Luận văn thạc sĩ inheritance a0bc type of saponin in wild soybean

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE THI THUY INHERITANCE NCE A0Bc TYPE OF SAPONIN IN WILD SOYBEAN BACHELOR THESIS Study Mode: Full-time Full Major: Biotechnology Faculty: Biology Technology – Food od Technology Batch: 2012 – 2016 Thai Nguyen, 04/07/2016 n THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE THI THUY INHERITANCE NCE A0Bc TYPE OF SAPONIN IN WILD SOYBEAN BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biology Technology – Food od Technology Batch : 2012 – 2016 Supervisors : Prof Lee Jeong-Dong Dr Pham Bang Phuong Thai Nguyen, 04/07/2016 n ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Bio Technology Student name Le Thi Thuy Student ID DTN1253140001 Thesis title Inheritance of A0Bc type of saponin in wild soybean Supervisors Prof Lee, Jeong-Dong Dr Pham Bang Phuong Saponins are plant secondary metabolites comprising a steroidal or triterpenoid aglycone with a number of carbohydrate moieties linked by glycoside linkages at one or more sites Since the genetic basis of soybean is very confined screening the genetic resource of wild soybean for the discovery of new saponins or mutation in saponin profile may have significant impacts on altering seed saponin composition or content of the soybean seeds Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents The saponin composition of F2 seeds derived from the cross of “Taekwang” (the “Group A saponin” was of Aa type) and “CW15095” (A0Bc type) was analyzed by TLC The type of saponin A0Bc in all the F2 seeds and the ratio of Aa type: Aa-A0Bc type: A0Bc type was : : in the F2 seeds From this results, it appeared that A0Bc was controlled by one allele Key words Number of pages Saponin, Thin layer chromatography, A0Bc type of saponin 28 pages Date of submission 14/8/2016 n ACKNOWLEDGMENT I would like to express my gratitude to my supervisor, Professor Jeong-Dong Lee who gave me a chance to the internship at Kyungpook National University, Korea, whose expertise, understanding, and patience, added considerably to my graduate experience Thanks for being my great teacher I would like to thank my supervisor, Park Cheolwoo for guidance, inspiring ideas and discussions, both academic and non-academic Thanks for helping me all the time when I was doing my internship Thank you for always being supportive and for taking care of me whenever I needed help I would like to thank all members Plant Genetics and Breeding Laboratory for great friendship and collegiality for their help and encouragement And I am grateful to my friend Ms AkiBotta, Thanks for taking the time to lay this out and providing me the inspiration to this thesis, Thanks for taking the time to help me find some needed correction Very special thanks to my teacher in charge PhD Pham Bang Phuong and PhD Duong Van Cuong always being interested, encourage, support and help me Thanks to all of my teacher in faculty: Bio Technology and Food Technology for teaching me how to become a good student, helped me how to get knowledge and how to become a useful person It is a great pleasure to thank all my great friends Thank you for all the laughs, scientific discussions and for making the four years as a student be worth every second Finally, I would like to thank my family for their love and support I could not have done this without you n TABLE OF CONTENTS LIST OF TABLES AND FIGURES i LIST OF ABBREVIATIONS ii PART I INTRODUCTION 1.1 Researcher rationale 1.1.1 Overview and benefits of soybeans 1.1.2 Saponin constituents and their functional benefits 1.1.3 Historical for finding group A0 saponin in soybean 1.1.4 Thin layer chromatography 10 1.2 Research’s objective 11 PART II MATERIALS AND METHODS 12 2.1 Materials 12 2.2 Methods .12 2.2.1 Extract saponin from soybean hypocotyl 12 2.2.2 Thin layer chromatographic condition 12 2.2.3 Statistical analysis 16 PART III RESULTS AND DISCUSSION 17 3.1 Saponin phenotypes of seed hypocotyls by thin layer chromatography (TLC) 17 3.2 Segregation of A0Bc in F2 populations and use statistical analysis 19 PART IV CONCLUSION 24 REFERENCE 25 n LIST OF TABLES AND FIGURES Table The segregation ratio of Aa : Aa – A0Bc : A0Bc types of the ninety three samples analyzed 20 Figure Soybean plants growing in a field (A) and grains of mature cultivated soybean (B) Figure Soybean plants growing in the glasshouse (C) and grains of mature wild soybean (B) Figure Chemical structures of group A, DDMP, group B and group E saponins Figure Structural variation of sugar chains