Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg / 药学学报

Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Cloning and expression analysis of JrGI gene in walnut / 生物工程学报

GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.

Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei / 生物工程学报

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Gene Cloning and Bioinformatics Analysis of Limonene-3-hydroxylase from Schizonepeta tenuifolia / 中国实验方剂学杂志

Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.

Cloning and analysis of CoDXR gene in Cornus officinalis / 中草药

Objective: To clone the full-length cDNA sequence of CoDXR, a key enzyme gene of Cornus officinalis, and provide a basis for further study of C. officinalis. Methods: In this study, we used the transcript sequence c147202_g1 from the transcriptome data of C. officinalis obtained in our laboratory as template, designed specific primers through Primer Premier 5.0, cloned the full-length cDNA sequence of C. officinalis DXR gene by RT-PCR technology, and the bioinformatics analysis and function prediction were carried out through the relevant bioinformatics software. Results: The results showed that the CoDXR gene was 1 505 bp in length and the ORF was 729 bp in length, encoding 242 amino acids. The results of predictive analysis of CoDXR protein by SignalP4.0Server and HMMTOP showed that the protein was a hydrophobic protein without signal peptide and transmembrane region. Phylogenetic tree analysis showed that the CoDXR protein had the highest similarity to the DXR protein sequence of Camellia sinensis. Conclusion: In this study, the key enzyme gene CoDXR was successfully cloned based on the sequencing of the C. officinalis transcriptome, and related bioinformatics analysis was carried out. The results of this study laid the foundation for further study on the function of CoDXR gene in the terpenoid synthesis pathway of C. officinalis.

Gene Clone and Bioinformatics Analysis of Subtilisin-Like Protease in Cucumis sativus

Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants. Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay. Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion. Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain. Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.

Cloning and Bioinformatics Analysis on CDS of SUS Gene in Dioscorea opposita / 中国实验方剂学杂志

Objective: To clone CDS sequence of Dioscorea opposita SUS gene and analyze the protein structure,in order to provide a theoretical basis for the regulation mechanism of SUS gene and the synthesis mechanism of D.opposita polysaccharides.Method: Total RNA in D.opposita was extracted and reverse-transcribed into first strand of cDNA.Specific primers were designed according to an annotated SUS gene sequence obtained from the laboratory D.opposita genome database,and the coding region of the SUS gene was obtained by gene cloning technique and the protein sequence characteristics were analyzed by protein prediction analysis software.Result: A 2 448 bp gene sequence was cloned with a complete open reading frame (ORF).The gene was named DoSUS1.The formula of protein encoded by DoSUS1 gene in D.opposita was C4209H6534N1115O1205S23,and the molecular weight was 9 788.32,the total number of amino acids was 815,the theory isoelectric point (PI) was 6.10,the extinction coefficient was 110 505,the aliphatic index (AI) was 94.15,the instability index was 32.18,and the grand average of hydropathicity (GRAVY) was-0.225.It was a stable and soluble acidic protein.There were phosphorylation sites in the DoSUS1 amino acid sequence,with no transmembrane region and signal peptide.The secondary structure and the tertiary structure showed that DoSUS1 was an α class protein.Functional domain predictions showed that DoSUS1 had sucrose synthesis domain and glycosyl transfer domain.The homology alignment showed that the amino acid sequence of DoSUS1 was more than 80% similar to the amino acid sequence of the aligned monocots.The phylogenetic tree showed that DoSUS1 was closely related to SUS of monocotyledon evolution.Conclusion: The coding sequence of SUS gene was cloned from D.opposita for the first time,and its protein structure was analyzed to lay a foundation for further studying the roles of SUS in the growth and polysaccharide synthesis of D.opposita.

