Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

A novel hypoxic lncRNA, LOC110520012 sponges miR-206-y to regulate angiogenesis and liver cell proliferation in rainbow trout (Oncorhynchus mykiss) by targeting vegfaa.

Long non-coding RNA (lncRNA) is a novel emerging type of competitive endogenous RNA (ceRNA) that performs key functions in multiple biological processes. However, little is known about the roles of lncRNA under hypoxia stress in fish. Here, vascular endothelial growth factor-Aa (vegfaa) was cloned in rainbow trout (Oncorhynchus mykiss), with the complete cDNA sequence of 2914 bp, encoding 218 amino acids. The molecular weight of the protein was approximately 25.33 kDa, and contained PDGF and VEGF_C domains. Time-course and spatial expression patterns revealed that LOC110520012 was a key regulator of rainbow trout in response to hypoxia stress, and LOC110520012, miR-206-y and vegfaa exhibited a ceRNA regulatory relationship in liver, gill, muscle and rainbow trout liver cells treated with acute hypoxia. Subsequently, the targeting relationship of LOC110520012 and vegfaa with miR-206-y was confirmed by dual-luciferase reporter analysis, and overexpression of LOC110520012 mediated the inhibition of miR-206-y expression in rainbow trout liver cells, while the opposite results were obtained after LOC110520012 silencing with siRNA. We also proved that vegfaa was a target of miR-206-y in vitro and in vivo, and the vegfaa expression and anti-proliferative effect on rainbow trout liver cells regulated by miR-206-y mimics could be reversed by LOC110520012. These results suggested that LOC110520012 can positively regulate vegfaa expression by sponging miR-206-y under hypoxia stress in rainbow trout, which facilitate in-depth understanding of the molecular mechanisms of fish adaptation and tolerance to hypoxia.

Functional Characterization of F3H Gene and Optimization of Dihydrokaempferol Biosynthesis in Saccharomyces cerevisiae.

The 1092 bp F3H gene from Trapa bispinosa Roxb., which was named TbF3H, was cloned and it encodes 363 amino acids. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbF3H with flavanone 3-hydroxylase from other plants. A functional analysis showed that TbF3H of Trapa bispinosa Roxb. encoded a functional flavanone 3-hydroxylase; it catalyzed the formation of dihydrokaempferol (DHK) from naringenin in S. cerevisiae. The promoter strengths were compared by fluorescence microscopy and flow cytometry detection of the fluorescence intensity of the reporter genes initiated by each constitutive promoter (FITC), and DHK production reached 216.7 mg/L by the promoter adjustment strategy and the optimization of fermentation conditions. The results presented in this study will contribute to elucidating DHK biosynthesis in Trapa bispinosa Roxb.

Rainbow screening: Chromoproteins enable visualized molecular cloning.

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E. coli) colonies by naked eyes, eliminating the need for additional genetic manipulations or costs. To illustrate the design, we select both E. coli 16s rRNA and sfGFP expression module as two inserted fragments. Using Rainbow Screening, false positive colonies can be easily distinguished on LB-agar plates. Moreover, both the assembly efficiency and the construct accuracy can exceed 80%. We anticipate that Rainbow Screening will enrich the molecular cloning methodology and expand the application of chromoproteins in biotechnology and synthetic biology.

In Vivo DNA Assembly Using the PEDA Method.

Simple and efficient DNA assembly methods have been widely used in synthetic biology. Here, we provide the protocol for the recently developed PEDA (phage enzyme-assisted in vivo DNA assembly) method for direct in vivo assembly of individual DNA parts in multiple microorganisms, such as Escherichia coli, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. PEDA allows in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and the efficiency is comparable to the prevailing in vitro DNA assembly, which will broadly boost the rapid progress of synthetic biology.

Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction.

Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%. In addition, up to 6 different mutations can be introduced in one reaction without causing any fortuitous change in the vector backbone. Using this strategy, we have introduced 32 S/T➔A substitutions in the N-terminus head and 13 changes in the C-terminus tail domain of vimentin.

Molecular cloning, tissue expression pattern, responses to different fatty acids and potential functions of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in large yellow croaker (Larimichthys crocea).

