An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica*

Zhongyi ZHANG, Hang LIU, Xiaohui PAN, Yanan ZONG, Leili FENG, Lixian LIU,Li GUO,**, Guanpin YANG,2,3,**

1 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

2 Institutes of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

3 Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003,China

Abstract 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.

Keyword: Nannochloropsis oceanica; genetic transformation; random insertional mutant library; zeocin pretreatment; forward genetics

1 INTRODUCTION

Microalgae have shown great potential as feedstocks for the production of biofuels and chemical intermediates (Dismukes et al., 2008; Li et al., 2008; Wijffels and Barbosa, 2010).In the last decade, numerous studies have made microalgal engineering one of the fastest-growing biotechnology fields.The microalgal species in the genusNannochloropsishave been considered industrial microalgae because they accumulate large quantities of neutral triacylglycerol lipid (up to 50% of dry weight) and polyunsaturated fatty acids such as eicosapentaenoic acid (Ma et al., 2014; Meng et al.,2015).Nannochloropsisspecies are unicellular and asexual (Pan et al., 2011).They range from 2 to 5 μm in size and widely habitat marine and freshwater (Pal et al., 2011; Georgianna and Mayfield,2012; Larkum et al., 2012).The developments of diverse genetic tools inNannochloropsisspecies have turned them into models for molecular biological research.Genome sequences ofNannochloropsisspecies are available (Radakovits et al., 2012; Wang et al., 2014; Guo et al., 2019).Electroporation (Kilian et al., 2011; Vieler et al.,2012), particle bombardment (Kang et al., 2015a, b),andAgrobacterium-mediated transformation (Cha et al., 2011; Beacham and Ali, 2016) have been established to deliver DNA fragments into the nuclear genome ofNannochloropsis.The established included also homologous recombination (Kilian et al.,2011), CRISPR/Cas9 (Wang et al., 2016; Verruto et al., 2018; Naduthodi et al., 2019) and transcription activator-like effector nuclease-mediated genome editing (Kurita et al., 2020) among others.These achievements should aid to promote the application and biological research ofNannochloropsisspecies including function deciphering of about 50% of the predicted but not annotated genes ofNannochloropsis.

Except for these tools, sequence isolation and function verification ofNannochloropsisgenes highly appreciate innovating a highly efficient method of creating variations, i.e., mutating a gene and tracing its position in the genome.In general,mutants are created through mutagenesis.Physical mutagens like ultraviolet light (UV) and chemical mutagens like ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) are widely used.For example, the genes involved in photosystem II and photosynthetic electron transport are cloned by screening UV-mutatedChlamydomonas reinhardtii(Levine, 1960).A large number of stable new variants with improved traits are generated through chemical mutation (Loppes, 1968; Davies and Plaskitt, 1971; Kamath et al., 2008; Anandarajah et al., 2012; Liang et al., 2017; Sivaramakrishnan and Incharoensakdi, 2017; Lee and Choi, 2018).Physical and chemical mutations are efficient in generating many mutants with (single nucleotide polymorphisms) SNPs and (insertions and deletions)Indels.However, these variations are instable due to either overlapping effects of the newly generated natural mutations or the easiness of loss due to reverse mutation.In contrast, random insertions of DNA into the genome may inactivate genes (Dent et al., 2015).The mutated genes can be identified using PCR-based screening and whole-genome resequencing (Gonzalez-Ballester et al., 2011).The mutant libraries obtained in this way have been widely used to clone genes and verify gene functions in species of a wide taxonomical range.For example, about 30 100 sequence-defined transposon insertion mutants ofPseudomonas aeruginosaare generated (Jacobs et al., 2003).TheAgrobacterium-mediated transformation has made TDNA insertions a viable method of genome-wide mutagenesis of high plants.A population of 60 480 T-DNA insertion lines ofArabidopsisis created aiming to establish a saturated T-DNA insertion mutant library (Krysan et al., 1999).Several insertional mutant libraries ofChlamydomonasreinhardtiihave been established for its genetic studies (Li et al.,2016, 2019; Cheng et al., 2017).Unfortunately,exogenous gene insertion heavily relies on insertion efficiency, which seems to be species dependent.

