Supported by Construction and Expression of Heterologous Biosynthesis Pathway for Resveratrol (BAK:201502bsh).

1 Introduction

Resveratrol is non-flavonoid polyphenol compound with a stilbene structure generated when the plant is under the stress of exotic species or abiotic environmental conditions. It can make plants resist mechanical damage, ultraviolet radiation, bacteria and pathogenic infection. Resveratrol mainly exists in plants such as Vitisvinifera L., Arachishypogaea Linn., Morusalba L. and Reynoutriajaponica Houtt.[1], and its chemical structure was shown in Fig.1. Resveratrol was originally known as plant spinosad. With constant deepening of studies, researchers found that resveratrol has biological activities of anti-aging, anti-tumor, preventing cardiovascular diseases, preventing neuropathic pathological changes, thus it has great application potential and broad development prospects in food, health care products, cosmetics, and bio-pharmaceutical industries[2-5].

我们很快上了一辆崭新的“快运”，事先表哥已经买了我们两个人的车票，并把我的车票钱塞到我的口袋里，说：“我每月都领着工资，这钱当然得由我来付。还有，我可告诉你，以后你去市里，如果不急，就不要坐‘黑出租’，不光违法，价格比‘快运’还高了一倍多。”见我唯唯诺诺一个劲儿地点头，表哥很是开心。

Fig.1 Structure of trans resveratrol (a) and cis resveratrol (b)

In plants, resveratrol is synthesized through phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), and resveratrol synthase (RS). As is a key enzyme in the synthetic pathway, RS catalyzes the formation of one molecule of resveratrol by one molecule of coumaroyl-CoA and three molecules of malonyl-CoA[6-7]. At present, resveratrol is mainly extracted from the tissue of grape skins, grape seeds and R. japonica Houtt. However, the content of resveratrol is low in plant tissues, in addition to limitations of raw material sources, seasons of plant growth, and regional conditions, as well as extraction processes, the costs for extracting and isolating high purity resveratrol. What’s more, due to complex reaction process, the chemical synthesis of resveratrol may pollute the environment, which also restricts its further application.

In recent years, with the rapid development of synthetic biology and metabolic Engineering, using microorganisms to synthesize plant-derived metabolites with great application value is receiving close attention of researchers[8]. Some scholars have made researches on biosynthesis of resveratrol, but there are relatively few studies on the RS gene related to V. vinifera[9-12]. In this experiment, taking total RNA of V. vinifera as the template, by RT-PCR method, we amplified a complete cDNA sequence of RS gene from V. vinifera, we made sequence alignment and bioinformatics analysis of RS gene, to lay a certain foundation for biosynthesis of resveratrol by the genetic engineering method.

2 Materials and methods

2.1.2 Instruments. TECHNE TC-512 PCR instrument (Bibby Scientific Limited from the UK); DYCP horizontal electrophoresis (Beijing Liuyi Biotechnology Co., Ltd.); Tanon 1600R automatic digital gel imaging analysis system (Shanghai Tanon Science & Technology Co., Ltd.); 5430R desktop high-speed centrifuge (Eppendorf Shanghai International Trade Co., Ltd.); YXQ-LS-50A vertical pressure steam sterilizer; BGZ-240 electric blast drying oven (Shanghai Boxun Industry & Commerce Co., Ltd.); DTY-5A smart thermostat circulator (Beijing Detianyou Technology Development Co., Ltd.); JE203 electronic balance (Shanghai Puchun Measure Instrument Co., Ltd.); PHS-3C pH meter (Shanghai Hongyi Instrument Co., Ltd.); DTY-900 clean bench (Suzhou Cleaning Equipment Co., Ltd.).

2.市场化程度。不管是对于城镇发展还是产业发展来说，要素资源的合理配置都起到重要作用，而资源如果要实现合理配置、达到理论上帕累托最优的状况，就必须推进市场化程度的提升。市场化水平的提高不但可以提升经济活力和发展效率，促进产业转型升级、使得劳动分工合理、加速劳动力的流动，而且也会使私有资本更多地参与到基础设施和公共服务领域的投资，对城市功能的完善起到重要的推动作用。

2.2.1 Extraction of total RNA from leaves of V. vinifera. First, picked tender leaves of V. vinifera with good growth, rapidly put them into small container filled with liquid nitrogen, and took the container to the laboratory. Took out leaves of V. vinifera from the container, put them into a triturator which has been soaked by Diethy pyrocarbonate (DEPC) and treated by high temperature, added proper amount of liquid nitrogen and quickly ground to powder, selected proper amount of powder and moved it to 1.5 mL centrifugal tube without ribozyme, then extracted the total RNA from leaves of V. vinifera in accordance with the operation instructions of total RNA extraction kit, and stored at -80℃ refrigerator for use.

3.4 Sequence analysis ofRS gene The sequencing results of the positive recombinant plasmids showed that the inserted fragment contained a complete open reading frame, and the fragment size was 1 179 bp, and it was named as RS. Sequence alignment of BLAST (https://blast.ncbi.nlm.nih.gov) in NCBI showed that the sequence similarity of this sequence with reported resveratrol synthase gene in NCBI reached 94%-99%, there were only 3 bases differences with NM_001281044, and the similarity with amino acid sequence was 96%-99%, indicating that the cloned fragment was resveratrol synthase gene of V. vinifera, its nucleic acid and translated amino acid sequence were shown in Fig.5.

