抗菌肽基因misgurin同向串连表达载体构建新策略

目的是将抗菌肽基因misgurin以同向串连方式连接成多拷贝基因,并克隆到甲醇酵母表达载体上。在设计和连接中采用新策略,运用常规分子克隆操作进行表达载体构建,结果表明,该策略能方便高效地进行基因多拷贝同向串连表达载体的构建。     

酿酒酵母基因编辑技术研究进展

酿酒酵母Saccharomyces cerevisiae是代谢工程中最重要的宿主之一,先进的基因编辑技术已经被广泛应用于酿酒酵母细胞工厂的设计和构建。随着基因编辑技术的飞速发展,早期基于重组酶和同源重组的基因编辑技术逐渐被新型基因编辑系统所替代。文中对酿酒酵母基因编辑技术的原理和应用进行了总结,包括经典的酿酒酵母基因编辑技术,基于核酸内切酶的MegNs、ZFNs和TALENs等基因组编辑系统,最后介绍和讨论了基于CRISPR/Cas系统、异源代谢途径多拷贝整合和基因组规模基因编辑的最新研究进展,并对酿酒酵母基因编辑技术的应用前景和发展方向进行了展望。     

融合有酸性片段的抗菌肽串连重复基因的构建

为提高抗菌肽的表达,设计在抗菌肽基因的N端融合一段编码酸性肽的片段以及减轻表达产物对宿主的毒性,通过含有酶切位点的接头将该融合肽基因以同向串连的方式连接成多拷贝基因,克隆至pUC19载体。为此,分段设计合成了编码天蚕素A-蜂毒素杂合肽和酸性肽的DNA片段。首先将其连接成融合肽全基因,然后分别与含相同粘性末端的前后接头连接。通过控制基因和接头加入的量及次序,可得到两侧有EcoRI和SalI酶切位点的同向串连的多拷贝基因。选取合适拷贝数的基因,将其克隆至pUC19载体,PCR扩增和DNA测序证明多拷贝基因构建成功且基因方向相同。结果表明,该方法能简捷高效地获得所需的多拷贝基因,为提高表达产物的量打下基因。     

家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定

目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

Catabolic Rate of Body Protein in Adult Rat over an Entire Period of Protein Depletion

In order to clarify the magnitude of different labile body proteins and the over-all catabolizable body protein, the catabolic rate of total body nitrogen in adult rat was measured by nitrogen balance method up to the time of death due to protein depletion.

The more labile body protein having 83.3% of fractional catabolic rate per day and occupying 2.8% of the whole body protein mainly represented the so-called protein reserves at the beginning of protein depletion. The less labile one, the remainder, namely 97.2%, having 0.79% of fractional catabolic rate almost wholly represented the exponential decrease of body protein during the first 40 days of protein depletion. Urinary and fecal nitrogen in this period showed a similar exponential decrease. In the next 40 days of the depletion, body protein decreased almost linearly giving the constant excretions of urinary and fecal nitrogen. In the last 40 days, it decreased drastically accompanied by a remarkable increase in urinary nitrogen.

After 118 days, on the average, the animals died of protein depletion at 35.7% level of the initial body nitrogen. Contributions of various organs to the total nitrogen deficit up to the time of death, were considerably different in different organs, where muscle was the greatest in total amount but with less catabolizability than the viscera, such as liver, pancreas and spleen.     

GUS基因拷贝数对转基因在受体植物烟草中表达的影响

GUS基因拷贝数对转基因在受体植物烟草中表达的影响