In a previous study (Zhang et al., 2022), we developed a high-efficiency transformation method forNannochloropsisoceanica, in which the transformation efficiency was significantly elevated by at least one order of magnitude when the parental algal cells were pretreated with zeocin.In this study,we attempted to use the method to construct a random insertional mutant library ofN.oceanicaand verified its feasibility in isolating new genes.This method may offer a new tool for obtaining random insertion mutants in species with low transformation efficiency.N.oceanicacells are treated ahead of transformation with zeocin in low concentration and then transformed with the insertion cassette (IC) that contains a selection marker and its expression-controlling elements.Double-strand DNA breaks (DSBs) caused by zeocin provide insertion sites for the IC, thus significantly improving the transformation efficiency,while the IC provides a marker for mutant screening and serves as a tag of mutation position in the genome.

2 MATERIAL AND METHOD

2.1 Strain and cultivation condition

The microalgal strain used in this study,N.oceanicaLAMB2011, was obtained from the Key Laboratory of Mariculture of Chinese Ministry of Education, Ocean University of China.The microalga was cultivated in f/2 liquid medium(Guillard, 1975) prepared with filtrated seawater and autoclaved at 121 °C for 20 min.The microalgal cells were cultivated in 250-mL Erlenmeyer flasks containing 100-mL liquid medium at 25 °C under 70-μmol/(m2·s) light intensity in 12-h light：12-h dark scheme.The solid f/2 medium was prepared by supplementing 1% (w/v) agar (Solarbio, China).After autoclaving, the medium was cooled to 60 °C and mixed with 400-μg/mL hygromycin B before distribution into Petri dishes.Algae-containing plates were incubated at 25 °C under a continuous irradiance of 70 μmol photons/(m2·s).

2.2 Insertion cassette

The plasmid pSELECT100 was used, which carries hygromycin B resistance geneaph7fromStreptomyceshygroscopicusunder the control of an endogenous promoter of lipid droplet surface protein (LDSP) gene.The detailed evolvement of pSELECT100 can be referred to the reported (Vieler et al., 2012).The plasmid was linearized before transformation withXmnI.

2.3 Zeocin pretreatment and transformation of N.oceanica

Nannochloropsisoceanicacells at the midlogarithm growth phase were incubated at the final cell density of ～2×106cells/mL in Erlenmeyer flasks containing fresh f/2 liquid medium with 0.2-μg/mL zeocin, and cultivated at 25 °C, 70 μmol photons/(m2·s)in 12-h light：12-h dark scheme in shaking incubator at speed of 125 r/min.After 48 h, cells were collected by centrifugation and used for transformation.

The electroporation method was used to generate random insertional mutants ofN.oceanica.Electroporation was performed according to published procedures (Vieler et al., 2012) with some modifications.Briefly, cells treated with zeocin were collected by centrifugation at 5 000×gand 4 °C for 10 min.Ice-cooled 375-mmol/L D-sorbitol(Solarbio, China) was used to wash the pellet, and then the cells were resuspended to a density of ～1×109cells/mL in ice-cooled 375-mmol/L D-sorbitol.A 2-mm cuvette with 200-μL cells and 1-μg linearized DNA was used for each electroporation.Electroporation was performed on a Bio-Rad GenePulser II system with the parameters adjusted to 600-Ohm shunt resistance, 50-μF capacitance,and 11 000-V/cm field strength.After the pulse, cells were resuspended in 5-mL ice-cooled f/2 medium and allowed to recover for 48 h in low light with shaking (120 r/min).The cells were collected by centrifugation at 7 000×gand 4 °C for 5 min and spread on agar containing 400-μg/mL hygromycin B.

2.4 Molecular analysis of transformants

ResistantN.oceanicacolonies were lysed to verify the existence of IC through PCR as described early (Zhang et al., 2022).In brief, cells in 1-mL suspensions were collected by centrifugation at 12 000 r/min for 10 min, which were then resuspended in 15-μL sterile and distilled water(SDW), mixed with 5-μL 1-mol/L NaOH, and left the mixture static overnight.The mixture was heated at 99 °C for 3 min, neutralized with 4-μL 1-mol/L HCL, stabilized with 5-μL SDW and 5-μL 1-mol/L Tris HCL (pH 8.0), heated at 99 °C for 3 min and centrifuged briefly.The upper liquid was diluted for PCR amplification of a fragment (205 bp in length)of the selection marker geneaph7with primers 5′-CGCGCTACTTCGAGCGGAGG-3′ (forward) and 5′-GCGCTTCTGCGGGCGATTTG-3′ (reverse).

