非特异性脂质转移蛋白(non-specific lipid transfer protein,nsLTPs)广泛存在于植物界中,参与许多关键的生物学过程,如花粉的发育、种子发育及细胞壁的延伸等。OsLTPL166基因编码区长度是465 bp,其编码的蛋白质由154个氨基酸组成;根据...非特异性脂质转移蛋白(non-specific lipid transfer protein,nsLTPs)广泛存在于植物界中,参与许多关键的生物学过程,如花粉的发育、种子发育及细胞壁的延伸等。OsLTPL166基因编码区长度是465 bp,其编码的蛋白质由154个氨基酸组成;根据跨膜结构域及信号肽预测,OsLTPL166在13~35氨基酸处含有跨膜结构域,N端存在一个32个氨基酸残基的信号肽,切割位点位于氨基酸残基32和33之间,产生具有122个氨基酸残基的成熟蛋白质。组织表达分析发现,OsLTPL166仅在种子中特异性表达,且种子发育后期表达量较高。为了研究水稻Os LTPL166在种子发育中的功能,本研究以‘浙辐粳83’为遗传背景构建了敲除载体,利用CRISPR/Cas9技术对OsLTPL166进行定点编辑,共获得24个独立的转基因株系,其中纯合突变株系有7株,编辑方式主要为碱基的插入和缺失,导致氨基酸序列发生移码,成功获得OsLTPL166功能缺陷型突变体。本研究为进一步探究该基因在种子发育进程中的生物学功能提供了遗传材料。展开更多
Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRIS...Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.展开更多
Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil.In the synthesis pathway of soybean fatty acids,the FAD2 gene family is the key gene that regulates the production of l...Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil.In the synthesis pathway of soybean fatty acids,the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid.In this study,CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression.Firstly,the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed.Then,the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation,and the mutant plants were obtained.Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out.The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties.The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.展开更多
以营养缺陷型筛选标记基因ura3为靶标,利用基于核糖核蛋白(ribonucleoprotein,RNP)的成簇规律间隔短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)...以营养缺陷型筛选标记基因ura3为靶标,利用基于核糖核蛋白(ribonucleoprotein,RNP)的成簇规律间隔短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术,对灵芝(Ganoderma lucidum)双核菌株‘沪农灵芝1号’G0119及从其分离的2个交配亲和的单核菌株L1、L2进行基因编辑,比较单双核的编辑效率;对编辑的2个单核,进行杂交,以获得ura3被编辑的双核基因位点纯合菌株。结果表明:利用CRISPR/Cas9基因编辑技术,获得G0119双核编辑株7个,经双单杂交实验鉴定,有6个G0119编辑株为L1核被编辑,1个为L2核被编辑,7个编辑株均为杂合子;获得L1、L2的单核编辑株分别为10、7个,其中L1核被编辑的效率为100%。将突变类型均为C碱基缺失的单核L1-2和L2-1编辑株以及A插入碱基的L1-5和L2-3编辑株分别进行单单杂交,获得2株ura3被破坏的双核纯合菌株。利用CRISPR/Cas9基因编辑与杂交技术靶向创制灵芝基因位点纯合菌株的方法,可为创制灵芝其他功能基因的基因位点纯合菌株提供新的方法,也可为灵芝双核菌株的基因表型分析研究提供新的途径。展开更多
基金supported by grants from the National Natural Science Foundation of China (Nos. 31872427 and 31572466)China Agriculture Research System (CARS-18)+1 种基金Chongqing Special Postdoctoral Science Foundation (XmT2018020)China Postdoctoral Science Foundation (2018M633309)
文摘Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.
基金This work was supported by the National Natural Science Foundation of China Project Nos.[31771817,31801381]National Key Research and Development Program[2019YFD1002603-1].
文摘Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil.In the synthesis pathway of soybean fatty acids,the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid.In this study,CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression.Firstly,the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed.Then,the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation,and the mutant plants were obtained.Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out.The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties.The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.
文摘以营养缺陷型筛选标记基因ura3为靶标,利用基于核糖核蛋白(ribonucleoprotein,RNP)的成簇规律间隔短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑技术,对灵芝(Ganoderma lucidum)双核菌株‘沪农灵芝1号’G0119及从其分离的2个交配亲和的单核菌株L1、L2进行基因编辑,比较单双核的编辑效率;对编辑的2个单核,进行杂交,以获得ura3被编辑的双核基因位点纯合菌株。结果表明:利用CRISPR/Cas9基因编辑技术,获得G0119双核编辑株7个,经双单杂交实验鉴定,有6个G0119编辑株为L1核被编辑,1个为L2核被编辑,7个编辑株均为杂合子;获得L1、L2的单核编辑株分别为10、7个,其中L1核被编辑的效率为100%。将突变类型均为C碱基缺失的单核L1-2和L2-1编辑株以及A插入碱基的L1-5和L2-3编辑株分别进行单单杂交,获得2株ura3被破坏的双核纯合菌株。利用CRISPR/Cas9基因编辑与杂交技术靶向创制灵芝基因位点纯合菌株的方法,可为创制灵芝其他功能基因的基因位点纯合菌株提供新的方法,也可为灵芝双核菌株的基因表型分析研究提供新的途径。
