Seed Storage Proteins

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Dec 11, 2012 (4 years and 8 months ago)

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1

Seed Storage Proteins


Biochemistry and Genetic Control of Zein Expression in Maize


READING
:



Schmidt, R.J. (1993).
Opaque
-
2
and zein gene expression. In: "Control of Plant Gene Expression".
Verma, D.P.S., ed. CRC Press, Inc., pp. 337
-
355.


INTRODUCTION



In cereals such as wheat, barley, rice and corn the bulk of the seed is made up of a storage organ
called the endosperm. Within the endosperm, starches, oils, and proteins are deposited during seed
development. Upon germination these complex organic mo
lecules will be broken down by the developing
seedling into sugars, fatty acids and amino acids that will be utilized during early seedling growth and
development.


In today's lab exercise we will concern ourselves with the deposition of the seed storage p
roteins.
The principal storage protein in the cereal endosperm is a group of related proteins collectively called
prolamins. The prolamins are characterized by their high content of proline and glutamine and low
content of lysine and tryptophan. In maiz
e the prolamins are called zeins, while in barley and wheat they
are called hordeins and glutelins, respectively.


During seed development, certain specialized regulatory and structural genes are turned on within
the endosperm cells. Often the expression
of these genes is restricted to this organ alone. The zein genes
are an example of structural genes of this type. Zein genes express proteins called zeins that accumulate
to high levels in special structures called protein bodies. They express a heterog
eneous group of proteins,
the majority of which fall into two size classes, 22
-
kDa and 19
-
kDa. The zein protein has no known
enzymatic properties and, like typical seed storage proteins, their sole purpose is to serve as a source of
amino acids for the de
veloping seedling following germination. The zeins make up about 60% of the total
endosperm protein fraction. These abundant proteins (called prolamins) are distinguished from other seed
proteins (the albumins and globulins) by their solubility propertie
s. Zeins are readily soluble in solutions
of alcohol, and very insoluble in aqueous solutions. The albumins are soluble in aqueous solutions while
the globulins become soluble in saline solutions.


The activation of zein genes is controlled by several
known regulatory loci. One of these, the
opaque
-
2

locus (
o2
), plays a central role in regulating the expression of a subset of the zein gene family.
Corn plants homozygous for mutations in
o2

produce seed with a dramatic reduction in total zein protein,
and generate kernel phenotypes that are opaque to transmitted light, as opposed to the normally
translucent endosperm of wild
-
type seeds.


An active area of research today is in understanding the mechanism by which regulatory loci
control the expression of

seed storage protein genes. Understanding how the product of regulatory loci
control the activation of other genes is central to unraveling the complex process of tissue
-

and temporal
-
specific gene expression. In addition, a thorough comprehension of th
e regulatory mechanisms
controlling seed storage protein gene expression may some day lead to the genetic engineering of seed
crops containing higher protein yields and better nutritional content.


In this lab exercise you will get a chance to observe th
e effect of the
opaque
-
2

regulatory mutation
on zein protein accumulation. You will compare the spectrum of total endosperm proteins in normal vs.

2

isogenic corn containing a mutation in the
Opaque2

(
O2
) regulatory gene that renders the O2 protein
inactive
. Because O2 regulates expression of a specific subset of the zein genes, the effect of the
o2

mutation on zein protein accumulation is most evident when the zein protein fraction is compared in
isolation from the other proteins. To this end you will iso
late the zein fraction and compare the wild type
zein profile to that in the o2 mutant.



Experimental Plan


1. Isolating and grinding the endosperm.



(a) Select 8
-
10 wild
-
type and 8
-
10 opaque seeds. These will be used in the endosperm protein
isolat
ion. Grind the seeds in a coffee mill to a coarse meal, then transfer the meal to a mortar and grind by
hand until you obtain the consistency of a
very fine

powder (note: this is not easily accomplished with the
wild
-
type sample, and may require the alter
nating efforts of each lab partner). If you do not obtain a fine
powder consistency, you will not get a good extraction of the proteins.


3

2. Total Endosperm Protein Isolation


a) Weigh 100 mg of the powdered endosperm meal (above) and transfer to a micr
ofuge tube.

b) Add 1 ml of 3X sample buffer. Vortex vigorously for 1 minute.

c) Put the microfuge tube at 65
o

C for 15 minutes, vortexing to resuspend the meal every 5
minutes.

d)

Pellet the insoluble material in the microfuge for 5 minutes and transfe
r the supernatant (avoid
the


pellet) to a clean microfuge tube.

e) To resolve the proteins on the basis of their size by SDS
-
PAGE (Poly
-
Acrylamide
-
Gel
-
Electrophoresis), each lab group should prepare two samples of wild
-
type and two of opaque
-
2 total
proteins as follows:



l of Extract




l 3X Sample Buffer



l of Water



30




20




100



50





0




100


Later, these samples must be boiled before loading on the gel to completely denature the protein in
the presence of the SDS. For now, plac
e these aside until the zein protein fraction is also ready to boil.