2.5 Construction of the random insertion mutant library and mutant screening

PCR-positive resistant strains were collected and used to construct a random insertion mutant library.Mutant strains were maintained in 24-well plates.The microalgal growth curves were drawn using OD750easured.N.oceanicamutants were inoculated into 100-mL flasks containing 20-mL f/2 medium at an initial OD750around 0.005, and wild-typeN.oceanicawas inoculated using the same method as the control.The OD750values of each strain were measured on the 5thand 7thdays, and the ΔOD750(the difference of OD between the 7thand 5thdays) was used to indicate the growth rate.Flow cytometry was used to evaluate the difference in cell size between wild type (WT) and mutants.The magnitude of the forward scattered (FSC) light is positively correlated with cell size.Cells in mid-log phases were washed with phosphate-buffered saline(PBS) and diluted to a defined cell concentration of 1×106cells/mL.The FSC was quantified by using the BD FACSAria III Flow Cytometer (BD Biosciences, USA) or CytoFLEX Flow Cytometer(Beckman Coulter, USA) equipped with a 488-nm laser.For standard fluorescence analyses, a minimum of 10 000 cells were screened.Total lipids were extracted from about 100 mg of lyophilized biomass with the chloroform-methanol solvent mixture (2：1, v/v) using a procedure described previously by Bligh and Dyer (1959).

2.6 DNA extraction and resequencing

Total DNA was extracted from cell pellets using HP Plant DNA Kit (OMEGA Bio-tek, USA) as per the manufacturer’s instructions.The DNA fragments about 350 bp in length were obtained by interrupting the total DNA with a Covaris focusedultrasonicator (Covaris, USA).The sequencing library was constructed by using Vazyme NDM607-01 (Vazyme-Biotech, China).The DNA fragments were end-polished, A-tailed, and ligated using the full-length adapters for Illumina sequencing, with further PCR amplification.All libraries were sequenced on the Hiseq 2000 platform.High-quality clean reads were mapped onto IC and assembled using software Spades (local assembling).The assembled sequence was aligned into the reference genome (GenBank accession numbers CP038106-CP03813; Guo et al., 2019) using software BLASTN to identify the insertion site.

2.7 Targeted gene knockdown with antisense RNA

The targeted gene was knocked down using antisense RNA.UsingN.oceanicagenomic DNA as templates, violaxanthin/chlorophylla-binding protein 1 (VCP1) promoter (Wang et al., 2016;Matsui et al., 2022) and lipid droplet surface protein(LDSP) terminator (Poliner et al., 2018) were amplified using a set of primers (Table 1) with PrimeSTAR HS Phusion High-Fidelity DNA polymerase (TaKaRa, Japan).A hygromycin B resistance cassette was also amplified using pSLECT100 as the template.Total RNA was isolated fromN.oceanicausing Trizol reagent(Invitrogen, USA).A total of 1-μg RNA ofN.oceanicawas reversely transcribed to cDNA using Evo M-MLV II (Accurate Biotechnology, China)according to the manufacturer’s instructions.Using the total cDNA as templates, a fragment of theC36p1gene was amplified using a set of primers.These DNA fragments were purified using Omega Gel Extraction Kit (Omega Bio-Tek, USA),and ligated to construct the plasmid pCEZero::Hygr-Pvcp1-C36p1-Tldsp using Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyma,China).The fragment ofC36p1was ligated in the antisense orientation into the site between the VCP1 promoter and LDSP terminator.Using the pCEZero::Hygr-Pvcp1-C36p1-Tldsp as the template, therecombinant fragments used for transformation were amplified with primers Hygr F (forward) and Tldsp R (reverse).

