Screening for Glutelin Subunit Mutants and Identification of altered GBSS Induced by Sodium Azide Mutagen

Young-Seop Shin1*, Yong-Weon Seo2, Ji-Ung Jeung3*, Hae-Yeong Ryu1, Young-Tae Lee11 National Institute of Crop Science, RDA, Suwon 441-100, Korea

2 Korea University, Seoul 136-701, Korea, 3 IRRI-Korea Office, RDA, Suwon 441-100, Korea

INTRODUCTIONINTRODUCTIONThe enhancement of seed storage protein quality is one of the import

ant breeding objectives in rice breeding for quality improvement. Since seed storage proteins are important in terms of nutritional value, the main objective of research was on the genetic research for improvement of seedstorage protein in rice. The purpose of this study was to obtain various types of mutations for glutelin subunits and GBSS using chemical mutagen,sodium azide (SA) in order to supply materials for improving rice seed quality.

MATERIALS AND METHODSMATERIALS AND METHODSPlant materialsPlant materials Dry seeds of rice, Oryza sativa L. cv. Suweon 472, which is non-glutinous, were treated with chemical mutagen, sodium azide (SA). Seeds of total 144 M2plants that had been selected for observable seed phenotypic variations were incorporated in screening of mutants.GlutelinGlutelin preparation preparation Two grains were crushed with mixed miller and placed insidea eppendorf tube filled with ethanol to exclude alcohol soluble fractions. The mixture was vortexed. After centrifugation, supernatant was removed. The pellet was rinsed twicewith DMSO and once with ethanol. After pellet dry, the mixture was added by 2X lane buffer and mixed gently. After adding dithioerythritol (DTE), the mixture was boiled. Aftercentrifugation, glutelin was transferred to a new eppendorf tube. The glutelin sample was mixed with 4-vinylpyridine solution and heated at 55 C. After adding DTE solution, themixture was heated at 55 C.1D SDS1D SDS––PAGEPAGE Samples were loaded onto the gel and were subjected to electrophoresis at 999 V, 20 W. Following electrophoresis, separated proteins were dyed with Coomasie Brilliant Blue R-250 or Silver-stained to observe either presence or absence ofglutelin subunits.Starch granule preparationStarch granule preparation Starch granules were extracted from flour samples.The samples mixture was incubated with shaking. After incubation, the mixture was centrifuged and supernatant was discarded. The second extraction was conducted as donein first extraction procedures. After second extraction, the pellet was washed twice withdistilled water. After centrifugation, pellet was washed twice with acetone and vacuum dried.GBSS preparation and separationGBSS preparation and separation In order to purify GBSS, dry starch was prepared and extracted using by lane. Di-sulfide bond formation was ceased by addition ofDTE solution. The GBSS was fractionated by 1D SDS-PAGE. The GBSS samples wereloaded in two gel formats and electrophoresis was done. After GBSS fractionation by electrophoresis, one part of gel which ran in the same running condition was silver stained. The other counter part of gel was used for immunoblotting assay.ImmunoblotImmunoblot analysis analysis After GBSS fractionation by electrophoresis, a transblot semidry electrophoretic transfer cell (BioRad) was constructed. Proteins of prepared blottinggel were transferred to nitrocellulose membrane by a semidry transfer cell set. After protein transfer, membrane was incubated and dried overnight. Gel attached area of membrane was blocked with blocking solution by constant agitation. After 2 hours of blocking,membrane was rinsed with TBS buffer in two times. Membrane was incubated with primary antibody (AWPab) diluted in incubation buffer.