at the C-22 position in Group A (Aa – series) saponin Figure Add a 10-fold volume (v/w) with 80% aqueous methanol for 24h at room temperature .14 Figure Developing solvent Chloroform : methanol : water (65:35:10/v/v/v) 14 Figure Developing chamber which was saturated with the lower phase of solvent .14 Figure Add 5µL sample extract were directly applied on silica gel plate .15 Figure Developed in the chamber for 55min and dried at 100℃ in dry oven .15 Figure 10 Develop in 10℃ sulfuric acid for 10min and dried at 115℃ for 4min in dry oven 15 Figure 11 Saponin pattern comparison of seed hypocotyl of CW15095 and Taekwang by thin layer chromatography.Lanes and denotes the phenotypes CW15095 (phenotype: A0Bc) and Taekwang (phenotype: Aa), respectively 18 Figure 12 Chromatograms of saponin extract of wild soybean accession in conventional TLC method Lane was hypocotyl of Taekwang, lane was hypocotyl of CW15095, lanes to were sevens hypocotyls seed of F2 generation (Taekwang x CW15095) 18 Figure 13 Chromatograms of saponin extract of F2 generation in conventional TLC method Lane was Taekwang, lane was CW15095, lanes to were homogenous Aa type (Taekwang), lanes 10 to 16 were the homogenous A0Bc type (CW15095), and lanes 17 to 23 were heterozygous .19 i n LIST OF ABBREVIATIONS CMV DDMP RT Chloroform : Methanol : Water 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one Room temperature SS-A Soyasaponin A SSC Seed saponin composition TLC Thin layer chromatography UV Ultraviolet ii n PART I INTRODUCTION 1.1 Researcher rationale 1.1.1 Overview and benefits of soybeans Soybean [Glycine max (L.) Merril ] is an agricultural crop of tremendous economic importance Soybean is the most important grain legume for the food such as the sprout, tofu, soy sauce, soy paste, forage, and used by pharmaceutical, cosmetic, and bio-fuel industries The health benefits of soybean have attached the attention of nutritionists as well as common people [1] The two main products derived from soybean seed after processing are oil is used as cooking oil and the protein-containing meal [2,12] The soybean meal is used primarily as a protein source for swine, poultry, beef, dairy and fish Soybean meal can also be used to make protein concentrate, texturized protein and protein isolates that are used in food products for human consumption [2] Soybean also contains several bioactive components such as isoflavones, saponins, and lecithin [14] G soja has mainly tiny, black seeds in contrast to the large yellow seeds of G max Wild soybean (Glycine soja Sieb and Zucc.) and cultivated soybean [G max (L.) Merr.] belong to the subgenus Soja within the genus Glycine wild of the family Leguminosae, which includes alfalfa (Medicago sativa), pea (Pisum sativum), common bean (Phaseolus vulgaris), peanut (Arachis hypogaea), and lentil (Lens culinaris) [3] G soja is generally considered to be the closest wild relative of G max G soja and G max both have 20 chromosomes (2n = 40), hybridize easily, exhibit normal meiotic chromosome pairing, and generate viable fertile hybrids [4] n A B Figure Soybean plants growing in a field (A) and grains rains of mature cultivated soybean (B) C D Figure Soybean oybean plants growing in the glasshouse (C) and grains of mature wild soybean (B) n However, wild and cultivated soybeans differ with respect to several plant morphological characteristics: wild soybean grows in the form of creepers with many lateral branches, flowers later than cultivated soybean, produces tiny black seeds rather than large yellow seeds, and its pod shatters easily, promoting long-distance seed dispersal [4] 1.1.2 Saponin constituents and their functional benefits Saponins are high-molecular-weight glycosides, consisting of a sugar moiety linked to a triterpene or steroid aglycone The classical definition of saponins is based on their surface activity; many saponins have detergent properties, give stable foams in water, show haemolytic activity, have a bitter taste and are toxic to fish Such attributes, while not common to all saponins, have frequently been used to characterize this class of natural products However, because of the numerous exceptions which exist, saponins are now more conveniently defined on the primary of their molecular structure, namely as triterpene of steroid glycosides Some saponin-containing plants have been employed for hundreds of years as soaps and this fact is reflected in their common names: soapwort (Saponaria officinalis), soaproot (Chlorogalum pomeridianum), soapbark (Quillaja Saponaria), soapberry (Sapindus saponaria), soapnut (Sapindus mukurissi) Indeed, the name 'saponin' comes from the Latin word sapo (soap) [5] The saponin distributions in soybean seed have been reported and the saponin levels were higher in hypocotyl than in cotyledon, and especially acetyl-soyasaponins and soyasaponins occur almost only in hypocotyl There were little saponins in the seed coat, but no study was attempted to find the saponin distributions in soybean plant organs [24] To date, several saponins in soybean seeds have been reported and characterized We considered acetylated saponins as intact constituents at least for saponins with a soyasapogenol A Acetyl-soyasaponins have some acetyl groups which are concentrated at one terminal sugar (glucose or xylose) of a sugar chain linked to the C22 position of soyasapogenol A Some reports was shown that these constituents occurred only in seed hypocotyls and that their levels were unusually high (1.2 - 2.9%) [25], thus these findings coincide with previous reports Behaviors of n hair drier All experiments were carried out in the rectangular developing chamber which was saturated with the lower phase of chloroform: methanol: water (CMW–65 : 35 : 10, v/v, first development) for 2h and the plates were developed in the sample phase for 50 In the conventional procedure, the plates were then dried at room temperature for 60 to evaporate the mobile phase and sprayed with 10 % H2SO4 While in the improved procedure, plates were dried at 100oC for 10 to completely evaporate the mobile phase Next, they have developed again with 10 % H2SO4 (second development) for 12 in a closed chamber The plates from both procedures were heated at 115oC for 13 to visualize the solute molecules The results were scanned using a Scanjet 5370c scanner and photoshopCS6 using Microsoft Windows 10 version TLC consists of six steps for separate hypocotyls from each dry matured seed • Separate hypocotyls from each dry matured seed and Add a 10-fold volume (v/w) with 80% aqueous methanol for 24h at room temperature • Developing solvent Chloroform : methanol : water (65:35:10/v/v/v) • Developing chamber which was saturated with the lower phase of solvent ã Add 5àL sample extract were directly applied on silica gel plate • Developed in the chamber for 55min and dried at 100℃ in dry oven • Develop in 10℃ sulfiric acid for 10min and dried at 115℃ for 4min in dry oven 13 n Describe more six steps by thin layer chromatographic from each dry matured seed through some pictures Filled with aqueous 80% methanol Hypocotyls Figure Add a 10-fold volume (v/w) with 80% aqueous methanol for 24h at room temperature Figure Developing solvent Chloroform : methanol : water (65:35:10/v/v/v) Figure Developing chamber which was saturated with the lower phase of solvent 14 n Figure Add 5µL sample extract were directly applied on silica gel plate Figure Developed in the chamber for 55min and dried at 100℃ in dry oven Figure 10 Develop in 10℃ sulfuric acid for 10min and dried at 115℃ for 4min in dry oven 15 n 2.2.3 Statistical analysis A chi-squared test, also referred to as a x2 test (or chi-square test), is any statistical hypothesis test wherein the sampling distribution of the test statistic is a chisquare distribution when the null hypothesis is true Chi-squared tests are often constructed from a sum of squared errors, or through the sample variance Test statistics that follow a chi-squared distribution arise from an assumption of independent normally distributed data, which is valid in many cases due to the central limit theorem A chi-squared test can be used to attempt rejection of the null hypothesis that the data are independent ( − ) = Df = n – x: Chi – square test O: Observed value e: Expected value Df: Degrees of freedom n: sample size 16 n PART III RESULTS AND DISCUSSION 3.1 Saponin phenotypes of seed hypocotyls by thin layer chromatography (TLC) In this study was extracted saponin from two kinds of soybean It was Taekwang and CW15095 by thin layer chromatography from ninety-three samples Each sample was compared with their parents based on the band appeared in the thin layer chromatography plates (Figure 11) Free sugars retained as black bands which often jeopardize the identification of saponin composition (Figure 9, 10) Free sugars in the extracts can be removed into the lower (water) phase by separating with a mixture of butanol: water (1:1, v/v) after drying the extracts Seven individuals hypocotyl from each line were separated from mature dry seeds Ten-fold volumes (v/w) of 80 % (v/v) aqueous