STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS

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1 STUDIES ON SEED STORAGE PROTEINS OF SOME ECONOMICALLY MINOR PLANTS THESIS SUBMITTED FOR THE DEGREB OF DOCTOR OF PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF CALCUTTA 1996 NRISINHA DE, M.Sc DEPARTMENT OF BIOCHEMISTRY BOSE INSTITUTE CALCUTTA

2 CONTENTS Page No INTRODUCTION Seed development Seed grain proteins Characteristic features of seed storage proteins Complexity of seed storage proteins Homologies between storage protein and their genes Storage protein synthesis and deposition Regulation of storage protein synthesis Regulatory sequences of storage protein genes Expression of seed protein genes in transgenic plants Implications of nutritional improvement of seeds by transgenesis Expression of plant genes in prokaryote Importance of economically minor seeds as food sources Aim and Scope of present study 28 MATERIALS AND METHODS Materials Methods RESULTS AND DISCUSSION SECTION -I characterization of 2S albumin from the seeds of Chenopodium album and its accumulation during seed development SECTION -II Characterization of storage proteins from seeds of sterculia foetida SECTION -III Cloning and sequencing of 2S protein gene of Chenopodium album GENERAL DISCUSSION SUMMARY REFERENCES PUBLICATIONS 119 1

3 The work mainly describes the importance of seed storage proteins of two economically minor plants, Chenopodium album and Sterculia foetida. Three nutritionally balanced seed storage proteins were isolated, characterized and their amino acid composition analysed. These balanced proteins may be used as protein food suppliments for animals and human beings. An attempt was made to isolate 2S protein gene of Chenopodium album by conventional method and the gene appears to be a potential candidate for plant improvement by using modern biotechniques. The work is summerised below: t Chapter - I ] 1. Fresh weight of growing Chenopodium album seeds increased linearly from the first week after anthesis upto third week then decreased slowly during dehydration stage. Dry weight of the seeds was found to increase more slowly at the begining and on the subsequent stages it followed a sigmodial pattern. 2. The amount of DNA was almost constant upto second week and then increased slowly until fifth week. The level of RNA increased linearly but slowly upto third week and then rapidly increased to its maximum level at the fourth week after flowering. The accumulation of 99

4 storage proteins followed almost a sigmodial pattern, begining at the second week and continuing upto fifth week. An analysis of SDS-PAGE pattern of seed storage proteins of C^. album at different stages of development revealed that deposition of major classes of seed storage protein started from the second week after flowering. In subsequent days most of the proteins accumulated showed the pattern that of matured processed proteins. A low molecular albumin from Chenopodium album was purified by molecular seive chromatography on Bio-gel P-60 column. It was characterized with respect to its sub-units structure, amino acid compositions and antigenecity. This albumin was found to contain nutritionally balanced amino acid composition comparable to that of recommended values of World Health Organisation (WHO) for an ideal protein. Again this albumin was found to be antigenically homologous with the seed storage proteins of some phylogenetically related species belonging to Chenopodiacae. Amaranthuceae and Basilaceae. but not to that of unrelated dicots. 100

5 [ Chapter II ] Gradual accumulation of major storage proteins in Sterculia foetida seeds were traced by SDS-PAGE analysis. Two proteins, an albumin and a globulin, were purified from S_;_ f oetida seeds through chromatography on sephadex G-150 column and their subunits composition patterns were studied on SDS-PAGE. Their amino acid composition matched closely to an ideal protein milk casein, and was also comparable to the recommended values by World Health Organisation (WHO) for an ideal balanced protein for human consumption. Antibody against the purified globulin was raised in rabbit. Sterculia globulin specific antibody was found to crossreact with seed proteins of phylogenetically related species of Sterculiaceae family, but not with other dicots. [ Chapter - III ] The gene for 2S albumin of Chenopodium album seeds, was chosen as a potential candidate gene for use in seed protein quality improvement by biotechnological approaches, with a view to clone the cdna of this, total RNA was extracted from mid-matured seeds of Chenopodium. Poly A + RNA was purified from extracted 101

6 total RNA by oligo dt-cellulose column chromatography. Poly A + RNA was reversed transcribed to corresponding complementary DNA (cdna) by using reverse transcriptase enzyme (RAV-2), Random hexamer primers were used for cdna synthesis. Complementary DNA (cdna) synthesis was monitored by autoradiography using radiolabled -P 32 datp. A plasmid expression vector pgex-2t was selected for library construction. This plasmid vector can efficiently synthesize forign polypeptide from the inserted gene or gene fragment as fusion protein with Glutathion S Transferase (GST) protein. Recombinant colonies were freshly grown on LB-amp plate overnight were transferred onto a nitrocellulose membrane and induced by 5 mm IPTG. Induced colonies on the membrane were lysed in presence of 5% SDS. The bound bacterial protein on the membrane then probed with antibody and developed according to the Western blot protocol. Two such immunopositive colonies (pgca2s-l and pgca2s-2) were identified by screening the colonies. One colony (pgca2s-l) was further studied in details. The cell lysate from this colony was prepared and run 102

7 on a SDS-PAGE. A fusion protein of around 45 Kd was located on the gel, which was found to crossreact with the antibodies raised against the 2S albumin of Chenopodium by Western blot analysis. Plasmid DNA from this colony was prepared. The insert DNA of 1.0 Kb was separated following EcoR 1/ BamH I digestion. The insert DNA was eluted from low melting agarose gel and cloned in pbsks (+) plasmid vector in corresponding restriction sites (EcoR I and BamH I).The The recombinant pbsks (+) clone was isolated and designated as pbsca2s-l. The sequencing of the DNA insert from the pbsca2s-l plasmid was done by dideoxy chain termination method us ing T 3 and T 7 primers and sequence o f the insert DNA was read. Deduced aminoacid sequence in all possible frames in the forward and reverse complementary sequences were obtained by computer analysis and was compared with published aminoacid sequences of several 2S albumin from different other dicotyledonous plant seeds to arrive at the correct alignment. This cdna clone will be useful in fishing out the desired gene from a Chenopodium genomic library. Further work along this line is in progress. 103