VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TITLE STUDY ON CHARACTERIZATIONS OF CHALCONE SYNTHAE (CHS) GENE FROM Pueraria lobata AND Pueraria mirifica IN VIETNAM Hanoi – 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TITLE STUDY ON CHARACTERIZATIONS OF CHALCONE SYNTHAE (CHS) GENE FROM Pueraria lobata AND Pueraria mirifica IN VIETNAM STUDENT: Nguyen Minh Phuong MAJOR: Biotechnology SUPERVISORS: Huynh Thi Thu Hue, PhD Vietnam Academy of Science and Technology Tran Thi Hong Hanh, M.S Vietnam National University of Agriculture Hanoi – 2021 COMMITMENT I hereby declare that I am the author of this thesis This thesis is original and is result of my own work This research is carried out without materials previously published or written by another person except where due reference has been made in the text I acknowledge that the copyright of all literature cited in my thesis resides with the copyright holders of that material is indicated the origin and all help is thankful I bear full responsibility for these reassurances Hanoi, February 1st , 2021 Student Nguyen Minh Phuong i ACKNOWLEDGMENTS This thesis would not have been possible if not for the help and support of many people First and foremost, I would like to express my appreciation to my principal supervisor, PhD Huynh Thi Thu Hue, who has directly guided and dedicated to supporting me in six months doing experiments, especially in writing this thesis My sincere thanks also go to MSc Tran Thi Hong Hanh for her assistance and giving me useful advice to complete this thesis In addition, I sincerely thank the principal of Vietnam National University of Agriculture, Faculty board, and teachers in the Faculty of Biotechnology for providing an active learning environment, as well as knowledge, and skills throughout four and a half academic years I want to thank Assoc Prof Nguyen Xuan Canh and Dr Dinh Truong Son, who helped me to complete administrative procedures, introduced me to a new scientific work environment Last but not least, special thanks go to all researchers at the Laboratory of Genetic Biodiversity, Institute of Genome Research, Vietnam Academy of Science and Technology (VAST) who supported me in the laboratory Student Nguyen Minh Phuong ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGMENTS ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT ix CHAPTER I INTRODUCTION 1.1 Introduction 1.2 Objectives CHAPTER II LITERATURE REVIEW 2.1 Overview of the P.lobata and P.mirifica 2.1.1 General introduction of the P.lobata and P.mirifica 2.1.2 Biological characteristics of Pueraria plants 2.2 Application of P.lobata and P.mirifica 2.3 Primary result and publications related to thesis 2.4 CHS gene encoding Chalcone synthase 2.4.1 Chalcone structure and biological activites 2.4.2 CHS gene encoding Chalcone synthase CHAPTER III MATERIALS AND METHODS 12 3.1 Time and place of study 12 3.2 Materials 12 3.2.1 Materials 12 3.2.2 Chemical reagents 12 3.2.3 Equipment 12 3.2 Methods 13 3.2.1 Total RNA extraction 13 3.2.2 Total RNA gel electrophoresis 14 iii 3.2.3 cDNA synthesis 14 3.2.4 Amplification P.lobata and P.mirifica CHS genes by using RT-PCR 16 3.2.5 RT-PCR products purification 17 3.2.6 Sanger DNA sequencing 18 CHAPTER IV RESULTS AND DISCUSSIONS 19 4.1 Total RNA extraction 19 4.2 Amplification CHS genes by using RT-PCR 20 4.3 Purification of PCR products 21 4.4 Sequence analysis of P.lobata and P.mirifica CHS gene 22 4.4.1 Sequence analysis of P.lobata CHS gene 22 4.4.2 Sequence analysis of P.mirifica CHS gene and published CHS gene on NCBI 25 4.4.3 Nucleotide and amino acid sequence comparisons of isolated P.lobata and P.mirifica CHS gene 28 4.4.4 Putative amino acid sequence analysis of CHS (P.lobata and P.mirifica) 32 CHAPTER V CONCLUSION AND SUGGESTION 37 5.1 