Cloning and Sequence Analysis of Cellulose Synthase-like Gene from Ampelopsis megalophylla / 中国实验方剂学杂志

Objective:To clone cellulose synthase-like(Csl)gene from ethnic medicinal plant Ampelopsis megalophylla,and analyze its sequence by bioinformatics. Method:Specific primers were designed for AmCsl gene sequences obtained from A. megalophylla transcriptome sequencing data. The full-length cDNA of AmCsl gene was amplified by PCR using cDNA of young leaves as template,and TA clone and sequencing was performed. The sequence was analyzed by bioinformatics. Result:The full length cDNA was 1 438 bp,containing a 561 bp open reading frame(ORF),and encoding 186 amino acids,the molecular formula of protein was C1011H1547N233O257S10,the theoretical relative molecular weight was 22.40 kDa,the theory isoelectric point(PI)was 7.59,and the aliphatic index(AI)was 116.88.There was a transmembrane region and no signal peptide,which may be located in the endoplasmic reticulum,the average hydrophobic coefficient was 0.670,and the instability index was 42.56.It belonged to a hydrophobic unstable protein. The conserved domain contained a cellulose synthase,and the secondary structure mainly was dominated by α-helix. Amino acid sequence alignment and phylogenetic tree analysis showed that AmCsl had a high homology with Vitis vinifera. Conclusion:The full length of AmCsl gene was obtained for preliminary bioinformatics analysis,which laid a necessary foundation for further study on the accumulation of polysaccharides and the regulation of biosynthesis.

Cloning and expression of key enzyme genes for chlorogenic acid synthesis in Chrysanthemum morifolium / 中草药

To investigate the effects of the expression of coumaroylquinate 3’-monooxygenase (C3’H) and shikimate O-hydroxycinnamoyltransferase (HCT) in the chlorogenic acid-producing pathway and active ingredients in Chrysanthemum morifolium under flooding stress, we cloned two C3’H genes which were CmC3’H1 and CmC3’H2 and two HCT genes which were CmHCT1 and CmHCT2 by the RT-PCR from Hangju and conducted bioinformatics analysis. During the flower bud differentiation stage, we flooded the C. morifolium and then used β-actin as the reference gene to detect the relative expression of the four genes by the qRT-PCR. Finally, the content of downstream products and other indicators of these four genes in C. morifolium were measured by HPLC. We obtained the four genes’ complete open reading frame and predicted the relative molecular mass of the amino acid sequence and the theoretical isoelectric point (pI). And the protein tertiary structure models were constructed. The qRT-PCR results showed that flooding the C. morifolium for 3 days during the flower bud differentiation stage resulted in significant expression changes of the four genes at different growth stages. The results of HPLC showed that chlorogenic acid, the downstream product catalyzed by the C3’H and the HCT, was significantly higher than that in the control group. It was also found that the content of luteoloside and 3,5-O-di-caffeoylquinic acid was also significantly higher than those of the control group. Therefore, C. morifolium regulates the synthesis of downstream products by regulating the expression of the four genes under flooding stress, thereby responding to flooding stress. And the flooding stress during flower bud differentiation can significantly enhance the accumulation of active ingredients of C. morifolium.

Cloning and bioinformatics analysis of SmMK gene in Swertia mussotii / 中草药

Objective: In order to identify the function of the mevalonate kinase (MK) which is a key enzyme of the mevalonate pathway (MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods: According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta (DE3) for expression. Results: The results showed that SmMK cDNA complete sequences had a length of 1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion: This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.

Cloning and expression analysis of LncRNA-RgAGT2 responding to consecutive monoculture in Rehmannia glutinosa / 中国中药杂志

The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334＿All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.

