Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba

Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.


Introduction
Gingko biloba L., which dates back to more than 200 million years, is the only surviving member of ginkgophyta in gymnosperm family and considered a "living fossil" [1]. Ginkgo leaf extracts are used to treat cardiovascular and cerebrovascular diseases because of their highly specific and potent platelet-activating factor receptor antagonists [2]. G. biloba contains terpene trilactones (TTLs), such as diterpenoid ginkgolides and sesquiterpenoid bilobalide, as the main bioactive components [3]. TTLs in G. biloba possess unique biological properties and promote high activities of anti-platelet-activation factors [4]. TTLs also function as a selective glycine receptor and participate as the main bioactive substance in G. biloba [5]. However, there are many difficulties with respect to supply of ginkgo leaves and chemical synthesis is far from of being applicable for commercial-scale production. Different biotechnological strategies to improve TTL production have been used, including screening and selection of in vitro ginkgo cultures, cell differentiation levels of these cultures, and optimization of culture conditions, feeding the elicitation strategies [6]. However, until now, the yields obtained from cell cultures have been low and new strategies such as the use of key genes for increasing TTL production by genetic engineering are imperative.
In the MVA pathway, acetyl-CoA C-acetyltransferase (AACT) is the first enzyme that catalyzes the conversion of acetyl-CoA into acetoacetyl-CoA. Mevalonate kinase (MVK) is the fourth enzyme that catalyzes the conversion of MVA into mevalonate-5-phosphate, which undergoes an enzymatic reaction catalyzed by phospho-mevalonate kinase to generate mevalonate-5-diphosphate. Mevalonate-5-diphosphate is then transformed into isopentenyl diphosphate, as catalyzed by MVD. AACT and MVK genes have been described in plants; while reports on these genes are few, what we do know is that AACT is found in Elaeis guineensis Jacq [21], Ganoderma lucidum [22], and Bacopa monnieri [23], and MVK is found in Eucommia ulmoides [24] and Hevea brasiliensis [25]. However, cloning and characterization of AACT and MVK from G. biloba have not been reported in the literature. In this study, two novel cDNAs of AACT and MVK were cloned and characterized from G. biloba. Yeast complementation assays were conducted to identify the function of these genes. The expression patterns of GbAACT and GbMVK in various tissues, including roots, stems, leaves, fruits, male and female flowers were also examined to describe TTL synthesis. In addition, our previous work showed that the transcripts of HMGR [26] and MVD [11] genes involved in the MVA pathway are positively responsive to methyl jasmonate (MeJA) and salicylic acid (SA) treatments in ginkgo. Therefore, the expression profiles of GbAACT and GbMVK as well as TTL contents under the induction by MeJA and SA were also investigated, which will facilitate future work to map and regulate these important steps involved in TTL biosynthetic pathway at the level of molecular genetics.

Isolation and Characterization of the cDNA of GbAACT and GbMVK
The full-length cDNA sequences of GbAACT and GbMVK were cloned through RT-PCR (Reverse transcription polymerase chain reaction). Using total RNA isolated from the young leaves of G. biloba, a

Isolation and Characterization of the cDNA of GbAACT and GbMVK
The full-length cDNA sequences of GbAACT and GbMVK were cloned through RT-PCR (Reverse transcription polymerase chain reaction). Using total RNA isolated from the young leaves of G. biloba, a 2028-bp and 2057-bp fragment was amplified through RT-PCR. After aligating these genes into pMD19-T vector and sequencing, the results of sequence analysis showed that GbAACT contained an open-reading frame (ORF) of 1215 bp and that GbMVK contained an ORF of 1194 bp. The GbAACT encoded a protein containing 404 amino acids with a 6.33 isoeletric point, and the calculated molecular weight was approximately 41.5 kDa. Meanwhile, GbMVK encoded a protein containing 397 amino acids with a 5.71 isoeletric point, and the calculated molecular weight was approximately 42 kDa. A BLASTn search of GbAACT and GbMVK with other plant species showed that GbAACT and GbMVK are highly homologous to AACT genes and MVK genes from other plant species. Therefore, these genes were designated as GbAACT (GenBank accession No. KX904942) and GbMVK (GenBank accession No. KX904944). Multiple alignments of GbAACT with AACTs from other plants indicated that the plant AACTs were the most similar ( Figure 2). The online InterPro result showed that the function of GbAACT harbors the activity of thiolase II, and the structure of GbAACT monomer contains three domains, including thiolase-like domain , N-terminal , and C-terminal (282-402). Based on the differences in catalytic activities, the thiolases are of two types: thiolase I (acetyl-CoA C-acyltransferase) and thiolase II (acetyl-CoA C-acetyltransferase). Thiolase I is a degradative thiolase, and thiolase II is a synthetic thiolase [27]. Residues of two cystines, one histidine, and one asparagine are present in GbAACT, which are highly conserved in AACT among the thiolases from different sources, and are important for catalytic activity ( Figure 2, marked with "asterisk") [28,29]. One highly conserved domain (NVHGGAVSIGHPIGCSG) at the C-terminal end is also present. The thiolase II active site GVAGVCNGGGGASA at the last position is specific for AACT [30]. This evidence confirms the similarity function of GbAACT to AACTs from other plants.