Conclusion 37 5.2 Suggestion 37 REFERENCES 38 iv LIST OF TABLES Table 3.1 Equipment used in the thesis 13 Table 3.2 Components of cDNA synthesis 15 Table 3.3 Components of cDNA synthesis (continue) 15 Table 3.4 Temperature and time for cDNA synthesis 15 Table 3.5 Nucleotide sequence of primer for RT-PCR 16 Table 3.6 RT-PCR reaction 16 Table 4.1 Quantitation of total RNA 20 Table 4.2 Number of nucleotide differences in P.mirifica CHS gene and reference on NCBI (JQ409456.1) 28 Table 4.3 Number of different nucleotides between P.lobata and P.mirifica CHS gene 32 v LIST OF FIGURES Figure 2.1 Overall reaction of Chalcone 10 Figure 2.2 Flavonoid biosynthetic pathway 11 Figure 3.1 RT-PCR program 17 Figure 4.1 Agarose gel electrophoresis of total RNA from P.lobata and P.mirifica 19 Figure 4.2 Agarose gel electrophoresis of CHS gene products 20 Figure 4.3 Agarose gel electrophoresis of purified PCR products 21 Figure 4.4 Sequence alignment of isolated P.lobata CHS gene and reference on NCBI (D10223.1) 24 Figure 4.5 Sequence alignment of isolated P.mirifica CHS gene and reference on NCBI (JQ409456.1) 27 Figure 4.6 Pairwise alignment of P.lobata and P.mirifica CHS gene at nucleotide and amino acid levels 31 Figure 4.7 Putative amino acid sequence alignment of the two isolated genes and CHS protein of the five most significant similarity species in Fabaceae family with full-length coding sequence 33 Figure 4.8 Multiple alignments of specific amino acid region of P.lobata , P.mirifica and other species in Fabaceae family 35 vi LIST OF ABBREVIATIONS Abbreviations Definitions aa Amino acid AD Anmo Domini BC Before Christ BLAST Basic Local Alignment Search Tool bp Base pair C164 Cysteine-164 cDNA Complementery DNA DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid dNTPs Deoxynucleotide triphosphates F215 Phenylalanine-215 G372 Glycine-372 H303 Histidine-303 K55, and K62 Lysine-55, Lysine-62 R58 Arginine-58 kb kilobase kDa kilodaltons L310, L317, L319 , L331 Leucine-310, Leucine-317, Leucine-319, Leucine-331 M Molar MCF-7 Michigan Cancer Foundation-7 mL Milliliter vii mM Milimolar Minute N336 Asparagine-336 ng Nanogram NCBI National Center for Biotechnology Information nm Nanometer P375 Proline-375 pT7-7 Plasmid pT7-7 rCHS Recombinant Chalcone synthase RNA Ribonucleic acid RNAase Ribbonuclease rpm Revolutions per minute rRNA Ribosomal RNA RT-PCR Reverse Transcription -Polymerase Chain Reaction S (28S, 18S, 5S) Svedberg UK The United Kingdom µL Microlitre US The United States VAST Vietnam Academy of Science and Technology viii Table 4.2 Number of nucleotide differences in P.mirifica CHS gene and reference on NCBI (JQ409456.1) Position Nucleotide P.mirifica Amino acid Nucleotide JQ409456.1 Amino acid 47 C P T L 51 55 57 60 63 69 77 78 A A C C C A A C A I L A G N C G T A T C C T A V L A G T 81 A P T P 84 A P C P 144 C H T H 281 G R A K 348 451 452 472 479 507 515 G C G A A G G R E M Y T R C G A C C C C D D L S T P 520 G A C P 523 A K C Q 533 C A T V 559 C V T V 627 T L A L 818 G G A E Note: Different amino acid positions are highlighted in yellow; others not mark up According to Dixon et al., 1999 and Pandith et al., 2019 (Dixon et al., 1999 and Pandith et al., 2019), the majority of CHS gene in legume species belonged to multigene families such as 6-8 members in green bean (Phaseolus vulgaris), seven in pea, six or seven in Pueraria lobata, at least nine in soybean (Glycine max), and other species Especially, Wani concluded that the two isoforms GaCHS1 (an ORF of 1176bp) and GaCHS2 (an ORF of 1170bp) (Grewia asiatica L) have appeared the differential expression pattern at different stages (Wani et al., 2017) Besides, P.mirifica CHS gene, with 1170 bp in length encoding 389 amino acid residues is also demonstrated its characteristics belonging to a