3. Zein Protein Isolation



a) Weigh 200 mg of the powdered endosperm meal and transfer into a microfuge tube.

b) Add l ml of 70% ethanol + l0mM DTT. Vortex vigorously for one min
ute.

c) Put the microfuge tube at 65°C for 15 minutes vortexing for 15 seconds every 3
-
5 minutes.

d) Pellet the insoluble material in the microfuge for 5 minutes and transfer the ethanol extract to a
clean microfuge tube.


e) To resolve the proteins o
n the basis of their size by SDS
-
PAGE, each lab group should prepare
two samples of wild
-
type and two of opaque
-
2 total proteins as follows:




l of Extract




l 3X Sample Buffer



l of Water



10




50




90



30




50




70


4. Preparing samples

for PAGE


a)

Boil the zein and total protein samples for 3 minutes. Let samples cool briefly at room
temperature, spin briefly in the microfuge to pull down moisture from the sides of the tube, and
briefly vortex to mix contents. Just before loading the gel
, gently rinse the wells of your gel
using a Pasteur pipet, to remove diffused salts and to better visualize the wells. Load 15

l per
sample onto the prepared 15% polyacrylamide/0.1% SDS gels. NOTE: include a molecular
weight standard! Load the equivalent amounts of wild
-
type and opaque
-
2 samples
side by side
and place the MW standard adjacent to one end of the samples
.


ZEIN Pr
otein Fraction



TOTAL Endosperm Protein

Loading order:
WT10/opaque10/WT30/opaque30/MWstand.
-----
WT30/opaque30/WT50/opaque50/MW


4


b) When all the samples are loaded, run the gels at 20 mA constant current until the dye enters the
separating gel (below t
he stacking gel) and then at 30 mA constant current until the dye reaches the
bottom (about 1.5 hours).


5. Visualizing the Zein Proteins



When the dye reaches the bottom, your protein gel should be ready to fix and stain.
Turn off the
Power Supply!

Fo
llow the instructions of the lab assistants and carefully separate the glass plates.
Gently lift the gel off the plate and place in the fixing solution.


a)

Fix the gel for 10 min at RT in 100 ml of FIXING SOLUTION. Put the container on the rocker
plat
form. This ensures sharp bands by preventing diffusion of proteins.


b)

Pour off the fixing solution and stain the gel over
-
night at room temperature in 100 ml of
STAINING SOLUTION and return it to the rocker platform. Be sure to cover your dish with sara
n
wrap to prevent evaporation.


During the Next Lab Class


c)

Destain in 10% acetic acid, 30% methanol at room temperature as long as necessary. Include a
Kimwipe in the dish to trap the stain. Change the destain a few times and continue as long as
neces
sary.


d)

Your gels can be photographed with a digital camera and each lab partner can include a color print
of the results in their notebook.



Can you identify the 22
-
kDa and 19
-
kDa zein proteins in the total protein samples and zein
samples?


Which

class is most affected by the opaque
-
2 mutation?


What about the other bands that appear above the 22
-
and 19
-
kDa bands?

Are they affected by the
mutation? Why did the extraction in alcohol preferentially isolate the zein proteins?


5

Solutions, Buffers,

and Reagents for Section 1
-

Proteins


70% Ethanol
-
10 mM DTT


3X SDS
-

PAGE Sample Buffer


0.2 M Tris
-
HCl, pH 6.8

4.5 % SDS

12 % ß
-
Mercaptoethanol

30% Glycerol

0.06% Bromophenol Blue


10x Laemmli Buffer



1L



Glycine



144.3 g


Tris Base



30.5 g


SD
S




10 g


FIXING SOLUTION








1L


Methanol








300 ml

Acetic Acid









100 ml

H
2
O









600 ml


STAINING SOLUTION






1L


Methanol








500 ml

Acetic Acid









100 ml

Coomassie Brilliant Blue R250






2.5 g

H
2
O









4
00 ml



DESTAIN








1L


Methanol








300 ml

Acetic Acid









100 ml

H
2
O









600 ml