Bioinformatics Analysis of the Deduced GbAACT and GbMVK Protein
Sequence alignment using Vector NTI 11.5.1 showed that the predicted GbMVK shared a high level of identity with MVKs from other plant species, implying that GbMVK harbors the activity of mevalonate kinase. The structure of GbMVK monomer contains ribosomal protein S5 2-type , GHMP kinase N-terminal domain (140-220), and GHMP kinase C-terminal domain (236-390). Ile 146 -Ala 157 is an ATP-binding conserved site ( Figure 3, marked with red box), and these findings indicated that GbMVK has a similar catalytic function to other plant MVKs.   I  V  I  I  I  I  I  I  I  I S  S   I  I  I  I  I  I  V  I  I  I   A  A  A  A  A  A  A  A  A  A   I  I  I  I  I  I  I  I  I  I T  T  T  T  T  T  T  T  T  T   T  T  T  T  T  T  T  T  T  T   I  I  I  I  I T  S  T  I  S  S  I  S  S  S   I  I  I  I  I  I  I  I  I  I GbAACT  399  NnAACT  396  AtAACT  394  HbAACT  394  RcAACT  400  MdAACT  398  MaAACT  396  EhAACT  400  PnAACT 396 NbAACT I  I  I  I  I  I  I  I  I  I I  I  I  I  I  I  I  I  I  I    Dark blue: identity = 100%; red: 75% ≤ identity < 100%; light blue: 50% ≤ identity < 75%.

Molecular Evolution Analysis
To investigate the evolutionary relations among deduced GbAACT with other AACTs, and among GbMVK with other MVKs from angiosperm, gymnosperm, fungus, and bacteria, two phylogenetic trees were constructed using the neighbor-joining (NJ) method with р-distance. Apparently, the bootstrap value is much high for each interior branch with a high identity (>90%). Therefore, the constructed phylogenetic trees of AACTs and MVKs are reliable. As shown in Figure 4A, AACTs from different species seemed to evolve into different groups, with fungus as an ancient group. Then the bacteria group containing AACTs diverged from Sphingobacterium and Mucilaginibacter paludis. The plant AACT group diverged later than the fungus and bacteria. Among the plant group, the GbAACT diverged a little earlier than AACT from other angiosperm plant species in the phylogenetic tree, which coincided with the evolutional position of G. biloba as the most ancient among gymnosperm plant species. As shown in Figure 4B, MVKs from different species evolved vertically from a common ancestor. The MVKs from  L  I  I  I  I  I  I  I   I  I  I  I  I  I  I  I P  P  P  P  P  P  P   I  I  I  I  I  I  I  I   K  G  G  G  G  G  G  T   R  R  R  K  K  R  R  R   I  I  I  I  L  I  I  I I  I  I  I  I  I  I  I   I  I  I  I  L  I  I  I H  I  I  I  I  I  I  V  I   T  T  T  T  T  T  T  T T  I  S  N  S   I  I  I  I  I  I  I  I   I  I  I  I  I  I  I T  T  T  T  T  T  T  T   T  T  T  T  T  T  T  T  AIO11226.1), Glycine max (GmMVK, Accestion No. NP_001276217.1). Dark blue: identity = 100%; red: 75% ≤ identity < 100%; light blue: 50% ≤ identity < 75%.