multigene family (Wiriyaampaiwong et al., 2012) In a recent study, Suntichaikamolkul and his colleagues (2019), based on transcriptome and phylogenetic analysis of P mirifica, showed that two CHS genes (CHS1, CHS2 encoding chalcone synthase) were involved in isoflavone biosynthesis in a total of 14 putative genes (Suntichaikamolkul et al., 2019) As mentioned before, multiple CHS genes of other plants have been found in different isoforms with many different nucleotides and amino acid positions 4.4.3 Nucleotide and amino acid sequence comparisons of isolated P.lobata and P.mirifica CHS gene The pairwise sequence alignments using BLAST (Figure 4.6) illustrated that CHS genes from P.lobata and P.mirifica shared a genetic similarity of 98.4%, and discovered only variants in total 19 different nucleotide positions (Table 4.3) leading different the amino acids For example, the nucleotide positions 28, 29, and 30 of P.mifica are G, G, G encode amino acid Glycine (G) which differs from amino acid Alanine (A) of P.lobata encode from the DNA codons „GCA‟ Similarly, nucleotide positions 31, 32, and 33 of P.lobata are C, A, and A, respectively, encoding amino acid Glycine (G), whereas the corresponding positions of P.mirifica are A, A, and C, 28 in that order encoding amino acid Alanine (A) Furthermore, these differences were observed much higher at the 5‟ and 3‟ end of encoding sequence and may able to create the own characteristics of P.mirifica compared to P.lobata species and others However, how different amino acid positions affect CHS activities and modify functions need to be further researched 29 CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica 10 20 30 40 50 60 | | | | | | | | | | | | ATGGTGAGCGTAGCTGAGATCCGCCAGGCACAAAGGGCAGAAGGCCCAGCAACCATCCTT M V S V A E I R Q A Q R A E G P A T I L GGA.C T T A C M V S V A E I R Q G N S A E S P A T I L 70 80 90 100 110 120 | | | | | | | | | | | | GCCATTGGAACTGCAAACCCACCAAACTGTGTTGATCAGAGCACCTATCCTGATTACTAC A I G T A N P P N C V D Q S T Y P D Y Y A I G T A N P P N C V D Q S T Y P D Y Y 130 140 150 160 170 180 | | | | | | | | | | | | TTCAGAATCACCAACAGTGAGCACATGACCGAGCTCAAAGAGAAATTCCAGCGCATGTGT F R I T N S E H M T E L K E K F Q R M C F R I T N S E H M T E L K E K F Q R M C 190 200 210 220 230 240 | | | | | | | | | | | | GACAAGTCTATGATCAAGAAGAGATACATGTACTTAACCGAAGAGATCTTGAAAGAGAAT D K S M I K K R Y M Y L T E E I L K E N D K S M I K K R Y M Y L T E E I L K E N 250 260 270 280 290 300 | | | | | | | | | | | | CCAAACATGTGTGCTTACATGGCACCTTCTTTGGATGCTAGGCAAGACATGGTGGTGGTG P N M C A Y M A P S L D A R Q D M V V V P N M C A Y M A P S L D A R Q D M V V V 310 320 330 340 350 360 | | | | | | | | | | | | GAGGTACCAAAACTAGGGAAAGAGGCTGCAACAAAGGCCATAAAGGAGTGGGGCCAGCCA E V P K L G K E A A T K A I K E W G Q P E V P K L G K E A A T K A I K E W G Q P 370 380 390 400 410 420 | | | | | | | | | | | | AAGTCAAAGATTACCCACTTGATCTTTTGCACCACAAGTGGTGTGGACATGCCTGGTGCT K S K I T H L I F C T T S G V D M P G A K S K I T H L I F C T T S G V D M P G A 430 440 450 460 470 480 | | | | | | | | | | | | GATTACCAACTCACCAAACAATTGGGCCTTCGCCCCTATGTGAAGAGGTACATGATGTAC D Y Q L T K Q L G L R P Y V K R Y M M Y D Y Q L T K Q L G L R P Y V K R Y M M Y 490 500 510 520 530 540 | | | | | | | | | | | | CAACAAGGTTGCTTTGCAGGTGGCACGGTGCTTCGTTTGGCCAAGGATTTGGCTGAGAAC Q Q G C F A G G T V L R L A K D L A E N Q Q G C F A G G T V L R L A K D L A E N 550 560 570 580 590 600 | | | | | | | | | | | | AACAAGGGTGCACGTGTGTTAGTTGTCTGTTCTGAGATCACTGCAGTCACATTCCGTGGC N K G A R V L V V C S E I T A V T F R G C N K G A R V L V V C S E I T A V T F R G 610 620 630 640 650 660 | | | | | | | | | | | | CCAAGTGACACTCACCTTGATAGTCTTGTGGGCCAAGCATTGTTTGGAGATGGAGCGGCT P S D T H L D S L V G Q A L F G D G A A C A P S D T H L D S L V G Q A L F G D G A A 30 CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica CHS P.lobata CHS P.mirifica 670 680 690 700 710 720 | | | | | | | | | | | | GCAGTAATTGTTGGTTCTGACCCAATTCCACAGGTTGAGAAGCCTTTGTATGAGCTGGTT A V I V G S D P I P Q V E K P L Y E L V .T A V I V G S D P I P Q V E K P L Y E L V 730 740 750 760 770 780 | | | | | | | | | | | | TGGACTGCACAAACAATTGCTCCAGACAGTGAAGGGGCTATTGATGGACACCTTCGTGAA W T A Q T I A P D S E G A I D G H L R E W T A Q T I A P D S E G A I D G H L R E 790 800 810 820 830 840 | | | | | | | | | | | | GTTGGCCTCACATTTCATCTCCTCAAGGATGTTCCTGGGATTGTCTCAAAGAACATTGAT V G L T F H L L K D V P G I V S K N I D .G .T V G L T F H L L K D V P G I V S K N I D 850 860 870 880 890 900 | | | | | | | | | | | | AAGGCACTTTTTGAGGCATTCAACCCACTGAACATCTCTGATTACAACTCCATCTTTTGG K A L F E A F N P L N I S D Y N S I F W K A L F E A F N P L N I S D Y N S I F W 910 920 930 940 950 960 | | | | | | | | | | | | ATTGCACACCCTGGTGGGCCTGCAATTTTGGACCAAGTTGAGCAGAAGTTGGGTCTCAAA I A H P G G P A I L D Q V E Q K L G L K T I A H P G G P A I L D Q V E Q K L G L K 970 980 990 1000 1010 1020 | | | | | | | | | | | | CCTGAGAAGATGAAGGCCACTAGAGATGTGCTTAGTGACTATGGTAACATGTCAAGTGCT P E K M K A T R D V L S D Y G N M S S A P E K M K A T R D V L S D Y G N M S S A 1030 1040 1050 1060 1070 1080 | | | | | | | | | | | | TGTGTTCTTTTCATCTTGGATGAGATGAGGAGGAAATCAGCTGAAAACGGACTTAAAACC C V L F I L D E M R R K S A E N G L K T C V L F I L D E M R R K S A E N G L K T 1090 1100 1110 1120 1130 1140 | | | | | | | | | | | | ACAGGTGAAGGACTTGAATGGGGTGTGTTGTTCGGTTTTGGACCTGGACTTACTATTGAG T G E G L E W G V L F G F G P G L T I E T G E G L E W G V L F G F G P G L T I E 1150 1160 1170 | | | | | | ACTGTTGTTCTGCGTAGTGTGGCCATCTGA T V V L R S V A I * T A .T A T V V L H S V A I * Figure 4.6 Pairwise alignment of P.lobata and P.mirifica CHS gene at nucleotide and amino acid levels 31 Table 4.3 Number of different nucleotides between P.lobata and P.mirifica CHS gene Position 29 30 31 33 Nucleotide C A C A P.lobata A Q Amino acid Nucleotide G G A C P.mirifica Amino acid G N 36 G R T S 39 A A T A 43 G G A S 60 559 621 657 684 786 804 909 1150 1154 1164 1167 T T T G A C C C C G C C L V L A P G L H V R A I C C C A T G T T T A T A L V L A P G L H V H A I Note: Different amino acid positions are highlighted in yellow; others not mark up 4.4.4 Putative amino acid sequence analysis of CHS (P.lobata and P.mirifica) The two CHS sequences and five sequences of relatives‟ species of Fabaceae family that have the full- length cds were aligned using BLAST and BioEdit (Figure 4.7), with 42 different amino acids, which may have directed specific characteristics and their functions, as well as increasing species diversity In fact, the highest genetic similarity was found between CHS gene of P.lobata and CHS8 Glycine max (NP_001304585.2) with 96.24% which was followed by that of CHS7 Glycine max (NP_001340309.1), Glycine soja (ACT32034.1), Phaseolus vulgaris (XP_007158815.1), and Vigna radiate (AJZ72655.1) with 96.15%, 95.98%, 91.81% and 89.49%, respectively Likewise, the most significant match was detected between CHS gene of P.mirifica and CHS8 Glycine max (NP_001304585.2), with a similarity of 95%, followed by Glycine soja (ACT32034.1) with 94,7%, the next Vigna radiate (AJZ72655.1) being 89.2%, CHS7 Glycine max of 74.4%, and Phaseolus vulgaris, with 73% - the lowest one among five selected species In conclusion, the two CHS gene sequences share similarities to the reference, ranging from 73% to 96.24% Amino acid sequences of the two genes are analyzed using Conserved Domains software (NCBI) to show the location of several key conserved domains such as Chalcone and stilbene synthase N-terminal, C-terminal, PLN03173, CHS-like, BH0617, and fabH and predict their function (Figure 4.7) 32 CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 10 20 30 40 50 60 | | | | | | | | | | | | MVSVAEIRQAQRAEGPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVAEIRQGNSAESPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVAEIRQAQRAEGPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVAEIRQGNSAESLATVLAIGTATPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVAEIRQAQRAEGPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVAEIRQAQRAEGPATILAIGTANPPNRVDQSTYPDYYFRITNSDHMTELKEKFQRMC MVSVAEIRQAQRAEGPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVSEIRQAQRAEGPANILAIGTATPSNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC MVSVYEIRQAQKAEGPATILAIGTATPPNCVDQSTYPDYYFRITNSEHMTDLKEKFQRMC CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 Chalcone N-terminal 110 70 80 and Stilbene 90 synthase,100 120 | | | | | | | | | | | | DKSMIKKRYMYLTEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAATKAIKEWGQP DKSMIKKRYMYLTEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAATKAIKEWGQP DKSMIKKRYMYLTEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAATKAIKEWGQP DKSMIKKRYMYLTEEILKENPNMCAYMAPSLDAKQDMVVVEVPKLGKEAATKAIKDWGQP DKSMIKRRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIKEWGQP DKSMIKTRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIKEWGQP DKSMIKRRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIKEWGQP DKSMIKKRYMHLNEEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEAAVKAIKEWGQP DKSMIKKRYMHVTEEILKENPNMSAYMAPSLDARQDIVVVEVPKLGKEAAVKAIKEWGQP CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 130 140 150 160 170 180 | | | | | | | | | | | | KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLDPYVKRYLMSQQGCFAGGTVLPLPQDLVEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN KSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 190 200 210 220 230 240 | | | | | | | | | | | | NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTRLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPISQIEKPLFELV NRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQIEKPLFELV CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 250 260 270 280 290 300 | | | | | | | | | | | | WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPEIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW WTAQTIAPDSDGAIDGHLREVGLTFHLLKDVPGIVSKNIGKALFEAFNPLNISDYNSIFW WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIGKALFEAFNPLNISDYNSIFW CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 310 320 330 340 350 360 | | | | | | | | | | | | IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSAENGHKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSDYGNMSSACVLFILDEMRRKSAENGLKT CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 NP_001340309.1 ACT32034.1 XP_007158815.1 AJZ72655.1 370 380 | | | | | TGEGLEWGVLFGFGPGLTIETVVLRSVAI TGEGLEWGVLFGFGPGLTIETVVLHSVAI TGEGLEWGVLFGFGPGLTIETVVLRSVAI TGEGLEWGVLFGFGPGLTIETVVLHSVAI TGEGLEWGVLFGFGPGLTIETVVLRSVAI TGEGLEWGVLFGFGPGLTIETVVLHSVAI TGEGLEWGVLFGFGPGLTIETVVLRSVAI TGEGLEWGVLFGFGPGLTIETVVLHSVAI TGEGLEWGVLFGFGPGLTIETVVLRSVTI Chalcone and Stilbene synthase, C-terminal Figure 4.7 Putative amino acid sequence alignment of the two isolated genes and CHS protein of the five most significant similarity species in Fabaceae family with full-length coding sequence 33 (NP_001304585.2 (Glycine max), NP_001340309.1 (Glycine max), ACT32034.1 (Glycine soja), XP_007158815.1 (Phaseolus vulgaris), AJZ72655.1 (Vigna radiate) are downloaded from NCBI web Chalcone and stilbene synthase N-terminal, C-terminal are shaded with right and left brackets The main conserved CHS active site residues are marked in yellow while the putative family signature is indicated by the blue box The black boxes shown the CoA binding active sites, whilst the leucine zipper motifs are indicated in the red boxes A filled black rectangle represents a unique residue The big black dot shows the different