methanol was added to extract saponins from an intact hypocotyl Extractions were carried out at room temperature for 12h TLC 10 µl from each sample extract was directly applied on silica gel (SiO2) coated TLC plates with an Eppendorf micropipette and slightly dried by using a hair drier The plates were developed in a rectangular developing chamber which was saturated with the lower phase of chloroform: methanol: water (65:35:10, v/v/v) for h Plates were dried at 100°C for 10 and then developed with 10% H2SO4 for ~12 in a closed chamber Saponins were visualized by heating the plates at 115°C for 13 The bands appeared on the TLC plate and the samples showed changes in the saponin banding pattern in TLC (Figure 12) 17 n } } Dehydrated sugars Saponin areas Figure 11 Saponin pattern comparison of seed hypocotyl of CW15095 and Taekwang by thin layer chromatography.Lanes and denotes the phenotypes CW15095 (phenotype: A0Bc) and Taekwang (phenotype: Aa), respectively } } Dehydrated sugars Saponin areas Figure 12 Chromatograms of saponin extract of wild soybean accession in conventional TLC method Lane was hypocotyl of Taekwang, lane was hypocotyl of CW15095, lanes to were sevens hypocotyls seed of F2 generation (Taekwang x CW15095) 18 n Figure 13 Chromatograms of saponin extract of F2 generation in conventional TLC method Lane was Taekwang, lane was CW15095, lanes to were homogenous Aa type (Taekwang), lanes 10 to 16 were the homogenous A0Bc type (CW15095), and lanes 17 to 23 were heterozygous The saponin composition of F2 seeds derived from the cross of "Taekwang" (the "Group A saponin" was of Aa type) and "CW15095" (A0Bc type) was analyzed by TLC The bands appeared on TLC plate and then compared with their parent's bands If the bands of each individual have appeared to two bands like land (Figure 11), it means that individual is homogenous Taekwang, if the bands of each individual were appeared one band like land (Figure 11), it means that individual is homogenous CW15095, in otherwise, all seven bands were not same one of their parent's band, seven band mixes homogenous Taekwang and CW15095, it means it was heterozygous 3.2 Segregation of A0Bc in F2 populations and use statistical analysis After segregating 93 individuals hypocotyl from each line were separated from mature dry seeds and use statistical analysis to know the segregation ratio of Aa : Aa – A0Bc : A0Bc types of the ninety three samples analyzed (Table 1) 19 n Table The segregation ratio of Aa : Aa – A0Bc : A0Bc types of the ninety three samples analyzed Parents and progenies X2 Value Number of seeds F2 individuals Phenotype Homozygous (1:2:1) Taekwang Heterozygous Homogenous CW15095 Observed Expected 30 23.25 46 17 46.5 23.25 3.645161 In this study, the ratio was 30 : 46 : 17 and base on a chi-squared test x2 value : 3.645161 to know about inheritance of A0Bc in F2 individuals Df = ( ) = 5.991 X2 value < ( ) = ! The genetic inheritance of saponins was examined in the F2 population derived from the cross: Taekwang x CW15095 In F2 seeds from each cross, a : : ratio was observed (Table 1) This suggests the unknown saponins were all inherited to gather by a single recessive gene 20 n DISCUSSION The accession of CW15095 (A0Bc) did not produce any group A saponins These wild soybean can be utilized in producing new soybean varieties with good tastes as well as in bio-synthetic studies The newly identified saponin gene in CW15095 can be utilized in producing soybean variety without bitter and astringent tastes Soybean also contains several bioactive components such as isoflavones, saponins and lecithin Soybean and its undomesticated progenitor wild soybean are both annual and similar in chromosome number (2n=40) Although soybean and wild soybean are phenotypically disparate in many ways, they readily cross with one another and give rise to normal fertile F1 hybrids [10] This makes wild soybean a primary gene pool (promising source of novel genes and alleles) for soybean breeding and improvement Group A disabled its function and caused the absence of acetylsaponins in soybean and found mutant (A0-S, A0Bc-S, A0Bc-ag, AuAeBc and AcAf) phenotypes (Krishnamurthy et al 2014a) All 12 accessions analysed in that study showed saponin mutant phenotype A0Bc-αg in TLC These accessions mainly contained saponin A0αg (belonging to A0 series) instead of saponin Aa (belonging to Aa series) or Ab (belonging to Ab series) The Aa, Ab and A0 series saponins have differed from one another by the terminal sugar of C-22 sugar chain of SS-A (Takada et