Table 1 Primers used to construct plasmid carrying antisense RNA

2.8 Quantitative real-time PCR

One microgram of total RNA in each sample was reversely transcribed into cDNA using Evo M-MLV II (Accurate Biotechnology, China) according to the manufacturer’s instructions.The cDNA was diluted to 10 ng/μL in SDW for mRNA abundance quantification.Quantitative real-time PCR was performed on the ABI StepOne system (Applied Biosystems, USA) with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA)according to the manual, and the data were collected and analyzed by the manager software.Housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control.Primers 5′-GTGTTGATCGCGGCAAAG-3′(forward) and 5′-AGCCTGGGAGGTAAGAGGG-3′ (reverse) were used to amplify a fragment of theC36p1gene.Primers 5′-TGACTTCATCACGGAC TCC-3′ (forward) and 5′-GTCATACCACGACAC CAGC-3′ (reverse) were used to amplify a fragment of the GAPDH gene.All assays were performed three times, and the reaction without reverse transcriptase was used as the negative control.

3 RESULT

3.1 Generation of a random insertional mutant library

The whole procedure is outlined in Fig.1.Mutants were obtained by electroporatingN.oceanicacells pretreated with 0.2-μg/mL zeocin.More than 1.5×103N.oceanicatransformants can be obtained from 1 μg of linearized DNA and ～2×108parental cells, corresponding to a transformation efficiency of ～7.5×10-6(Zhang et al., 2022).Compared with transformation using WT cells, the transformation efficiency was significantly elevated by at least one order of magnitude.At last, about 2 000 PCR-positive colonies were collected and used as a random insertional mutant collection (library).

3.2 Screening and characterization of slowgrowing mutants

Fig.1 Generation of a random insertional mutant library of N.oceanica

A strain named F4-3 showed the minimum growth rate in the mutant library.F4-3 had very low biomass in 24-well plates compared with others under the same cultivation condition.At the same initial inoculation concentration, the WT cells declined in 30 days while F4-3 cells still grew well(Fig.2a).The growth curve showed the growth difference between WT and F4-3 (Fig.2b).Flow cytometry was used to evaluate the difference in cell size between WT and F4-3.The magnitude of the forward scattered (FSC) light is positively correlated with cell size.As shown in Fig.2c, the FSC-A of F4-3 cells peaked at the right of WT cells, indicating that F4-3 cells are larger on average than WT cells.It was also found that the total lipid content of F4-3 was significantly decreased (Fig.2d).

3.3 Identification of the cassette insertion site

The existence of the IC in F4-3 was verified through PCR and resequencing.A PCR product,205 bp in length, was amplified from F4-3, but not from WT cells using primers specific for IC,indicating that IC has been integrated into the F4-3 genome successfully (Fig.3b).

We sequenced the genome of F4-3.In total, 2.69-G clean base (147 bp in average length, ～84-fold of the genome) was generated, and 18 617 632 clean reads were obtained.About 96.73% of the clean reads can be mapped onto the reference genome.F4-3 had 188 InDels in the coding sequence (CDS),which was more than that in mutant transformed from WT cells (146 InDels in CDS).According to the local assembling results, a single copy of IC was integrated at position 488 of chromosome 21(GenBank No.CP038119.1), and 617 bp upstream of the initiation codon of a hypothetical proteincoding geneC36p1(evm.model.Contig36_pilon.1).TheC36p1gene has two exons (Fig.3a) and encodes a protein of 96 amino acids.However, no functional information about this protein and its coding gene can be found in the database.

3.4 Knockdown of C36p1 gene in wild N.oceanica

Fig.2 The growth status of F4-3 and others in 24-well plates and flasks (a); the growth curve of WT and two mutants (b); the forward scatter area (FSC-A) density plot of WT and F4-3 cells (c); and the total lipid content of WT and F4-3 (d)

Fig.3 Schematic of the insertion site of IC in the genome of F4-3 (a) and PCR verification of F4-3 and WT cells using primers specific for the IC (205 bp) (b)

Since mutant F4-3 showed a significant difference from WT, we selected this mutant to further confirm the connection between its genotype and phenotype.IC insertion site was very close to theC36p1gene,which may affect the transcription of theC36p1gene and then change the phenotype of F4-3.The influence on the transcription of theC36p1gene was verified by knocking down the transcription of theC36p1gene.A fragment of theC36p1open reading frame (ORF) was amplified fromN.oceanicacDNA, inserted into pCEZero::Hygr-Pvcp1-C36p1-Tldsp (Fig.4a), and oriented to synthesize an antisense RNA of theC36p1gene.The transcript of this fragment will anneal with the transcript of theC36p1gene, forming a doublestrand RNA that hinders the protein translation either directly or via RNA interference.