In farmed fish, diets rich in palm oil have been observed to promote abnormal lipid build-up in the liver, subsequently leading to physiological harm and disease onset. Emerging research suggests that integrating phospholipids into the feed could serve as a potent countermeasure against hepatic impairments induced by vegetable oil consumption. Phosphatidylcholine is the most abundant type among phospholipids. In the metabolic processes of mammal, lysophosphatidylcholine acyltransferase 1 (LPCAT1), crucial for phosphatidylcholine remodeling, demonstrates a marked affinity towards palmitic acid (PA). Nonetheless, aspects concerning the cloning, tissue-specific distribution, and affinity of the LPCAT1 gene to diverse oil sources have yet to be elucidated in the large yellow croaker (Larimichthys crocea). Within the scope of this study, we successfully isolated and cloned the cDNA of the LPCAT1 gene from the large yellow croaker. Subsequent analysis revealed distinct gene expression patterns of LPCAT1 across ten different tissues of the species. The fully sequenced coding DNA sequence (CDS) of LPCAT1 spans 1503 bp and encodes a sequence of 500 amino acids. Comparative sequence alignment indicates that LPCAT1 shares a 69.75 % amino acid similarity with its counterparts in other species. Although LPCAT1 manifests across various tissues of the large yellow croaker, its predominance is markedly evident in the liver and gills. Furthermore, post exposure of the large yellow croaker's hepatocytes to varied fatty acids, PA has a strong response to LPCAT1. Upon the addition of appropriate lysolecithin to palm oil feed, the mRNA expression of LPCAT1 in the liver cells of the large yellow croaker showed significant variations compared to other subtypes. Concurrently, the mRNA expression of pro-inflammatory genes il-1ß, il-6, il-8, tnf-α and ifn-γ in the liver tissue of the large yellow croaker decreased. Interestingly, they exhibit the same trend of change. In conclusion, we have cloned the LPCAT1 gene on fish successfully and find the augmented gene response of LPCAT1 in hepatocytes under PA treatment first. The results of this study suggest that LPCAT1 may be associated with liver inflammation in fish and offer new insights into mitigating liver diseases in fish caused by palm oil feed.

Characterization, Expression, and Functional Analysis of the Northern Snakehead (Channa argus) Hepcidin.

Hepcidin, an antimicrobial peptide (AMP), is a well-conserved molecule present in various species such as fish, amphibians, birds, reptiles, and mammals. It exhibits broad-spectrum antimicrobial activity and holds a significant role in the innate immune system of host organisms. The northern snakehead (Channa argus) has become a valuable freshwater fish in China and Asia. In this investigation, the cDNA encoding the hepcidin gene of northern snakehead was cloned and named caHep. The amino acid sequences and protein structure of caHep are similar to those of hepcidins from other fish. The eukaryotic expression product of the caHep gene showed broad-spectrum antibacterial activity. Scanning electron microscope analysis indicated that the caHep peptide inhibited bacterial growth by damaging their cell membranes. Lipopolysaccharide (LPS) injection induced significant expression of caHep, implying the involvement of caHep in the innate immune response of northern snakeheads. This investigation showed that the caHep peptide is potentially a robust antibacterial drug against bacterial diseases in aquaculture animals.

Molecular Cloning, In Silico Analysis, and Characterization of a Novel Cellulose Microfibril Swelling Gene Isolated from Bacillus sp. Strain AY8.

A novel cellulose microfibril swelling (Cms) gene of Bacillus sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that the gene consisted of 679 bp sequences encoding 225 amino acids. Further in silico analysis unveiled that the Cms gene contained the NlpC/P60 conserved region that exhibited a homology of 98% with the NlpC/P60 family proteins found in both the strains, Burkholderialata sp. and Burkholderia vietnamiensis. The recombinant Cms enzyme had a significant impact on the reduction of crystallinity indices (CrI) of various substrates including a 3%, a 3.97%, a 4.66%, and a substantial 14.07% for filter paper, defatted cotton fiber, avicel, and alpha cellulose, respectively. Additionally, notable changes in the spectral features were observed among the substrates treated with recombinant Cms enzymes compared to the untreated control. Specifically, there was a decrease in band intensities within the spectral regions of 3000-3450 cm-1, 2900 cm-1, 1429 cm-1, and 1371 cm-1 for the treated filter paper, cotton fiber, avicel, and alpha cellulose, respectively. Furthermore, the recombinant Cms enzyme exhibited a maximum cellulose swelling activity at a pH of 7.0 along with a temperature of 40 °C. The molecular docking data revealed that ligand molecules, such as cellobiose, dextrin, maltose 1-phosphate, and feruloyated xyloglucan, effectively bonded to the active site of the Cms enzyme. The molecular dynamics simulations of the Cms enzyme displayed stable interactions with cellobiose and dextrin molecules up to 100 ns. It is noteworthy to mention that the conserved region of the Cms enzyme did not match with those of the bioadditives like expansins and swollenin proteins. This study is the initial report of a bacterial cellulose microfibril swellase enzyme, which could potentially serve as an additive to enhance biofuel production by releasing fermentable sugars from cellulose.