RESULTSRESULTS Among 144 M2 mutant lines that showed Among 144 M2 mutant lines that showed phenotypicallyphenotypically different seed tr different seed traits with aits with SuweonSuweon 472, about 9% exhibited 472, about 9% exhibited glutelinglutelin subunit changes ( subunit changes (TableTable11).).•• The screening was performed by comparing the profiles of sodium The screening was performed by comparing the profiles of sodium dodedodecylsulfatecylsulfate polyacrylamidepolyacrylamide gel electrophoresis with that of the original culti gel electrophoresis with that of the original cultivar, var, SuweonSuweon 472. With some exceptions, we have been able to select lacki 472. With some exceptions, we have been able to select lacking or decreased mutant lines for each of the bands of major proteins withng or decreased mutant lines for each of the bands of major proteins withmolecular weights of 80 molecular weights of 80 kDakDa. . ElectrophoreticElectrophoretic analysis of analysis of glutelinsglutelins reveale revealed that the range of 97.4 d that the range of 97.4 kDakDa and 45 and 45 kDakDa were considered as subunit variat were considered as subunit variation occurrence region. Selected 13 ion occurrence region. Selected 13 glutelinglutelin mutant lines were classified in mutant lines were classified into 3 types on the basis of variations of the relative contents of the polypeto 3 types on the basis of variations of the relative contents of the polypeptides (ptides (Fig1 and Fig2Fig1 and Fig2).).•• GBSSsGBSSs were detected about 60 were detected about 60 kDakDa with observable two fractions (44109 with observable two fractions (441095, 442297). One was a more dense and the other was a faint upper band. L5, 442297). One was a more dense and the other was a faint upper band. Line (440874) that was dull type showed missing upper band. ine (440874) that was dull type showed missing upper band. SuweonSuweon 472 472was identical to two floury mutant rice (441095, 442297). Other lanes werewas identical to two floury mutant rice (441095, 442297). Other lanes werenot detected any GBSS bands. This result was corresponded to the phennot detected any GBSS bands. This result was corresponded to the phenotypic observation or iodine staining, which determine floury, dull and waotypic observation or iodine staining, which determine floury, dull and waxy (xy (Fig3Fig3).).•• In order to identify for GBSS specific bands, In order to identify for GBSS specific bands, immunoimmuno assay using assay using AWPaAWPabb was conducted (Fig. 4). was conducted (Fig. 4). AWPabAWPab was hybridized to about 60 was hybridized to about 60 kDakDa GBSS. GBSS.Although SDS-PAGE was detected different density, the separated GBSSAlthough SDS-PAGE was detected different density, the separated GBSSfractions were equally bound with fractions were equally bound with AWPabAWPab. . AWPabAWPab reacted only fragment reacted only fragmentof 60 of 60 kDakDa GBSS in dull type mutant (440874). Group A, B, and C associate GBSS in dull type mutant (440874). Group A, B, and C associated fragment presented in SDS-PAGE were not detected in d fragment presented in SDS-PAGE were not detected in immunoblottingimmunoblotting((Fig4Fig4).).

ConclusionConclusion

Treatment of the mature seed with sodium Treatment of the mature seed with sodium azideazide was found to be an was found to be aneffective method to induce mutations for storage proteins in protein bodieeffective method to induce mutations for storage proteins in protein bodies of rice. The use of SDS-PAGE techniques has enabled the arranging of as of rice. The use of SDS-PAGE techniques has enabled the arranging of asystem by which the changes in the various seed storage protein componsystem by which the changes in the various seed storage protein components can be extracted and detected in a relatively simple manner and theents can be extracted and detected in a relatively simple manner and theselection of seed storage protein mutants has been facilitated by a methoselection of seed storage protein mutants has been facilitated by a method in which the endosperm of the M3 seed is examined directly. Altered Gd in which the endosperm of the M3 seed is examined directly. Altered GBSS could also be induced by mutagen treatment. In this experiment, GBBSS could also be induced by mutagen treatment. In this experiment, GBSS was successfully identified and mutants were screened with altered GSS was successfully identified and mutants were screened with altered GBSS.BSS.

* Corresponding authors (shinys@rda.go.kr, jju@rda.go.kr)

Table 1. Types and frequencies of induced mutants by SA treatment

Figure 1. Diagram of glutelin subunit profiles as fractionated by 1D SDS- PAGE.lane 1: Molecular size markerlane 2: Suweon 472,lane 3: type A mutantlane 4: type B mutantlane 5: type C mutant.

Figure 2. 1D SDS-PAGE profiles of M3seed derived by individual M2 mutant.lane 1: Suweon 472lane 2: molecular size marker.Type A mutants : lanes 3, 5, 10, 13, 14, 18, 19.Type B mutants : lanes 8, 9, 12, 15, 17Type C mutant : lane 7.

Figure 3. SDS-PAGE patterns of starch granule-bound proteins from mutated lines.

The arrowsThe arrows indicated GBSS fraction which wasindicated GBSS fraction which was61 61 kDakDa. A, B, and C were. A, B, and C were other protein subunitsother protein subunitswhich were only observed in waxy lines.which were only observed in waxy lines.

Figure 4. Immunoblotting of GBSS from mutated lines.

lane 1, 3, 19, and 20; Suweon 472,lane 4-16; mutant lines having altered GBSSlane 17-18; mutant lines having non-altered GBSS.

The arrows were GBSSThe arrows were GBSSfraction hybridized witfraction hybridized with h AWPabAWPab..

lane 1-2; molecular size markerlane 3, 19, and 20; Suweon 472lane 4-16; mutant lines having altered GBSSlane 17-18; mutant lines having non-altered GBSS.