The fragments used to construct the plasmid pCEZero::Hygr-Pvcp1-C36p1-Tldsp were shown in Fig.4b.The recombinant plasmid was used to transform wild-typeN.oceanica.Eight positive mutants were screened out by PCR.We examined the transcription level of theC36p1gene in these mutants and wild-typeN.oceanicathrough qPCR.Due to the possible position effect of integration,3 showed downregulation of theC36p1gene expression among 8 transformants with C36p1Ri6 as the most downregulated (Fig.4c).Compared with the WT, the growth rate of C36p1Ri6 also decreased, but it grew faster than F4-3 (Fig.2b).C36p1Ri6 also showed some changes in cell size(Fig.4d).These findings partially verified the function of theC36p1gene.To some extents, it may control the growth rate ofN.oceanicaand its cell size and lipid content.

4 DISCUSSION

As one of the best ways of determining gene function, random insertional mutant libraries have been constructed in some model organisms includingN.oceanica.For example, a random insertional mutant library ofN.oceanicawas constructed by inserting a transposition complex Tn5 and used to obtain mutants with altered phenotypes in the accumulation of intracellular lipids (Osorio et al., 2019).High-lightresistance1(HLR1) gene was cloned by isolating anN.oceanicamutant that was able to grow under high irradiance(Lu et al., 2021).Several novel genes associated with an elevated neutral lipid content were discovered by constructing an insertional mutation library ofN.oceanicaand selecting lipid-rich mutants (Südfeld et al., 2021).However, this method can only be used in species with high transformation efficiency.A large number of mutants ensure the coverage of mutation sites in the genome.The efficient development of random insertional mutant libraries will facilitate the studies of gene function.TakingN.oceanicaas the subject,we tried to establish an efficient construction method of the random insertional mutant library forN.oceanica.

Zeocin is a member of the bleomycin family of antibiotics, which causes double-strand DNA breaks(Gatignol et al., 1988).It can induce DSBs inC.reinhardtiiCW15, and the abundance of DSBs induced by 100-μg/mL zeocin is similar to that induced by 250 Gy of gamma-ray irradiation(Chankova et al., 2007).It has been approved that mutatingN.oceanicawith zeocin is effective (Lin et al., 2017) and it caused abundant InDels in the genome ofN.oceanica(Liang et al., 2019).DNA damage repairing mechanisms will join the broken ends of DNA, during which introduce free DNA into the broken site or generate nucleotide insertion and deletion (Liu et al., 2022).Our previous study has proved that pretreatment ofN.oceanicawith 0.2-μg/mL zeocin can improve the transformation efficiency significantly (Zhang et al., 2022).A library containing 2 000 mutants was obtained using this method and one mutant in low growth rate, big cell size, and low lipid content was screened.According to the location of IC, we found that theC36p1gene was potentially the mutated gene determining the phenotype of F4-3.We chose to use antisense RNA to verify the gene function.TheC36p1gene knockdown strain C36p1Ri6 grew more slowly than WT, but faster than F4-3.The reason for this phenomenon may be the position effect of foreign gene insertion.The cell size of the C36p1Ri6 strain also showed some variation.Accordingly, we speculated that theC36p1gene partially controlled the phenotype of F4-3.However,methods to verify the gene function such as homologous recombination and CRISPR-Cas9 are more direct and reliable than antisense RNA.The decrease in total lipid content may relate to the changes in cell size, the larger the cell size, the lower the content of cell membrane lipids.

In our previous study, we verified that the effects of zeocin treatment on the number of InDels and SNPs and the stability of mutants among others are less (Zhang et al., 2022).In this study, we tried to use zeocin pretreatment to construct a random insertional mutant library ofN.oceanica.Our results evidenced that mutants with changed traits can be created.We believe this method can be used to establish a random insertional mutant library for diverse microalgal species, especially those with low transformation efficiencies.

5 DATA AVAILABILITY STATEMENT

The data of this study are available from the corresponding author upon request.

6 ACKNOWLEDGMENT

The authors highly appreciate the valuable comments from the anonymous reviewers for their constructive comments and suggestions.