al 2013) Previously, it has been reported that saponins Aa and Ab are the major components while others are minor components among Aa and Ab series saponins, respectively (Tsukamoto et al 1993) Because saponin A0-ag is an intermediate precursor for the major saponins (saponins Aa and Ab), the Korean wild soybean mutants predominantly contained saponin A0-ag and subordinately contained other A0 series saponins The results suggest that the 12 Korean G soja mutant accessions might have mutation in their Sg-1 locus which controls the addition of terminal sugar of C-22 sugar chain of SS-A Triterpene saponins are a diverse group of biologically functional products in plants Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions In the group A saponins of soybean (Glycine max), 21 n differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1 Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-ag Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products Triterpenoid saponins are common secondary metabolites in soybean seeds, and these are a group of diversified chemicals that are the consequence of a variety of hydroxylations and subsequent glycosylations Soybean saponins are divided into group A and DDMP saponins based on aglycon structures The decrease or elimination of group A saponins in soybean seeds is an important target of soybean breeding, because they cause a bitter and astringent taste in foods prepared from the seeds (Okubo et al 1992) While there have been earlier reports that soyasapogenol Aconjugated saponins (group A saponins) were exclusively localized in hypocotyls (Taniyama et al 1988; Shimoyamada et al 1990), recent results have demonstrated the presence of group A saponins in cotyledons (Tsukamoto et al 2011) As a considerable amount of partially and fully acetylated group A saponins was detected in cotyledons, genetic elimination of group A saponins from cotyledons could improve the taste of soy foods A wild soybean accession, CW15095, was identified that lacks group A saponins It is unable to synthesize soyasapogenol A, the discriminative aglycone of group A saponins Two mutant lines have been already identified from cultivated and wild soybeans that are free from ordinary group A saponins such as Aa and Ab series (Tsukamoto et al 1993; Kikuchi et al 1999) A single recessive allele, sg-1o, causes the accumulation of group A deacetyl saponins (A0 series saponins) instead of the group A acetyl saponins, and has been successfully incorporated into the breeding program to yield a new commercial variety, ‘Kinusayaka’ ‘Kinusayaka’ does not produce a bitter or astringent taste in traditional soy-based foods (Kato et al 2007) However, group A saponins, including group A deacetyl saponins, have almost no beneficial effects on human health (Tsukamoto and Yoshiki 2006) Thus, an increase 22 n of DDMP (group B) saponins achieved simultaneously with a decrease of group A saponins has become an objective in our soybean breeding program To achieve this objective, the genetic trait responsible for the lack of soyasapogenol A in B01082 was successfully introduced into a new breeding line ‘Tohoku 152’ ‘Tohoku 152’ and its progeny accumulated an increased amount of DDMP saponin in response to eliminating soyasapogenol A from the seed These breeding lines exhibited no detectable adverse agronomic traits such as poor germination, sterility and decreased seed production They are therefore very promising germplasm for use in generating soybeans destined for the soy-based food production 23 n PART IV CONCLUSION The saponin composition of F2 seeds derived from the cross of “Taekwang” (the “Group A saponin” was of Aa type) and “CW15095” (A0Bc type) was analyzed by TLC The type of saponin A0Bc in all the F2 seeds and the ratio of Aa type: AaA0Bc type: A0Bc type was : : in the F2 seeds From this results, it was appeared that A0Bc was controlled by one allele Use a segregation population derived Taekwang x CW15095 Taekwang has normal Aa saponin was conduct to confirm segregation ratio In this study was found A0 saponin type in CW15095 was control by a single recessive gene Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents This result will be used to develop soybean variety with A0 saponin type which will foster soybean food industry This study can be helpful for improvement of soybean cultivars to use food industries and breeding programs as well Further study identified polymorphism of A0 saponin control which kind of alleles and 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