2.1 Materials and instruments

2.2 Experimental methods

2.1.1 Materials. Leaves of V. vinifera were collected from plantation of Shandong Jiuzhou Biological Industry Park Co., Ltd.; DP432 plant total RNA extraction kit, E. coli DH5a competent cells, molecular weight standard D2000 DNA Marker (TIANGEN® Biotech (Beijing) Co., Ltd.); PrimeScriptTM II 1st Strand cDNA Synthesis Kit, TaKaRa LA Taq®, Takara MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0, Takara pMDTM 18-T Vector Cloning Kit, TaKaRa MiniBEST Plasmid Purification Kit Ver. 4.0, T4 DNA Ligase (TaKaRa Biotechnology (Dalian) Co., Ltd.); LB medium peptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1 000 mL; other reagents were analytical reagents.

另外，为妥善处置大量淤泥，金坛区还与东南大学合作开发，引进欧洲先进技术研制淤泥制备烧结砖，并开展试生产。淤泥制砖过程中加入10%～15%的页岩烧制成为陶砖，砖块强度得到大幅提高，透气性很强，具有自保温、防渗漏等特点，而且每平方米建筑面积比免烧砖节省50～80元。同时，东南大学材料学院研究结果表明，经高温烧制的陶砖，重金属全部固化在砖块中，不会形成二次污染。

2.2.2 Design and synthesis of PCR amplification primer. According to the published sequences of RS gene of V. vinifera (GenBank accession number: NM001281044.1), we designed the amplification primer using GeneTool, the upstream primer RS-1F: 5′ATGGCTTCAGTCGAGGAAT3′, downstream primer R: RS-1R: 5′ATTTGTAACCGTAGGAATGC3′. Primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

2.2.3 RT-PCR amplification of RS gene. Taking the total RNA extracted from leaves of V. vinifera in accordance with Section 2.2.1 as the template, reverse transcription was carried out in accordance with operation instructions of PrimeScriptTM II 1st Strand cDNA Synthesis Kit, and the first strand cDNA was synthesized; taking the synthesized cDNA as the template, RS-1F: 5’-ATGGCTTCAGTCGAGGAAT-3’, RS-1R: 5’-ATTTGTAACCGTAGGAATGC-3’ as primers, PCR amplification of target gene was carried out, and the reaction system was 50.0 μL (including 5.0 μL buffer, 8.0 μL dNTPs, 5.0 μL RS-1F, 5.0 μL RS-1R, and 5.0 μL cDNA template, 21.5 sterilized ddH2O and 0.5 μL LA Taq polymerase); PCR amplification conditions: pre-denatured at 94℃ for 5 min; denatured at 94℃ for 4 s, annealed at 56℃ for 40 s, extended at 72℃ for 110 s, repeated 35 cycles; finally extended at 72℃ for 10 min; after completion of reaction, took proper amount of PCR amplification product and detected by 1.0% agarose gel electrophoresis.

3.5 Bioinformatics analysis ofRS gene

3.5.1 Primary structure prediction and analysis. The resveratrol synthase gene encoded RS protein consisted of 392 amino acid residues, with predicted molecular weight of 42.78 kDa, and isoelectric point of 6.57. The calculated unstable parameter was 35.92, belonging to the stable protein in the classification. This is favorable for further research in the future.

3 Results and analyses

3.1 Extraction of total RNA from leaves ofV. vinifera The electrophoresis results of total RNA extracted from leaves of V. vinifera were shown in Fig.2. From the top to bottom, it was RNA of 28S, 18S, and 5S respectively. From Fig.2, it can be seen that strips of 28S and 18S were complete and clear, while the strip of 5S was dim, indicating that the extracted total RNA from leaves of V. vinifera has no degradation and the quality is high, and can meet the experimental requirement of the next step target gene amplification.

Fig.2 Electrophoresis detection of total RNA of leaves ofV. vinifera

3.2 Cloning of resveratrol synthase geneRS The total RNA of leaves of V. vinifera ha was reversely transcribed to synthesize into the first strand cDNA, taking RS-1F and RS-1R as primers and cDNA as the template, and a specific 1 200 bp electrophoresis strip was obtained through PCR amplification, as shown in Fig.3, and the size conformed to the expected amplification products (1 179 bp).

Fig.3 RT-PCR amplification of Resveratrol SynthaseRS gene

3.3 Building and screening of recombinant plasmids Under the gel imaging system, specific amplification electrophoresis strip was cut, purified, recovered, and ligated with pMD18-T carrier, transformed into E. coli DH5α and coated onto screening plate containing IPTG, X-gal and ampicillin resistance. After culturing, white monoclonal clones were picked and transferred to the liquid LB containing ampicillin resistance for culturing overnight and then plasmids were extracted. Taking the extracted plasmid as the template, RS-1F and RS-1R as primers, through PCR amplification, an electrophoresis strip with the same size as the specific strip in Fig.3 was obtained (Fig.4), indicating that the target fragments were successfully inserted into the cloned carriers and the recombinant plasmids were successfully built.