amino acid positions) Based on Conserved Domains software (NCBI), PLN03173 (from 1-388aa) seemed to be a provisional chalcone synthase, while BH0617 (from 17-388aa) region is expected for naringenin-chalcone synthase CHS_like (from 16-384aa) are described as Chalcone and stilbene synthases; plant-specific polyketide synthases (PKS) and related enzymes FabH (from 17-385aa), which is well-known for 3-oxoacyl-(acylcarrier-protein) synthase III, is a determining factor in branched-chain fatty acid biosynthesis (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) In addition, some motifs supported more understanding of isolated CHS genes in this study For instance, the conserved CHS active site residues (C164, F215 H303, and N336) were identified functions as the active site of enzyme, which is essential for the catalytic activity of both enzymes (Chalcone and stilbene) and probably represents the binding site for the 4-coumaryl-CoA group (Ferrer et al., 1999) Especially, C164 acts as the active site nucleophile and the attachment site for polyketide intermediate in polyketide formation, whereas H303 and N336 are known as the decarboxylation of malonyl CoA F 215 is emphasized with the aim of binding substrate through elongation of the polyketide intermediate (Jez et al., 2000) and the other residues K55, R58 and K62 are introduced as the CoA binding active sites on the figure (Dao et al., 2011; Ferrer et al., 1999) The leucine zipper motif, L310, L317, L319 and L331 (Claudot et al., 1999) plays an important role as the functional active site and was also found in the Pueraria candollei var mirifica CHS gene product (Wiriyaampaiwong et al., 2012) The results shown in Figure 4.7 that all residues remained unaltered in the two sequences of P.lobata and P.mirifica And another, it is interesting 34 to note that a family signature “GVLFGFGPGLTI” motif (Martin, 1993; Dao et al., 2011) of CHS genes including the unique amino acid Pro-375 (P375) in the G372FGPG loop appears as a strictly conserved in all members of CHS superfamily, without other condensing enzymes (Suh et al., 2000) As shown in Figure 4.7, this motif has also been observed in the two isolated P.lobata and P.mirifica CHS gene As indicated above, the “GVLFGFGPGLTI” motif is the family signature of CHS gene and therefore the only signature region that belongs to Chalcone –stilbene synthase C-terminal of P.lobata, P.mirifica and 24 species in Fabaceae family is used to check the conserved sequence of this family (Figure 4.8) CHS P.lobata CHS P.mirifica D10223.1 P lobata JQ409456.1 P mirifica NP_001304585.2 CAA10131.1 AJZ72657.1 PNY03318.1 ABF59866.1 ABM66532.1 AAA67701.1 AAB81987.1 AAU43217.1 BAA01512.1 CAA52819.1 RDY14063.1 ACH67480.1 ACB78187.1 XP_015971138.1 KEH27377.1 AEF14414.1 AFA55180.1 QCE15713.1 XP_020230031.1 TKY62964.1 XP_028780252.1 XP_028189397.1 AYE88587.1 360 370 380 | | | | | | | | RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-RSVAI -RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-RSVAI -RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-RSVAI -KKSAKDGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -RKSAENRLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVTI -RKSKEDGLATTGEGLEWGVLFGFGPGLTVETVVL-HSVAT -RKSKEDGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -KKSAQDGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -KKSAQNGLKTTGEGLDWGVLFGFGPGLTIETVVL-HSVAI KSAQNGLKTTGEGLEWGVLFGFGPGLTIETVVLLRSVAI -NKSLQQGLQTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -KKSTQDGLNTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -EKSVENGLKTTGKDLEWGVLFGFGPGLSLETVVL-HSVAI -RKSAQNGQKTTGEGLEWGVLFGFGPGLTIETVVL-RSVAI -KKSAQNGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI KSIEDGLKTTGEGLDWGVLFGFGPGLTVETVVL-RSVGVN EEGKATTGEGFDWGVLFGFGPGLTVETVVL-HSLPLENRS