重组人乳铁蛋白对大鼠生长发育作用的实验研究

目的 研究从转基因牛乳中分离纯化的重组人乳铁蛋白是否具有促进动物生长发育的作用.方法 给大鼠灌胃重组人乳铁蛋白共8周,设低、中、高3个剂量组(0.375,0.75和2.25 g/kg BW·d)以水为对照组.检测指标包括动物体重、身长、摄食童和食物利用率.结果 高剂量组体重、身长和食物利用率指标均显著优于对照组,低、中剂量组的体格发育指标较对照组有轻度改善.结论 在本研究条件下,重组人乳铁蛋白具有促进大鼠生长发育的作用.     

国内外紫苏研究进展概述

本文概述了紫苏在开发利用、种质资源收集评价、栽培与生理、活性物质提取、组织培养、转基因及基因克隆表达分析等方面的国内外研究进展,以期为紫苏的深入研究和进一步开发应用提供参考,有效促进紫苏产业的发展。     

Accumulative gene integration into a pre-determined site using Cre/loxP

Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.     

甜菜转基因植株抗性表现及种子获得

用农杆菌介导方法，将甜菜坏死黄脉病毒外壳蛋白（BNYVV CP）基因转化甜菜不同品系叶柄外植体。以卡那霉素作为筛选标记基因，获得抗卡那霉素再生植株。再生植株经PCR扩增，获得目的基因大小的DNA片段，初步证明为转基因植株。部分转基因TO代植株在病土中实验表明，其抗病性明显高于对照。将移栽田间所获得的转基因甜菜母根进行单株套罩采种，获得不同品系单株自交种。     

三角褐指藻LPAAT基因在酵母中表达对脂肪酸的影响

从三角褐指藻（Phaeodactylum tricornutum）中克隆到1 632bp的溶血磷脂酸酰基转移酶（LPAAT）全长cDNA,其中包含的开放阅读框被命名为PtLPAAT。在毕赤酵母中异源表达PtLPAAT基因,诱导培养45h时随机选取的3个酵母转化子总脂肪酸含量分别提高了2.68%、11.14%、31.28%;诱导培养72h时同样的转化子总脂肪酸含量分别提高11.59%、18.72%、46.63%。用薄层层析法分离诱导培养48h后的酵母菌体的甘油三酯,并用气相色谱法测定其脂肪酸,结果显示：与非转基因对照菌株相比,其油酸提高了0.9倍、亚油酸提高了5倍。用荧光定量PCR检测发现PtLPAAT基因的表达量随三角褐指藻油脂不断积累逐渐升高。表明在酵母中表达PtLPAAT基因不仅能够明显地提高其脂肪酸含量,而且还能选择性结合不饱和脂肪酸到甘油三酯中。     

Effects of BmKIT3R gene transfer on the development and survival of silkworm Bombyx mori

To verify the effects of gain-of-function mutation of the BmKIT3^R gene (from the Chinese scorpion Buthus martensii Karsch) on the development and survival rate of insects and to explore a novel strategy for pest control, the effects of BmKIT3^R gene transfer on the development and survival rate of silkworms were investigated. A novel transgenic vector derived from the piggyBac transposon with the BmKIT3^R gene controlled by the Bmhsp20.4 promoter was transferred into silkworm eggs. Transgenic silkworms were obtained after screening with GFP and G418 antibiotics and verification by PCR and dot hybridization. The results showed that the oviposition number decreased by 18.9%, and the hatching and final survival rates were approximately 63% and 47.5%, respectively. Some 18.9% of surviving pupae died before developing into moths in the G3 generation. A specific band corresponding to BmKIT3^R was detected for transgenic silkworms by Western blotting. This indicates that the Bmhsp 20.4 promoter has constitutive expression activity. The significant decrease in the survival rate suggests that pest population numbers could be effectively controlled by using BmKIT3^R gene transfer. Furthermore, it can be speculated that pupal development to moths could be blocked if BmKIT3^R were specially expressed in the pupal stage and reeling with fresh cocoons was performed.     

实时定量PCR法检测转基因CHO细胞中外源抗体轻重链基因的拷贝数

目的建立实时定量PCR法检测转基因CHO细胞中外源抗体轻重链基因的拷贝数。方法以分别带有抗体轻链和重链的质粒作为标准品,进行实时定量PCR反应,建立标准曲线。提取转染抗体轻重链基因的CHO细胞基因组DNA,进行定量PCR反应,通过标准曲线,再根据10 ng CHO基因组中含有的单拷贝基因的数量,分别计算得到抗体的轻链和重链基因在CHO细胞中的拷贝数。结果分别建立了抗体轻链和重链基因的拷贝数标准曲线,标准曲线的相关系数均在0.99以上,PCR扩增效率分别为91.6%和91.8%,具有良好的特异性。随着细胞培养代次的增加,轻链基因和重链基因的拷贝数均出现降低的现象。结论成功建立了实时定量PCR法检测转基因CHO细胞中外源抗体轻链和重链基因的拷贝数,可用于外源抗体基因在CHO细胞中的遗传稳定性研究,也为高表达细胞株的获得提供了一种检测方法。