Cloning of the Murine Na+-K+-2Cl-Cotransporter Gene Promoter and the Effect of 20-HETE on Its Transcriptional Activity / 中国医科大学学报

Objective To clone the murine Na+-K+-2Cl-cotransporter (Nkcc2) gene promoter and analyze 20-HETE regulation of the murine Nkcc2 gene transcriptional activity. Methods A fragment of the murine Nkcc2 gene promoter was analyzed using bioinformatics software AliBaba and TRANSFAC TESS. The murine Nkcc2 gene promoter fragment (-1 462 bp-+40 bp) was amplified by PCR using murine genomic DNA as a template and then cloned into a pGL3-Basic vector to generate a luciferase reporter construct. The recombinant reporter construct was transiently transfected into HEK293 T cells using Lipofectamine 2000 for 24 h. The transfected HEK293 T cells were treated with 20-HETE for 2 h followed by measurement of the luciferase activity using the Dual-Luciferase Reporter Assay system. Results A luciferase reporter construct containing the murine Nkcc2 gene promoter was successfully generated. The results showed that 20-HETE significantly reduced the transcriptional activity of the construct. Conclusion 20-HETE may reduce the expression of the murine Nkcc2 gene through transcriptional regulation.

Clone and expression of CpGAPDH gene in Codonopsis pilosula / 中国中药杂志

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.

Cloning and analysis of a powdery mildew responsive gene CmSAMDC from Cucumis melo L / 生物工程学报

In order to investigate the effect of S-adenosylmethionine decarboxylase (SAMDC) gene in melon resistance to powdery mildew, according to the previous results of EST sequences from cDNA-AFLP library and the melon genome sequence data, the SAMDC gene was isolated from Chinese wild melon clone 'Yuntian930' by RT-PCR and designated as CmSAMDC (GenBank Accession No. KF151861). The mORF (main open reading frame) was 1 095 bp encoding 364 amino acids with a molecular mass of 40 kDa. The predicted protein sequence showed high similarity with Cucumis sativus and Citrofortunella microcarpa. The SDS-PAGE showed that the expression protein was a fusion protein with the molecular weight of 40 kDa, which perfectly matched the mass calculated from the amino acid sequence. Quantitative real-time PCR analysis indicated that the expression level of CmSAMDC reached a maximum at 48 hpi (hours post inoculation) that over 7-fold to 0 hpi, and the expression of CmSAMDC was also detected in tendril, root, leaf and stem. These results indicate that CmSAMDC gene may play protective roles in melon resistance to powdery mildew infection.

Cloning and Expression Analysis of Nine Major Facilitator Superfamily Encoding Genes in Polyporus umbellatus / 中国药学杂志

OBJECTIVE: To clone and isolate the major facilitator superfamily(MFS)genes of Polyporus umbellatusand carry out bioinformatic analysis. METHODS: Nine major facilitator superfamily(MFS)genes were cloned fromPolyporus umbellatus sclerotia by RT-PCR and the expression analysis of the nine genes in different parts ofPolyporus umbellatus sclerotia was carried out using quantitative Real-time PCR.RESULTS: The full open reading frame cDNA sequence of these nine genes was between 1 321 and 1 860 bp, the putative encoding proteins were between 441 and 620 amino acids, the molecular weight was between 48.45×103 and 64.79×103 and the theoretical pI was between 6.59 and 9.56. The amino acids of these nine genes possessed 11 to 14 membrane-spanning domains. Phylogenetic tree analysis indicated that Comp34750, Comp34832, Comp29252, Comp42895, Comp32579 and Comp27555 had the highest similarity with MFS general substrate transporter, Comp28872 andComp26306 had the highest similarity with MFS monosaccharide transporter, and Comp33117 had the highest similarity with MFS sugar transporter. Quantitative real-time PCR showed that these nine genes were expressed in both the symbiotic part and non-symbiotic part. Meanwhile, the expressions of seven genes were significantly up-regulated in the symbiotic part except Comp34382 and Comp32579. CONCLUSION: The investigated nine genes might play an important role during the defense response and nutrient absorption of P.umbellatus.