Molecular Evolution Analysis
To investigate the evolutionary relations among deduced GbAACT with other AACTs, and among GbMVK with other MVKs from angiosperm, gymnosperm, fungus, and bacteria, two phylogenetic trees were constructed using the neighbor-joining (NJ) method with p-distance. Apparently, the bootstrap value is much high for each interior branch with a high identity (>90%). Therefore, the constructed phylogenetic trees of AACTs and MVKs are reliable. As shown in Figure 4A, AACTs from different species seemed to evolve into different groups, with fungus as an ancient group. Then the bacteria group containing AACTs diverged from Sphingobacterium and Mucilaginibacter paludis. The plant AACT group diverged later than the fungus and bacteria. Among the plant group, the GbAACT diverged a little earlier than AACT from other angiosperm plant species in the phylogenetic tree, which coincided with the evolutional position of G. biloba as the most ancient among gymnosperm plant species. As shown in Figure 4B, MVKs from different species evolved vertically from a common ancestor. The MVKs from different species evolved into various groups, with bacteria as an ancient group, and then the fungus group containing MVKs diverged from Fusarium fujikuroi and Candida dubliniensis. The plant species divided into angiosperm and gymnosperm species, and the GbMVK diverged earlier than MVK from other plant species. These results correspond with the fact that G. biloba is the most ancient among gymnosperm plant species. Given that both the AACTs and MVKs can be found in fungus, bacteria, and higher plants, the bioinformatics analysis also indicated that GbAACT was a plant AACT protein with AACT activity and that GbMVK was a plant MVK protein with GHMP kinase activity. Both GbAACT and GbMVK can also be inferred to be a class of highly conservative ancient genes. different species evolved into various groups, with bacteria as an ancient group, and then the fungus group containing MVKs diverged from Fusarium fujikuroi and Candida dubliniensis. The plant species divided into angiosperm and gymnosperm species, and the GbMVK diverged earlier than MVK from other plant species. These results correspond with the fact that G. biloba is the most ancient among gymnosperm plant species. Given that both the AACTs and MVKs can be found in fungus, bacteria, and higher plants, the bioinformatics analysis also indicated that GbAACT was a plant AACT protein with AACT activity and that GbMVK was a plant MVK protein with GHMP kinase activity. Both GbAACT and GbMVK can also be inferred to be a class of highly conservative ancient genes.

Functional Complementation of GbAACT and GbMVK in Saccharomyces Cerevisiae
The ergosterol synthesized from MVA pathway is essential for yeast survival [31,32]. A disruption of the MVA pathway genes is lethal in yeast [33,34]. To determine the function of GbAACT and GbMVK, two ergosterol auxotrophic strains of Saccharomyces cerevisiae that lacked the AACT or the MVK allele, named YPL028W (ΔERG10) and YMR208W (ΔERG12), respectively, were used for experiment. The pYES2 vectors, containing a yeast galactose-dependent promoter, were used as carrier for target genes in this study. The disrupted strains that harbored empty pYES2 could not grow on either the YPG expression medium or the YPD non-expression medium. Two expression vectors, namely, pYES2-GbAACT and pYES2-GbMVK, were constructed and transformed into strains YPL028W and YMR208W, respectively. YPL028W harbored pYES2-GbAACT, and YMR208W harbored pYES2-GbMVK, which grew well on the YPG medium. However, neither the YPL028W harbored with pYES2-GbAACT nor the YMR208W harbored with pYES2-GbMVK grew on the YPD medium ( Figure 5). This proves that the transformed GbAACT can fix the functional loss of the AACT knockout yeast and that the transformed GbMVK can

Functional Complementation of GbAACT and GbMVK in Saccharomyces Cerevisiae
The ergosterol synthesized from MVA pathway is essential for yeast survival [31,32]. A disruption of the MVA pathway genes is lethal in yeast [33,34]. To determine the function of GbAACT and GbMVK, two ergosterol auxotrophic strains of Saccharomyces cerevisiae that lacked the AACT or the MVK allele, named YPL028W (∆ERG10) and YMR208W (∆ERG12), respectively, were used for experiment. The pYES2 vectors, containing a yeast galactose-dependent promoter, were used as carrier for target genes in this study. The disrupted strains that harbored empty pYES2 could not grow on either the YPG expression medium or the YPD non-expression medium. Two expression vectors, namely, pYES2-GbAACT and pYES2-GbMVK, were constructed and transformed into strains YPL028W and YMR208W, respectively. YPL028W harbored pYES2-GbAACT, and YMR208W harbored pYES2-GbMVK, which grew well on the YPG medium. However, neither the YPL028W harbored with pYES2-GbAACT nor the YMR208W harbored with pYES2-GbMVK grew on the YPD medium ( Figure 5). This proves that the transformed GbAACT can fix the functional loss of the AACT knockout yeast and that the transformed GbMVK can compensate the functional lack of the MVK knockout yeast. These results confirmed that GbAACT and GbMVK have AACT and MVK activity respectively.

