解真江蓠成功入侵的潜在机制,文章对其入侵起源地——日本北部的真江蓠及非入侵种——绳状龙须菜(Gracilariopsis chorda)进行了同质园实验(common garden experiment)处理后的比较转录组研究,以探究该地区入侵属性不同的两种红藻间的基因表达差异。结果表明,真江蓠和绳状龙须菜共有基因序列集(Universal Gene,unigene)主要集中在核糖体、嘌呤和嘧啶代谢等通路。其中,在真江蓠中光系统II反应中心蛋白D1(photosystem II reaction center protein D1)、细胞色素P450单加氧酶(cytochrome P450 monooxygenase)和核酮糖1,5二磷酸羧化酶大亚基(Ribulose bisphosphate carboxylase large subunit,rbcL)等基因的表达量显著上调,而逆转录转座子蛋白(retrotransposon protein)、细胞壁相关的水解酶(cell wall-associated hydrolase)和金属离子转运蛋白Nramp5的表达既上调也下调。与光合作用过程相关基因的大量表达可能有助于真江蓠应对逆境胁迫,特别是光系统ⅡD1反应中心蛋白表达量升高可能有助于藻体修复光系统Ⅱ复合体,从而制造更多的有机物以备藻体生长所需。而金属离子转运蛋白Nramp5等的上调和下调则表明江蓠等红藻可能通过某些基因表达量的增减对不同的环境变动作出响应。总体而言,代谢过程中的资源再分配很可能是驱动真江蓠适应和耐受新的生境的主要分子机制。
基金Shanghai Science and Technology Commission’s“Belt and Road Initiative”International Cooperation Project,China(No.19410741800)。
文摘Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.