Cloning, Sequence Analysis and Expression Analysis of Eleven Cytochrome P450 Encoding Genes in Polyporus umbellatus / 中国药学杂志

OBJECTIVE: To clone the cytochrome P450 genes from a medicinal fungus Polyporus umbellatus and carry out bioinformatic analysis. METHODS: The full length cDNAs of the genes were obtained by RT-PCR. Some bioinformatic tools were used to characterize the physiochemical properties of the 11 deduced protein. The analyses of multiple alignment and phylogenetic trees were performed with MEGA 6.0 software. RESULTS: Eleven P450 genes were cloned using RT-PCR method from the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of these eleven genes was between 1 326 and 1 635 bp, the putative encoding proteins were between 442 and 545 amino acids, the molecular weight was between 48.9×103 and 61.5×103, and the theoretical pI was between 6.3 and 8.9. Protein sequence analysis found that there was conserved P450 domain in the 11 genes. Phylogenetic tree analysis indicated that all these 11 P450 genes belonged to basidiomycetes. Quantitative real-time PCR showed that these 11 genes were expressed in both the infected partand non-infectedpart. Meanwhile, the expressions of seven genes were significantly up-regulated in the infected part except comp18720, comp26906, comp32003 or comp33717. CONCLUSION: Molecular characterization of the 11Cytochrome P450 genes will be useful for further functional determination of the genes involved in the defense progress of Polyporus umbellatus.

Gene cloning of farnesyl pyrophosphate synthase in Atractylodes lancea and its expression pattern analysis / 中草药

Objective: To clone the full-length cDNA encoding farnesyl pyrophosphate synthase (FPPS) from Atractylodes lancea and analyze its expression. Methods: The full-length cDNA of FPPS in A. lancea was cloned via homology-based cloning and rapid amplification of cDNA ends approach. Also, the characterization of gene was revealed by bioinformatic analysis. The expression of FPPS was determined by qRT-PCR while the content of sesquiterpenes of rhizome in different growth stages in A. lancea was measured by GC-MS. Meanwhile the correlation between them was analyzed. Results: The full-length cDNA (1320 bp) of FPPS gene was obtained (AlFPPS, GenBank accession number KX443242), with an open reading frame of 1029 bp, encoding 342 amino acids. The deduced AlFPPS protein sequence contained five conserved motifs, two of which is full of Asp (DDXXD). qRT-PCR and GC-MS results showed a significant positive correlation between the content of sesquiterpenes and AlFPPS expression level. Conclusion: It can be primarily deduced that AlFPPS gene should be an important control point in the biosynthetic pathway of sesquiterpenes in A. lancea. This work provides scientific basis for clarifying the biosynthetic pathway of sesquiterpenes and application of biological engineering.

Cloning and expression of squalene epoxidase from Paris polyphylla var. yunnanensis / 中草药

Objective: To clone and express a full-length cDNA encoding squalene epoxidase which is key to the biosynthetic pathway of triterpenoid saponins in Paris polyphylla var. yunnanensis. Using real-time PCR method to detect the relative content of SE1 gene and SE2 gene from the cDNA sample. Methods: Total RNA was extracted from the roots of P. polyphylla var. yunnanensis and transcription was reversed. Using the reversed transcription of cDNA as template, specific primer was designed according to the transcriptome data about two groups of SE gene sequence from the HiSeq2500 sequencing platform, and then SE gene was cloned. The production was inserted to pEASY-T1 Simple Cloning Vector, and pEASY-E1-SE expression vector was built after sequencing appraisal right. The ArtMedia protein Expression/Amp+ medium was used to induce expression automatically. Using real-time PCR method to detect the relative contents of SE1 gene and SE2 gene from the cDNA sample. Results: Two SE genes of P. polyphylla var. yunnanensis were obtained, which were named as ppSE1 and ppSE2, repectively. cDNA was 1 598 bp of ppSE1 and 1 509 bp of ppSE2, respectively. The full length for ppSE1 was 1 932 bp, length of ORF was 1 578 bp, coding 525 AA; The full length for ppSE2 was 1 828 bp, length of ORF was 1 548 bp, coding 515 AA. Fluorescence quantitative PCR results showed that the expression of ppSE1 and ppSE2 genes had significant differences in stem and leaf, and the expression of ppSE1 was most pronounced in the leaf. Enzyme digestion and sequencing results showed that the prokaryotic expression vector pEASY-E1-SE was built successfully. SDS-PAGE analysis showed that two fusion proteins of SE genes were induced expression successfully in BL21 (DE3) express competent cells. Conclusion: The SE gene of P. polyphylla var. yunnanensis has been cloned, and the SE protein with biological activity in vitro has been obtained. The ppSE1 and ppSE2 genes have different expression patterns in P. polyphylla var. yunnanensis, and play a different role in the synthesis of secondary metabolites.