Transcript Level of the Gene Expression Pattern of GbAACT and GbMVK in Different Tissues of G. biloba
To present, reports corresponding to the AACT and MVK of G. biloba have been inexistent. To determine the expression patterns of GbAACT and GbMVK genes among different tissues in G. biloba, total RNA was extracted from roots, stems, leaves, female flowers, male flowers, and fruit, and cDNA was synthesized as mentioned above. qRT-PCR (Real-time quantitative reverse transcription PCR) was performed for GbAACT and GbMVK ( Figure 6).
qRT-PCR revealed that the expression profile of GbAACT is distributed throughout all ginkgo tissues. Among different tissues, GbAACT had highest expression in fruit, followed by the leaf and the male flower. The GbAACT expression in the roots was significantly lower than that in the other tissues ( Figure 6A). AACT genes were reported to be tissue-specific genes in other plants [27]. In Bacopa monnieri, the BmAACT gene was highly expressed in the root, followed by the stem and leaf [23], whereas results of a recent study in Isodon rubescens corresponded with this study in that AACT gene is more significantly expressed in leaves and flowers than in roots and stems [35]. Our data revealed that the transcript of GbAACT was detected in all ginkgo organs, including the root, stem, leaves, flowers, indeed overlapped with those of GbLPS (Levopimaradiene synthase) [20], GbIDS (1-Hydroxy-2-methyl-2-(E)-butenyl 4diphosphate reductase) [19] and GbMVD (Mevalonate diphosphate decarboxylase) [11] in the roots and flowers. This result verifies the roots as the preferential site of TTL biosynthesis. However, one unexpected observation of this study is that the transcript level of GbAACT was higher in aerial tissues than roots. The possibility is that GbAACT in aerial parts of ginkgo is involved in the biosynthesis of yet to be identified terpenoids.
As shown in Figure 6B, GbMVK was highly expressed in leaves, roots, and stems, and the highest GbMVK expression was detected in roots and leaves. The expression levels of GbMVK in floral organs and fruits were significantly low. A similar expression pattern of the MVK gene was found in Panax notoginseng, though in a lesser degree, because PnMVK is significantly expressed in roots, flowers, and leaves but is seldom expressed in stems [36]. The expression profile is consistent with the TTL distribution from earlier reports [37], showing that higher contents of TTLs were found in the leaves and roots than other tissues of ginkgo. A correlation exists between the transcript level of GbMVK and the content of TTLs, thereby suggesting that GbMVK plays an important role in the production of TTLs in ginkgo.
The biosynthesis organ of the TTL in G. biloba was not clearly reported, and long-distance transport was reported to be involved in the translocation of various compounds [38]. Therefore, considering the results of the present study and given that both GbAACT and GbMVK are highly expressed in the leaf and GbMVK is highly expressed in the root, we infer that the root and leaf could be most the important tissues