Molecular cloning, expression, and functional analysis of a putative lectin from the pearl oyster (Pinctada fucata, Gould 1850).

Marine lectins are a group of proteins that possess specific carbohydrate recognition and binding domains. They exhibit various activities, including antimicrobial, antitumor, antiviral, and immunomodulatory effects. In this study, a novel galectin-binding lectin gene named PFL-96 (GenBank: OQ561753.1) was cloned from Pinctada fucata. The PFL-96 gene has an open reading frame of 324 base pairs (bp) and encodes a protein comprising 107 amino acids. The protein has a molecular weight of 11.95 kDa and an isoelectric point of 9.27. It contains an N-terminal signal peptide and a galactose-binding lectin domain. The sequence identity to lectin proteins from fish, echinoderms, coelenterates, and shellfish ranges from 31.90 to 40.00 %. In the phylogenetic analysis, it was found that the PFL-96 protein is closely related to the lectin from Pteria penguin. The PFL-96 recombinant protein exhibited coagulation activity on 2 % rabbit red blood cells at a concentration of ≥8 µg/mL. Additionally, it showed significant hemolytic activity at a concentration of ≥32 µg/mL. The PFL-96 recombinant protein exhibited significant antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Candida albicans, and Vibrio alginolyticus, with minimum inhibitory concentrations (MIC) of 4, 8, 16, and 16 µg/mL, respectively. The minimum bactericidal concentrations (MBC) were determined to be 8, 16, 32, and 32 µg/mL, respectively. Furthermore, the PFL-96 recombinant protein exhibited inhibitory effects on the proliferation of Hela tumor cells, HepG2 tumor cells, and C666-1 tumor cells, with IC50 values of 7.962, 8.007, and 9.502 µg/mL, respectively. These findings suggest that the recombinant protein PFL-96 exhibits significant bioactivity in vitro, contributing to a better understanding of the active compounds found in P. fucata. The present study establishes a fundamental basis for further investigation into the mechanism of action and structural optimization of the recombinant protein PFL-96. The aim is to develop potential candidates for antibacterial and anti-tumor agents.

PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs.

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids of interest is becoming increasingly important. Unfortunately, current cloning platforms fall short regarding affordability, ease of combinatorial assembly and, above all, the ability to iteratively remove individual cassettes at will. Herein we construct, implement and make available a broad set of cloning vectors, called PlayBack vectors, that allow for the expression of several different constructs simultaneously under separate promoters. Overall, this system is substantially cheaper than other multicomponent cloning systems, has usability for a wide breadth of experimental paradigms and includes the novel feature of being able to selectively remove components of interest at will at any stage of the cloning platform.

A Novel Antimicrobial Peptide, Dermaseptin-SS1, with Anti-Proliferative Activity, Isolated from the Skin Secretion of Phyllomedusa tarsius.

The emergence of multidrug-resistant bacteria has severely increased the burden on the global health system, and such pathogenic infections are considered a great threat to human well-being. Antimicrobial peptides, due to their potent antimicrobial activity and low possibility of inducing resistance, are increasingly attracting great interest. Herein, a novel dermaseptin peptide, named Dermaseptin-SS1 (SS1), was identified from a skin-secretion-derived cDNA library of the South/Central American tarsier leaf frog, Phyllomedusa tarsius, using a 'shotgun' cloning strategy. The chemically synthesized peptide SS1 was found to be broadly effective against Gram-negative bacteria with low haemolytic activity in vitro. A designed synthetic analogue of SS1, named peptide 14V5K, showed lower salt sensitivity and more rapid bacteria killing compared to SS1. Both peptides employed a membrane-targeting mechanism to kill Escherichia coli. The antiproliferative activity of SS1 and its analogues against lung cancer cell lines was found to be significant.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method.

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.

Discovery of α-Linolenic Acid 16(S)-Lipoxygenase: Cucumber (Cucumis sativus L.) Vegetative Lipoxygenase 3.