Molecular cloning and sequence analysis of two GA2ox genes in Pseudostellariae Radix / 中草药

Objective: To clone the gibberellin 2-oxidase (GA2ox) genes from Pseudostellaria heterophylla and perform the bioinformatic and expression mode analysis. Methods: According to P. heterophylla transcriptome annotation, two transcript codings of GA2ox were identified. The full-length cDNA PhGA2ox1 and PhGA2ox8 were determined using RT-PCR. Then the bioinformatic analysis of these genes and encoded proteins were performed. The coding sequences of PhGA2ox1 and PhGA2ox8 were amplified by PCR. And then analyzed the bioinformation of these sequences. The expression levels of PhGA2ox1 and PhGA2ox8 were analyzed using qPCR. Results: Bioinformatic analysis showed that PhGA2ox1 contained 981 bp and encoded a predicted protein of 326 amino acids with molecular weight of 36 871.3 and isoelectric point (PI) of 8.66; PhGA2ox8 contained 1 056 bp and encoded a predicted protein of 351 amino acids with molecular weight of 40 169.9 and PI of 6.91. Both PhGA2ox1 and PhGA2ox8 contained GA2ox conserved domains DIOX_N and 20G-Fell_Oxy, without obvious hydrophobic region, transmembrane domain and signal peptide. Phylogenetic analysis showed that the GA2ox of plant could be divided into three classes, PhGA2ox1 bolongs to Class I and PhGA2ox8 belongs to Class III. The qPCR. showed that the expression of PhGA2ox1 in different tissues and organs of P. heterophylla was constant, but the expression of PhGA2ox8 in root xylem was significantly higher than that in other tissues and organs (P < 0.05). Conclusion: The PhGA2ox1 and PhGA2ox8 genes are cloned for the first time, and provide a foundation for they may play an important role in the growth and development process of P. heterophylla.

Cloning and expression analysis of MYB transcription factor gene involved in regulation of flavonoids biosynthesis in Carthamus tinctorius / 中草药

Objective To clone the MYB transcription factor gene in safflower, the sequence information and gene expression were analyzed to preliminarily identify the MYB transcription factor gene involved in regulation of flavonoid biosynthesis. Methods All the sequences of MYB transcription factor gene that was reported to involved in regulation of flavonoid biosynthesis were analyzed. The degenerate primers were designed to clone the core sequence. The full length of MYB transcription factor genes were cloned by RACE. The bioinformatics were used to analyze the sequences. The expression of MYB transcription factor genes in different tissues and different developmental stages of flower were analyzed by semi-quantitative PCR. Results Three candidate MYB transcription factors were cloned, named as CtFRMYB1, CtFRMYB2 and CtFRMYB3. The full lengths of the sequence were 1 223 bp, 1 080 bp and 1 348 bp, respectively. And the molecular weight were 17 878.15, 28 766.45 and 27 987.89, respectively. All three transcription factors have DNA binding domain and belong to the MYB family. Homologous analysis showed that CtFRMYB1 and CtFRMYB2 were closely related to AtMYB12. Expression analysis showed that CtFRMYB1 and CtFRMYB2 were only expressed in flowers, and the expression level was higher in flowering stage 3. Conclusion Three candidate MYB transcription factor genes involved in regulation of flavonoid biosynthesis (CtFRMYB1, CtFRMYB2 and CtFRMYB30) were successfully cloned. Two MYB transcription factor genes (CtFRMYB1 and CtFRMYB2) were identified by bioinformatics and expression analysis. These result laid a foundation for the molecular mechanism analysis of flavonoid biosynthesis.