Transcript Level of the Gene Expression Pattern of GbAACT and GbMVK in Different Tissues of G. biloba
To present, reports corresponding to the AACT and MVK of G. biloba have been inexistent. To determine the expression patterns of GbAACT and GbMVK genes among different tissues in G. biloba, total RNA was extracted from roots, stems, leaves, female flowers, male flowers, and fruit, and cDNA was synthesized as mentioned above. qRT-PCR (Real-time quantitative reverse transcription PCR) was performed for GbAACT and GbMVK ( Figure 6).
qRT-PCR revealed that the expression profile of GbAACT is distributed throughout all ginkgo tissues. Among different tissues, GbAACT had highest expression in fruit, followed by the leaf and the male flower. The GbAACT expression in the roots was significantly lower than that in the other tissues ( Figure 6A). AACT genes were reported to be tissue-specific genes in other plants [27]. In Bacopa monnieri, the BmAACT gene was highly expressed in the root, followed by the stem and leaf [23], whereas results of a recent study in Isodon rubescens corresponded with this study in that AACT gene is more significantly expressed in leaves and flowers than in roots and stems [35]. Our data revealed that the transcript of GbAACT was detected in all ginkgo organs, including the root, stem, leaves, flowers, indeed overlapped with those of GbLPS (Levopimaradiene synthase) [20], GbIDS (1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase) [19] and GbMVD (Mevalonate diphosphate decarboxylase) [11] in the roots and flowers. This result verifies the roots as the preferential site of TTL biosynthesis. However, one unexpected observation of this study is that the transcript level of GbAACT was higher in aerial tissues than roots. The possibility is that GbAACT in aerial parts of ginkgo is involved in the biosynthesis of yet to be identified terpenoids.
As shown in Figure 6B, GbMVK was highly expressed in leaves, roots, and stems, and the highest GbMVK expression was detected in roots and leaves. The expression levels of GbMVK in floral organs and fruits were significantly low. A similar expression pattern of the MVK gene was found in Panax notoginseng, though in a lesser degree, because PnMVK is significantly expressed in roots, flowers, and leaves but is seldom expressed in stems [36]. The expression profile is consistent with the TTL distribution from earlier reports [37], showing that higher contents of TTLs were found in the leaves and roots than other tissues of ginkgo. A correlation exists between the transcript level of GbMVK and the content of TTLs, thereby suggesting that GbMVK plays an important role in the production of TTLs in ginkgo.
The biosynthesis organ of the TTL in G. biloba was not clearly reported, and long-distance transport was reported to be involved in the translocation of various compounds [38]. Therefore, considering the results of the present study and given that both GbAACT and GbMVK are highly expressed in the leaf and GbMVK is highly expressed in the root, we infer that the root and leaf could be most the important tissues for forming TTLs. To further determine which specific part of plant is involved in TTL biosynthesis, an isotopic tracer technique might be helpful.

Transcript Level of GbAACT and GbMVK and TTL Content Changes in Ginkgo biloba under the Induction of MeJA and SA Elicitors
To understand the expression pattern of GbAACT and GbMVK, GbAACT and GbMVK transcript levels and TTL content were measured after seedlings at 4-5-leaf stage were treated with the elicitors MeJA and SA. qRT-PCR experiments clearly showed that SA and MeJA induction significantly increased GbAACT and GbMVK expression, and the TTL content was significantly enhanced after SA and MeJA treatments (Figure 7). The expression level of GbAACT continuously increased after MeJA treatment and peaked at 64 h with an 3.73-fold increase compared with that of control seedlings. The SA-treated seedlings showed the highest level of GbAACT expression at 32 h, and then slowly decreased ( Figure 7A). With regard to the GbMVK gene, the expression level quckly increased after the seedlings were treated with MeJA for 8 h, and expression level of GbMVK gene peaked at 24 h after MeJA treatment. The GbMVK expression slightly changed from 16 h to 32 h, and the GbMVK expression decreased from 32 h to 96 h after treatment. Under SA treatment, GbMVK was highly expressed 8 h after the treatment, and GbMVK expression peaked until 96 h, with a 3.34-fold increase compared with that of the control ( Figure 7B). TTL increased by 5.8% 8 h after SA treatment, and TTL content continuously increaesed from 0 h to 64 h. A similar increase was observed among MeJA-treated seedlings ( Figure 7C).