Fig.4 Verification of positive clones by PCR amplification

数据采集是大数据分析的第一步，真实可靠的数据有利于快速进行分析，数据采集了华北油田采油三厂近800余口油井的生产数据，包括井号、井数、电动机负载率、抽油机载荷利用率、单井日耗电，并对其中明显错误的数据进行了处理，得到可以进行数据分析的分析数据集。

2.2.4 TA cloning and sequencing of target genes. After electrophoresis, the target fragment was purified and recovered by agarose gel recovery kit. The ligation system of pMDTM 18-T and purified fragment was: 1.0 μL of pMDTM 18-T Vector, 4.0 μL of purified target fragment and 1 μL of ligation solution I. The above reaction system was slowly and evenly mixed and ligated overnight at 4℃. Added 10 μL of the ligation product into E. coli DH5α competent cells, transformed through heat shock, coated on ampicillin-resistant plate, and cultured overnight at 37℃; white monoclonal cells were picked for liquid culture and the plasmids were extracted and verified by PCR amplification and correct size of fragments were inserted into recombinant plasmids, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

2.2.5 Bioinformation analysis of RS gene sequence. Sequence alignment and conserved domain search of RS gene were carried out in NCBI using BLAST and online search of Conserved Domain Search (CD Search); multiple sequence alignment was carried out using biological software such as DNAMAN 5.22 and Clustal X; prediction of primary structure, secondary structure and physical and chemical properties of RS protein was carried out by online server ExPASY ProtParam Server, and tertiary structure prediction was carried out SWISS-MODEL server in the manner of automatic modeling.

Note: single line sequences denote the site of stilbene synthase activity, sequences with * are conserved amino acids, and sequences within the square frame are characteristic sequences of stilbene synthase family.

Fig.5 Nucleic acid and translated amino acid sequences ofRS gene

3.5.2 RS gene domain analysis. Resveratrol synthase belongs to the stilbene synthase family. The domain analysis results of the amino acid sequence of RSgene by NCBI online software were shown in Fig.5. Thisamino acid sequence contained complete characteristic sequence GVLFGPGLT and active center sequence GCYAGGTVLR of stilbene synthase family; Cys164 is a conserved, catalytically active amino acid in the stilbene synthase family and plays an important role in the catalysis of enzymes.

3.5.3 Secondary structure prediction and analysis. RS protein secondary structure was predicted by the online server ExPASY ProtParam Serve. The results were shown in Fig.6. From Fig.6, it can be seen that the secondary structure of the RS protein was mainly α-helix, random coil and β-folding, and the α-helix content was 44.13%, random coil content was 26.53%, and β-folding content was 17.66%.

结合本工程强蚀变岩的物理力学特性、注浆模拟试验成果及工程实践，总结出了一套适合于强蚀变岩TBM洞段的施工方法，其处理工艺流程见图7。主要步骤如下：

3.5.4 Tertiary structure prediction and analysis. The RS protein sequence was searched by BLAST in the SWISS-MODEL server to obtain a template 3tsy.1.A with sequence similarity of 96.43% (Fig.7). Using automatic modeling, tertiary structure of RS protein was predicted with 3tsy.1.A as the template, shown in Fig.8. According to Fig.8, this structure contained more α-helices and random coil, which was consistent with the secondary structure prediction results of RS protein in Section 3.5.3.

对于路桥工程，其特点多现场，工序繁多，工艺复杂，在施工现场要用到大量不同的机械设备，且不同班组相互交叉，使施工管理难度极大，对管理人员提出了极高的要求，实际工作中必须完善技术准备。

Note: h:α-helix; c: random coil; e: β-folding

Fig.6 Secondary structure prediction of RS protein

Fig.7 Tertiary structure of 3tsy.1.A template

Fig.8 Predicted tertiary structure of RS protein

4 Conclusions

Resveratrol synthase (EC 2.3.1.95), also known as 3,4,5-trihydroxystilbene synthase, is a member of the family of Stiibene Synthase (STS), and it synthesizes resveratrol with 4-Coumaroyl CoA and malonyl CoA as substrates[13]. In this experiment, a gene fragment of 1 179 bp in length was obtained by RT-PCR amplification from leaves of V. vinifera, named as RS, and its accession number in GenBank was KX688208. The similarity with reported resveratrol synthase gene reached 94%-99%, and the similarity with amino acid sequence reached 96%-99%; it contained complete characteristic sequence GVLFGPGLT and active center sequence GCYAGGTVLR of stilbene synthase family. These indicated that the isolated RS gene is a resveratrol synthase gene from V. vinifera. This experiment is expected to lay a certain foundation for biosynthesis of resveratrol by the genetic engineering method.

相对会计模拟实训的成熟和完善，审计专业实训长期以来是被纳入“大会计”专业教学范畴，很多职业院校没有单独的审计模拟实训室，一般是采用在会计模拟实训的基础上，增加一定数量的审计案例和练习来进行审计模拟实训的。对审计实训教学认识不充分，重视程度不高。

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