The GC-MS profiling of the endogenous oxylipins (Me/TMS) from cucumber (Cucumis sativus L.) leaves, flowers, and fruit peels revealed a remarkable abundance of 16-hydroxy-9,12,14-octadecatrienoic acid (16-HOT). Incubations of homogenates from these organs with α-linolenic acid yielded 16(S)-hydroperoxide (16-HPOT) as a predominant product. Targeted proteomic analyses of these tissues revealed the presence of several highly homologous isoforms of the putative "9S-lipoxygenase type 6". One of these isoenzymes (CsLOX3, an 877 amino acid polypeptide) was prepared by heterologous expression in E. coli and exhibited 16(S)- and 13(S)-lipoxygenase activity toward α-linolenic and linoleic acids, respectively. Furthermore, α-linolenate was a preferred substrate. The molecular structures of 16(S)-HOT and 16(S)-HPOT (Me or Me/TMS) were unequivocally confirmed by the mass spectral data, 1H-NMR, 2D 1H-1H-COSY, TOCSY, HMBC, and HSQC spectra, as well as enantiomeric HPLC analyses. Thus, the vegetative CsLOX3, biosynthesizing 16(S)-HPOT, is the first 16(S)-LOX and ω3-LOX ever discovered. Eicosapentaenoic and hexadecatrienoic acids were also specifically transformed to the corresponding ω3(S)-hydroperoxides by CsLOX3.

Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning.

Megaprimer-based polymerase chain reaction (PCR) strategies allow the versatile and fast assembly and amplification of a myriad of tailor-made or random DNA sequences readily available for conventional or restriction-free (RF) cloning.In this chapter, we present a megaprimer-based PCR protocol that enables the expeditious construction of customized fusion genes ready for cloning into commercial expression plasmids. With the expanding use of protein tag technology in the most diverse application fields, this protocol remains a versatile and affordable solution for the synthesis and fusion of peptide tags/domains of interest.

Phylogeny of Alisma (Alismataceae) revisited: implications for polyploid evolution and species delimitation.

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter.

Ship Hull-Fouling Diatoms on Korean Research Vessels Revealed by Morphological and Molecular Methods, and Their Environmental Implications.

Ship biofouling is one of the main vectors for the introduction and global spread of non-indigenous organisms. Diatoms were the early colonizers of ship hulls; however, their community composition on ships is poorly understood. Herein, we investigated the diatom community on the hull samples collected from two Korean research vessels Isabu (IRV) and Onnuri (ORV) on September 2 and November 10, 2021, respectively. IRV showed low cell density (345 cells/cm2) compared to ORV (778 cells/cm2). We morphologically identified more than 15 species of diatoms from the two research vessels (RVs). The microalgae in both RVs were identified as Amphora, Cymbella, Caloneis, Halamphora, Navicula, Nitzschia, and Plagiogramma. Of them, the genus Halamphora was found to be predominant. However, both RVs had a varied dominant species with a significant difference in body size; Halamphora oceanica dominated at IRV, and Halamphora sp. at ORV, respectively. Molecular cloning showed similar results to morphological analysis, in which Halamphora species dominated in both RVs. The hull-attached species were distinct from species found in the water column. These results revealed diatoms communities that are associated with ship hull-fouling at an early stage of biofilm formation. Moreover, ships arriving from different regions could show some variation in species composition on their hull surfaces, with the potential for non-indigenous species introduction.

Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an essential traditional Chinese herb that is commonly used to treat colds, fever, and influenza. Indole alkaloids, such as indigo and indirubin, are the primary active constituents of B. cusia. The indole-producing reaction is crucial for regulating the flow of indole alkaloids metabolites along the pathways and coordinating primary and secondary product biosynthesis in plants. The tryptophan synthase alpha-subunit (TSA) can catalyse a process that produces indole, which is free to enter secondary metabolite pathways; however, the underlying potential mechanism of regulating indigo alkaloids synthesis remains unknown. Here, a BcTSA was cloned from the transcriptome of B. cusia. The BcTSA has a significant degree of similarity with other plant TSAs according to bioinformatics and phylogenetic analyses. Quantitative real-time PCR (RT-qPCR) research showed that BcTSA was dramatically enhanced in response to treatment with methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA), and was predominantly expressed in the stems as opposed to the leaves and rhizomes. Subcellular localization revealed that BcTSA is localized in chloroplasts, which is compatible with the fact that the conversion of indole-3-glycerol phosphate (IGP) to indole occurs in chloroplasts. The complementation assay results showed that BcTSA was functional, demonstrating that it was capable of catalyzing the conversion of IGP to indole. BcTSA was shown to stimulate the manufacture of indigo alkaloids including isatin, indigo, and indirubin when the gene was overexpressed in the hairy roots of Isatis indigotica. In conclusion, our research provides novel perspectives that might be applied to manipulating the indole alkaloid composition of B. cusia.