Transcript Level of GbAACT and GbMVK and TTL Content Changes in Ginkgo biloba under the Induction of MeJA and SA Elicitors
To understand the expression pattern of GbAACT and GbMVK, GbAACT and GbMVK transcript levels and TTL content were measured after seedlings at 4-5-leaf stage were treated with the elicitors MeJA and SA. qRT-PCR experiments clearly showed that SA and MeJA induction significantly increased GbAACT and GbMVK expression, and the TTL content was significantly enhanced after SA and MeJA treatments (Figure 7). The expression level of GbAACT continuously increased after MeJA treatment and peaked at 64 h with an 3.73-fold increase compared with that of control seedlings. The SA-treated seedlings showed the highest level of GbAACT expression at 32 h, and then slowly decreased ( Figure 7A). With regard to the GbMVK gene, the expression level quckly increased after the seedlings were treated with MeJA for 8 h, and expression level of GbMVK gene peaked at 24 h after MeJA treatment. The GbMVK expression slightly changed from 16 h to 32 h, and the GbMVK expression decreased from 32 h to 96 h after treatment. Under SA treatment, GbMVK was highly expressed 8 h after the treatment, and GbMVK expression peaked until 96 h, with a 3.34-fold increase compared with that of the control ( Figure 7B). TTL increased by 5.8% 8 h after SA treatment, and TTL content continuously increaesed from 0 h to 64 h. A similar increase was observed among MeJA-treated seedlings ( Figure 7C). GbAACT and GbMVK expression level (A,B) and total terpene trilactone (TTL) content (C) changes in ginkgo by salicylic acid (SA) and methyl jasmonate (MeJA). The expression levels were normalized to the house-keeping gene GAPDH (glyceraldehyde-3-phospate dehydrogenase). Data from qRT-PCR were analyzed as expression ratios relative to the level of control (CK), and are shown as the mean ± SD of triplicate assays. Means with different letters from each time of post-treatments are significantly different at p < 0.05 by one-way ANOVA, with Tukey's honestly significant difference test.
With the regulation of SA and MeJA, plants were capable of adjusting to both abiotic and biotic stress [39]. SA and MeJA interact antagonistically aginst each other to induce transcription of defense-related genes by producing certain compunds and proteins [40][41][42][43]. Therefore, challenging ginkgo with SA and MeJA would activate defense-related genes in plants concomitantly increasing secondary metabolites. Indeed, the exogenous application of SA and MeJA has been performed to improve the biosynthesis of ginkgolide A and B and bilobalide in cell cultures of G. biloba [44]. In addition, some genes involved in TTL biosynthesis are upregulated after SA and MeJA treatments are administered in ginkgo. The transcript levels of GbDXS (1-deoxy-D-xylulose 5-phosphate synthase) [13], GbCMK2 (4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase) [15], GbHMGR [26], GbIDS2 [45], and GbMVD [11] are induced by SA and MeJA and are positively involved in TTL biosynthesis in G. biloba. Similar to these reports, our data showed that the expression of GbAACT and GbMVK was upregulated and the TTL content was increased after SA and MeJA treatments, implying that the transcription of these genes responsible for TTL biosynthesis was enhanced. However, TTLs comprise a wide range of compounds and an integrated reaction of a cluster of genes related to TTL biosynthesis. The SA or MeJA-induced increase in TTL production in the present study may be arrtributed to an integrated effect on multiple genes related to TTL biosynthesis. Hence, it is interesting to unveil the expresssion profiling of other genes With the regulation of SA and MeJA, plants were capable of adjusting to both abiotic and biotic stress [39]. SA and MeJA interact antagonistically aginst each other to induce transcription of defense-related genes by producing certain compunds and proteins [40][41][42][43]. Therefore, challenging ginkgo with SA and MeJA would activate defense-related genes in plants concomitantly increasing secondary metabolites. Indeed, the exogenous application of SA and MeJA has been performed to improve the biosynthesis of ginkgolide A and B and bilobalide in cell cultures of G. biloba [44]. In addition, some genes involved in TTL biosynthesis are upregulated after SA and MeJA treatments are administered in ginkgo. The transcript levels of GbDXS (1-deoxy-D-xylulose 5-phosphate synthase) [13], GbCMK2 (4-(cytidine 5 -diphospho)-2-C-methyl-D-erythritol kinase) [15], GbHMGR [26], GbIDS2 [45], and GbMVD [11] are induced by SA and MeJA and are positively involved in TTL biosynthesis in G. biloba. Similar to these reports, our data showed that the expression of GbAACT and GbMVK was upregulated and the TTL content was increased after SA and MeJA treatments, implying that the transcription of these genes responsible for TTL biosynthesis was enhanced. However, TTLs comprise a wide range of compounds and an integrated reaction of a cluster of genes related to TTL biosynthesis. The SA or MeJA-induced increase in TTL production in the present study may be arrtributed to an integrated effect on multiple genes related to TTL biosynthesis. Hence, it is interesting to unveil the expresssion profiling of other genes involved in TTL biosynthesis under SA or MeJA treatments in ginkgo, which will provide more insights into the regulatory role of SA and MeJA in TTL biosynthesis.

