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UDC 635.656:581.19
POLYMORPHISM OF PEA STORAGE PROTEIN CONVICILIN
Selikhova T.N., Senior Scientist of Plant Physiology and Biochemistry Laboratory All-Russia Research Institute of Legumes and Groat Crops, Orel City, Russia
E-mail: tat.selihowa@yandex.ru
Bobkov S.V., Head of Plant Physiology and Biochemistry Laboratory All-Russia Research Institute of Legumes and Groat Crops, Orel City, Russia
E-mail: svbobkov@gmail.com
ABSTRACT
Pea convicilin could hinder physico-chemical properties of protein isolates. Pea accessions without convicilin or with its unusual isoforms could be used in pea breeding on high quality of storage proteins. Polymorphism of convicilin was studied with use of 60 accessions of genus Pisum L. Standard SDS-PAGE electrophoresis was used for separation of storage proteins. In total, four isoforms of convicilin were revealed. They were located on positions 14, 15, 16 and 17 of electrophoretic spectra. Isoform 15 was found with high frequency (0.35). The frequency of isoform 17 was equal to 0.32. Differences between cultivated and wild pea accessions on frequency of convicilin isoforms were determined. Among 6 breeding lines and varieties of P. sativum only two (14 and 17) isoforms were revealed. Isoform 17 was represented with the highest frequency (0.83). In wild subspecies of P. sativum (24 accessions) only three (15, 16, 17) isoforms were found. Isoform 15 had the highest frequency (0.6). In accessions of wild species P. fulvum all four revealed isoforms of convicilin were represented. Isoform 15 was found preferably (0.59). In variety Aest electrophoretic analysis revealed one seed which did not contained convicilin. This seed was characterized by distinguished from other seeds spectrum.
KEY WORDS
Pea; SDS-PAGE; Storage protein; Accession; Pea breeding.
The genus Pisum L. contains the two species: P. sativum L., the cultivated pea, and P. fulvum Sibth. et Smith, the red-yellow pea. Species P. sativum L. includes 6 subspecies: elatius (Bieb.) Schmalh., syriacum (Boiss. et Noe) Berger, abyssinicum (A. Br.) Berger, transcaucasicum Makash., asiaticum Govorov u sativum [11].
Electrophoresis of pea storage proteins was used for the identification of various genotypes, characterization of genetic diversity and in taxonomy [9, 12]. The species P. fulvum has recently considered as a perspective object of research [4, 14]. In compare to P. fulvum interest in wild subspecies of cultivated pea was underestimated. Comparative studies of storage proteins in various pea taxa were limited by small number of accessions. Polymorphism of main pea storage proteins was not studied enough and, consequently, not used in pea breeding.
Pea storage proteins are represented by albumins and globulins which includes approximately 80% of their total content [15]. Convicilin, vicilin, and legumin are the main globulin proteins. Vicilin and legumin produce proteins gels of high quality. But convicilin could hinder physico-chemical properties of protein isolates. Actual isophorms of convicilin restrict using of pea protein isolates in food industry. Innovative varieties of pea should be with high protein quality. Null mutations of convicilin or its new isoforms are considered as a tool for use in pea breeding on high storage protein quality.
Objective of current research was to study polymorphism of convicilin isoforms in wide range of pea (Pisum L.) accessions.
MATERIALS AND METHODS
Polymorphism of convicilin isoforms was studied with use of 60 accessions of pea Pisum L. Cultivars and lines of P. sativum ssp. sativum - Aest (normal plant type), Stabil (tendril leaf, af), 109b (tendril leaf, af), PAP-485/4 ("parsley leaf", aftl), VI 9402 ("acacia leaf", tl), RAS-type (changed shape of leaves) were used. Also it was used 20 accessions of P. sativum wild subspecies - elatius (K1851, K2173, K2524, K3115, K3370, K4014), transcaucasicum (K296, K2365, K2376, K3249, K3980), asiaticum (K1923, K1974, K1975, K2645, K5322, K1915), abyssinicum (K2759, I565496), syriacum (K2521). In experiment 34 accessions of pea wild species P. fulvum - I609881, I609885, K2523, K6070, K2529, K1878, I592579, I592608, I592602, I592597, I592595, I592583, I592884, I592607, I592618, I592577, I592613, I592609, I592598, I592610, I592614, I592604, I592623, I592612, I592575, I592611, I592626, I592882, I592603, I592573, I592590, I592589, I592582, I592592 were studied. Wild accessions of pea were received from the collection of Vavilov Institute of Plant Industry (St. Petersburg, Russia).
Polymorphism of convicilin was estimated as heterozygosity (H), the probability that any randomly chosen individual is heterozygous for any two alleles at a locus having allele frequencies pi [7]. The H was calculated for data of 60 pea accessions. This value estimates the number of polymorphic pairs among all possible ones in a population. The H was calculated with use the next equation:
Hj=1-Zipij2, where pij is the frequency of the ith allele at locus j.
Standard SDS-PAGE electrophoresis was used for separation of storage proteins [8]. Proteins were extracted from 4 mg flour of each seed for 20 h at 3-4°C with Tris-glycine buffer (Tris, glycine, sodium dodecylsulphate), pH 8.3. After centrifugation, 10 ml of the extract were placed into the cell of the plate and mixed with an equal volume of application buffer (sodium dodecylsulphate, Tris-HCl, glycerol, p-mercaptoethanol, bromphenol blue). Electrophoresis was run in polyacrylamide gel using a VE-4 unit for vertical electrophoresis (Helicon, Russia). Concentrations of the separating and stacking gels were 12.5% and 5%, respectively.
