Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.     

转兔角蛋白基因改良棉纤维品质研究

通过花粉管通道转基因技术,将E6启动子驱动的兔角蛋白基因导入高产棉花品种苏棉16号。所用转基因表达载体还含有选择标记基因NPTⅡ(卡那霉素抗性基因)及Gus报告基因。对转化体后代的检测结果表明,T1代有2.1%呈现Gus阳性,在Gus阳性株中84.6%具有卡那霉素抗性。用依据E6启动子序列和兔角蛋白基因序列设计的两对引物,对经过上述筛选的植株进行PCR检测,多次重复,最终确定3株结果稳定的转兔角蛋白基因棉株。从品质分析结果看,这3个株系成熟棉纤维的品质部分得到改良,尤其比强度有较大幅度提高,与转基因受体相比平均提高6.3cN·tex 1。     

Genetic issues and pitfalls in transgenic plant breeding

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional breeding. In order to effectively apply the technology to breeding, we need to understand how transgenes behave in plants. Transgenes may or may not follow Mendelian segregation; their expression can be significantly affected by integration positions and structures of the transgenic DNA in host genomes; transgenes may become unstable over generations, genetic background sand environmental conditions; and they may have significantly negative impact on expression of endogenous genes. If not well understood, the sehurdles could become significant barriers in transgenic breeding. This paper reviews some genetic issues and pitfalls that are often encountered in transgenic breeding. Because of the necessity of being brief, transgene expression, silencing, and breeding are the three areas of focuses in this discussion. While molecular mechanisms underlying many of the transgenic phenomena have not been completely understood, some practical ‘rules’ are now available for creating, evaluating and selecting desirable transgenic transformants. It can be certain that with more transgenic plants generated and characterized our knowledge of transgene genetics at both molecular and plant levels will continue to accumulate. This revised version was published online in July 2006 with corrections to the Cover Date.     

离子注入在生命科学中的应用研究

本文概述了近十几年来离子注入技术在机理研究、生物品种改良及介导外源基因转移等方面的研究进展     

粳稻转基因技术体系的研究与应用进展

水稻是我国60%以上人口的主食,在保障国家口粮安全中起着举足轻重的作用。近年来,随着人们生活水平的提高、城镇化进程的加快和消费习惯的改变,我国粳稻消费需求快速增长。但是,受耕地减少、生物与非生物因素胁迫日益严重的影响,粳稻增产的难度越来越大。基因工程技术的发展和应用为打破这一僵局提供了可能。回顾了粳稻转基因技术的发展,对粳稻转基因体系建立、体系优化、育种应用和安全转基因技术研发等方面进行了综述,并对目前粳稻转基因技术存在的问题和未来的发展进行了分析和展望。     

利用卡那霉素筛选野罂粟转基因植株的技术研究

待供试转基因和非转基因野罂粟植株长至5～7片叶时,用微型喷雾器将不同浓度的卡那霉素溶液均匀喷施于叶片上,结果显示浓度为6.5g/L的处理表现效果最好。对于大田筛选转基因野罂粟,在苗期对叶片喷施6.5g/L卡那霉素溶液喷雾,并对产生黄斑的野罂粟植株进行剔除后,拔节期用7.5g/L卡那霉素溶液连续5d涂抹后,对产生黄斑的植株进行再次剔除,最终得到30株未见黄斑的野罂粟植株,经PCR进行检测后,最终得到8株野罂粟转基因植株。     

沙冬青AmCSDP基因特性及转基因烟草的抗寒性分析

冷休克结构域蛋白(CSPs)是含有不同数量冷休克结构域(cold shock domain,CSD)的低温响应蛋白,在低温胁迫中发挥重要作用。以沙冬青(Ammopiptanthus mongolicus)为材料,克隆得到了冷休克结构域蛋白基因AmCSDP。生物信息学分析显示AmCSDP(Gen Bank登录号:KX756575)全长540bp,编码180个氨基酸,二级结构由8.9%的α–螺旋、47.8%的β–折叠和43.3%的无规则卷曲组成,其N端含有冷休克结构域。进化分析表明沙冬青与花生同源性最高。构建了AmCSDP真核表达载体Pcambia2300-35S-AmCSDP-OCS,经农杆菌介导转入本氏烟中。对转基因T1代和野生型烟草进行4℃低温胁迫处理,结果证实转基因本氏烟比野生型的耐冷能力强。     

RESEARCH PROGRESS ON GENETIC ENGINEERING OF STRESS TOLERANCE IN SUGARCANE

病虫害、干旱、低温是甘蔗生产中主要的生物和非生物胁迫因素,给甘蔗生产造成巨大损失。提高甘蔗抗逆性是甘蔗育种的重要目标。基因工程技术在作物遗传改良中应用广泛,为甘蔗抗逆育种提供了有效途径。本文概述了转基因技术在甘蔗抗病虫害、抗旱、抗寒方面的研究进展,并提出了该领域尚存在的一些问题及其应用前景展望。     

植物基因工程中存在的问题及对策

从外源基因进入植物细胞到功能的表达，存在许多不确定性，如外源基因拷贝数、结构的完整性、插入位点的选择以及整合方式。而且外源基因进入受体细胞后，与受体细胞的基因组间相互作用、相互影响从而使基因植物中存在的这些问题，近几年有了一些相应的对策，如对外源基因的密码子优化，使用从植物中克隆的具有组织与发育特异性调控作用的增强子、使外源基因带上合适的5‘端先导序列和3‘端URT区、去除标记基因、共转化系统的重新应用、多自－动转化系统的应用、筛选单拷贝转基因个体，采用Agrolistic转化法，同时对转基因与常规育种、农业资源遗传多样性的保护和环境生态平衡问题做了相应的研究。