Plant Materials
Plant samples were obtained from 18-year-old trees of G. biloba growing on the ginkgo garden of Yangtze University, Hubei, China (around N30.35, E112.14). The roots, stems, leaves, female and male flowers were collected in April 2014, and the fruits were collected in June 2014. Primer synthesis and DNA sequencing were performed by Sangon Biotechnology Company (Shanghai, China). Agarose Gel DNA Extraction Kit Ver. 4.0, pMD19-T vector kit, first-strand cDNA synthesis kit, dNTPs, RNasin, and Taq DNA polymerase were purchased from Takara Company (Dalian, China). A PrimeScript RT reagent kit with a gDNA (genomic DNA) Eraser and AceQ qPCR SYBR Green Master Mix were purchased from Vazyme Bio Inc. (Nanjing, China).

Cloning of the Full-Length cDNA of GbAACT and GbMVK
The RNA of G. biloba was extracted with TaKaRa MiniBEST Plant RNA Extraction Kit (Takara Bio Inc., Dalian, China) according to the manufacturer's instruction. The extracted RNA was purified and used as templates to constructed RNA-seq library according to the manufacturer's protocol. The transcriptome sequencing libraries were generated using a using a NEB Next ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, Ipswich, UK). Sequencing run was performed at Biomarker Co., Beijing, China using Illumina Hiseq 2500 platform. In our previous work, we assigned the functional annotations of 35,113 unigenes [46]. The specific cDNA isolations were obtained through the PrimeScript first-strand cDNA synthesis kit. Primers were designed on the basis of the initial data of AACT and MVK unigenes in the transcriptome (GenBank accession numbers SRR3985386 and SRR3989536 for male and female strobilus, respectively). The sequences of all primers were shown Table 1. The PCR products were purified and ligated into pMD19-T vector (Takara Bio Inc., Dalian, China). The recombined plasmids were transformed into E. coli DH5α competent cells (Vazyme Bio Inc., Nanjing, China) and sequenced.  [52] with minor modification. In detail, an appropriate amount of purified methanolic fraction was evaporated to dryness and trimethylsilylated by adding 100 µL of a silylating agent (Trisil BSA formula D, Sigma-Aldrich, Darmstadt, Germany). This mixture was vortex and heated for 2 h at 80 • C. Analysis by Gas chromatography-flame ionization detection (GC-FID) was performed with a Shimadzu GC-14B (Shimadzu, Japan) equipped with a 30 m × 0.53 mm × 1.5 µm TC-1 capillary column (HP-5, Agilent, San Francisco, CA, USA). The temperatures of the column, injector, and detector were maintained at 290 • C, 320 • C, and 320 • C, respectively. He was used as the carrier gas at a flow rate of 3.1 mL/min. TTL content was calculated as the sum of ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide contents by dry weight percentages. Each sample was evaluated in triplicate, and data were represented as means ± SD (n = 3).

Statistical Analysis
Data were analyzed with using the statistical software SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA). Comparisons between multiple treatment groups were performed using one-way ANOVA, with Tukey's honestly significant difference test. p < 0.05 was considered to be statistically significant.

Conclusions
The genes GbAACT and GbMVK encoding AACT and MVK of the MVA pathway and the enzymes catalyzing the first and fourth steps in this pathway, respectively, were cloned and characterized. Bioinformatics analysis revealed that the deduced GbAACT and GbMVK harbored a highly similar identity to AACTs and MVKs of other plants. Functional complementation experiments in yeast demonstrated that GbAACT and GbMVK encode functional AACT and MVK, respectively. The transcript levels of GbAACT and GbMVK significantly increased in response to MeJA and SA treatments, and this increase corresponded to the increase in the TTL content after SA or MeJA treatment was performed. qRT-PCR analysis showed that GbAACT and GbMVK are tissue-specific genes. The transcript level of GbAACT is highly expressed in fruits, leaves, and floral organs, whereas the transcript level of GbMVK is highly expressed in leaves, roots, and stems. For further unveiling of the function of GbAACT and GbMVK, a plant expression vector containing the GbAACT or GbMVK has been constructed and a study of the genetic transformations of ginkgo is underway in order to investigate the potential role in enhancing TTL accumulation by genetic engineering.