KRSAQEGLETTGEGLKWGVLFGFGPGLTIETVVL-HSMVI -RKSKENGLATTGEGLEWGVLFGFGPGLTVETVVL-RSVAA -RRSVKEGKATTGEGLEWGVLFGFGPGLTVETVVL-HSVPV -EEGKSSTGEGLKWGVLYGFGPGLTMETIVL-HSATIDTNN RKSAKDGLKTTGEGLEWGVLFGFGPGLTIETVVL-HSVAI -SKEEGKGTTGEGLEWGVLFGFGPGLTVETVVL-HSVPLEG-KRSMNGGKATTGEGLEWGVLFGFGPGLTVETVVL-HSVPV -RKSAENGLKTTGEGLEWGVLFGFGPGLTIETVVL-RSVAI -EEGKSTTGEGLNWGVLFGFGPGLTMETIAL-HSANIDTGY Figure 4.8 Multiple alignments of specific amino acid region of P.lobata , P.mirifica and other species in Fabaceae family The family signature region of CHS gene taken from full length cds CHS NP_001304585.2 (Glycine max); CAA10131.1 (Cicer arietinum); AJZ72657.1 (Vigna radiate); PNY03318.1 (Trifolium pretense); ABF59866.1 (Lupinus luteus); ABM66532.1 (Glycyrrhiza uralensis); AAA67701.1 (Trifolium subterraneum); AAB81987.1 (Onobrychis viciifolia); AAU43217.1 35 (Arachis hypogaea); BAA01512.1 (Pisum sativum); CAA52819.1 (Vigna unguiculata); RDY14063.1 (Mucuna pruriens); ACH67480.1 (Glycyrrhiza inflate); ACB78187.1 (Senna tora); XP_015971138.1 (Arachis duranensis); KEH27377.1 (Medicago truncatula); AEF14414.1 (Onobrychis viciifolia); AFA55180.1 (Acacia confuse); QCE15713.1 (Vigna unguiculata); XP_020230031.1 (Cajanus cajan); TKY62964.1 (Spatholobus suberectus); XP_028780252.1 (Prosopis alba); XP_028189397.1 (Glycine soja); AYE88587.1 (Caragana korshinskii) are accession number from NCBI There are 16 out of 26 species including Pueraria genus (P.lobata and P.mirifica) were all included “GVLFGFGPGLTI ” motif in the Chalcone –stilbene synthase C terminal However, the last amino acid position of putative CHS family signature was changed to Valine (V) instead of Isoleucine (I) in PNY03318.1 Trifolium pretense, ACB78187.1 (Senna tora); XP_015971138.1 (Arachis duranensis), AEF14414.1 (Onobrychis viciifolia), AFA55180.1 (Acacia confuse), TKY62964.1 (Spatholobus suberectus), XP_028780252.1 (Prosopis alba), while only AYE88587.1 (Caragana korshinskii), QCE15713.1 (Vigna unguiculata) was modified to Methionine (M) Moreover, CAA52819.1 (Vigna unguiculata) has two amino acid Threonine- Ileucine (TI) at the end of „GVLFGFGPGLTI‟ motif but has none of Serine – Leucine (SL) Consequently, „GVLFGFGPGLTI‟ amino acid residue region is a family signature of CHS gene, which was also observed in P.lobata and P.mirifica, but it was not included for all members in Fabaceae family With this in sillico analysis, the P.lobata and P.mirifica CHS cds sequences have highly conserved regions to maintain their structures and functions, however, it needs further studies to clarify these points 36 CHAPTER V CONCLUSION AND SUGGESTION 5.1 Conclusion In general, this is the first study to analyze the characteristics of P.lobata and P.mirifica CHS genes in terms of nucleotide and amino acid levels in Vietnam -After extraction, total RNA was obtained in large quantities of high purity and integrity -The optimal temperature for P.lobata and P.mirifica CHS genes was determined and amplified with high specificity at 59°C -CHS genes from P.lobata and P.mirifica have been identified and analyzed in the length of 1170bp encoding a 389 amino acids residues -Both predicted amino acid sequences of CHS gene from P.lobata and P.mirifica were compared with published CHS sequences on GenBank having high significant similarity at 100% and 97.78%, respectively Finally, these CHS genes have highly conserved regions to maintain their structures and functions 5.2 Suggestion These results are very promising to apply to further studies: -Cloning and bacterial expression of putative Chalcone synthase from P.lobata and P.mirifica -Culture scale up and purification of recombinat CHS protein -The successful expression of CHS activity in P.lobata and P.mirifica will 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