Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally leth...Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally lethal. To verify such function of Zeocin, we mutated Nannochloropsis oceanica by increasing the concentration of Zeocin in medium gradually, and isolated a N. oceanica strain(single cell culture) which survived Zeocin up to 10.0μg mL^(-1). The Zeocin-tolerant strain entered the exponential growth phase later and grew slower than the wild strain. Transcriptome profiling showed that the Zeocin-tolerant N. oceanica strain survived Zeocin mainly by adapting(heritable), rather than acclimating(plastic) to Zeocin. Hence mutating N. oceanica with Zeocin was approved effective. Meanwhile, the physiological characteristics of this Zeocin-tolerant strain were demonstrated. As we proposed, N. oceanica tolerated Zeocin by strengthening its protein degradation and antioxidation. The genes controlling cell division and cellular response to stimuli may also have played important roles in the reduction of growth and the tolerance to Zeocin. Our findings evidenced that Zeocin can serve as an appropriate mutagen of microalgae. Creating variations through mutation with Zeocin may help to study the genetic basis of the traits of this monoploidy and asexual microalga, as well as improve its production.展开更多
Zeocin can cause double strand breaks of DNA and thus may be employed as a mutagen. In this study, two strains of Nannochloropsis oceanica, the wild and the Zeocin-tolerant strains, were re-sequenced to verify such fu...Zeocin can cause double strand breaks of DNA and thus may be employed as a mutagen. In this study, two strains of Nannochloropsis oceanica, the wild and the Zeocin-tolerant strains, were re-sequenced to verify such function of Zeocin, The results showed that Zeocin can mutate the N. oceanica genome and cause the structural variation. Zeocin either swept away or selected the alleles of genes functioning in ubiquitin-mediated proteolysis, alpha-linolenic acid metabolism, ascorbate and aldarate metabolism, ribosome biogenesis, and circadian rhythm, indicating that N. oceanica may have adjusted its metabolic performances for protein, carbohydrate, and lipid, and changed its ribosome biosynthesis and living rhythm to survive in Zeocin containing medium. In addition, Zeocin caused mutation may have influenced the expression of a set of tanscription factors. It was concluded that Zeocin effectively caused the structural variation of the genome of N. oceanica, and forced the microalgae to select out the alleles of a set of genes around these variations in order to adapt to Zeocin containing medium. Further studies on the genetic basis of the phenotypic adaptation of this haploid and asexual microalga and the application of Zeocin to its genetic improvement are very important.展开更多
Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
基金funded by the National Natural Science Foundation of China (No. 31270408)National High Technology Research and Development Program (863 Program) of China (No. 2014AA022001)
文摘Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally lethal. To verify such function of Zeocin, we mutated Nannochloropsis oceanica by increasing the concentration of Zeocin in medium gradually, and isolated a N. oceanica strain(single cell culture) which survived Zeocin up to 10.0μg mL^(-1). The Zeocin-tolerant strain entered the exponential growth phase later and grew slower than the wild strain. Transcriptome profiling showed that the Zeocin-tolerant N. oceanica strain survived Zeocin mainly by adapting(heritable), rather than acclimating(plastic) to Zeocin. Hence mutating N. oceanica with Zeocin was approved effective. Meanwhile, the physiological characteristics of this Zeocin-tolerant strain were demonstrated. As we proposed, N. oceanica tolerated Zeocin by strengthening its protein degradation and antioxidation. The genes controlling cell division and cellular response to stimuli may also have played important roles in the reduction of growth and the tolerance to Zeocin. Our findings evidenced that Zeocin can serve as an appropriate mutagen of microalgae. Creating variations through mutation with Zeocin may help to study the genetic basis of the traits of this monoploidy and asexual microalga, as well as improve its production.
基金funded by the National Natural Science Foundation of China(No.31270408)the National High Technology Research and Development Program(863 Program) of China(No.2014AA022001)
文摘Zeocin can cause double strand breaks of DNA and thus may be employed as a mutagen. In this study, two strains of Nannochloropsis oceanica, the wild and the Zeocin-tolerant strains, were re-sequenced to verify such function of Zeocin, The results showed that Zeocin can mutate the N. oceanica genome and cause the structural variation. Zeocin either swept away or selected the alleles of genes functioning in ubiquitin-mediated proteolysis, alpha-linolenic acid metabolism, ascorbate and aldarate metabolism, ribosome biogenesis, and circadian rhythm, indicating that N. oceanica may have adjusted its metabolic performances for protein, carbohydrate, and lipid, and changed its ribosome biosynthesis and living rhythm to survive in Zeocin containing medium. In addition, Zeocin caused mutation may have influenced the expression of a set of tanscription factors. It was concluded that Zeocin effectively caused the structural variation of the genome of N. oceanica, and forced the microalgae to select out the alleles of a set of genes around these variations in order to adapt to Zeocin containing medium. Further studies on the genetic basis of the phenotypic adaptation of this haploid and asexual microalga and the application of Zeocin to its genetic improvement are very important.
基金the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