For identification of convicilin isoforms on electrophoretic gels molecular mass markers 6.5-200 kDa (SIGMA, USA) were used [15].
RESULTS AND DISCUSSION
Both vicilin and legumin in various compositions are able to form good protein gel. But convicilin can hinder physico-chemical properties of pea protein isolates [13]. Therefore, pea seeds without convicilin would be served as desirable material for food industry [5, 15]. Various isoforms of convicilin could also have possibility to improve the quality of pea storage proteins. Convicilin is characterized by molecular weight approximately 70 kDa [13, 15]. Polymorphism of convicilin isoforms was studied with use of 60 accessions of pea Pisum L. Polymorphic bands were revealed on electrophoretic spectra of pea storage protein (Fig. 1).
HHuK> conviaim
f _ 5b kDa
—45 kDa
12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 1 - Protein spectra in accessions of P. sativum: 1, 2 - elatius (K2524); 3, 4 - asiaticum (K2645);
5, 6 - abyssinicum (K2759); 7, 8 - elatius (K3115); 9, 10 - transcaucasicum (K3249); 11, 12 - elatius (K4014); 13, 14 - asiaticum (K5322); 15, 16 - transcaucasicum (K2376); and 17, 18 - P. fulvum (2523).
Isoforms of convicilin was localized on four positions (14, 15, 16 and 17) of electrophoretic spectra (Table 1). Isoform 15 was found with high frequency (0.35). The frequency of isoform 17 was equal to 0.32. Among 6 lines and varieties of P. sativum only two (14 and 17) isoforms were revealed (Table 2).
Table 1 - Frequency of convicilin isoforms in Pisum L.
Isoform Frequency
14 0.12
15 0.35
16 0.21
17 0.32
Total 1.00
Isoform 17 was represented with the highest frequency (0.83). In wild subspecies of P. sativum (24 accessions) only three (15, 16, 17) isoforms were found. Isoform 15 had the highest frequency (0.6). In accessions of wild species P. fulvum all revealed isoforms of convicilin were represented. Isoform 15 was found preferably (0.59).
Table 2 - Frequency of convicilin isoforms in various representatives of genus Pisum L.
Taxa Number of accessions Isoform Frequency
P. sativum - lines and varieties 6 14 0.17
17 0.83
P. sativum - wild subspecies 20 15 0.60
16 0.35
17 0.05
P. fulvum 34 14 0.18
15 0.59
16 0.17
17 0.06
Early it was studied that convicilin was controlled by one single locus [6]. Composition of convicilin bands (isoforms) in parental plants and F2 hybrids was analyzed in the PAP-485/4 x I609885 (P. fulvum) cross [1, 2]. Analysis of spectra revealed genetic segregation of convicillin isoforms 15 and 16. It was determined that isoforms of convicilin I609885 and PAP-485/4 were encoded by two codominant alleles of the same locus. Consequently, variation of convicilin could be determined by various alleles of single locus. But interesting results were obtained after dissecting the proteome of pea variety Cameor mature seeds [3]. The 33 spots related to convicilin were identified on 2D electrophoretic gels. It was known that convicilin do not exhibit post-translational processing and glycosylation [5]. Hence, such diversity of protein spots related to convicilin could be explained with presence of paralogous genes or unknown processing that remains to be elucidated [3].
If we proposed that existing bands represented four isophorms (alleles) the level of convicilin locus heterozygosity was Hj=1-Zipij2=1-0.5668=0.43. This value characterized moderate polymorphism which might be used in pea breeding. This value (0.43) was comparable with polymorphism (0.63) of pea microsatellites markers [10].
In pea variety Aest electrophoretic analysis revealed one seed which did not contained a band with molecular weight approximately 70 kDa (Fig. 2). This band represented storage protein convicilin. It was seem that among typical seeds of variety Aest we found the seed with distinguished spectrum. Genotype without convicilin could accelerate producing of pea innovative varieties with high quality of protein isolates.
m
66 kDa
Figure 2 - Electrophoretic spectra of P. sativum L. storage proteins: 1-3 - elatius 5322, 4-6 - Aest, 718 - F2 Deviz x elatius 5322, 19 - soybean variety Lantsetnaya. Absent of convicilin band in spectrum
of variety Aest (arrow).
CONCLUSION
Vicilin and legumin alone and in various compositions are able to form good protein gel. But convicilin can hinder physico-chemical properties of protein isolates. Therefore, pea with absence of convicilin would be served as desirable material for food industry. On the other hand, new unusual isoforms of convicilin could also have possibility to improve the quality of pea storage proteins. Polymorphism of convicilin was studied with use of 60 accessions of genus Pisum L. In total four isoforms of convicilin were revealed. Differences on convicilin isoform frequency between cultivated and wild pea accessions were revealed. The index of convicilin polymorphism in representatives of genus Pisum L. was equal to 0.43. This value characterized moderate level of polymorphism which might be used in pea breeding. In pea variety Aest electrophoretic analysis revealed one seed which did not contained convicilin. Use of pea accessions without convicilin in breeding could accelerate producing of innovative pea varieties with high quality of protein isolates.
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