The Protein Expression Differences Between Opaque2 NILs In Maize

As an important food and forage crop, corn has the largest growing area in our country. With the improvement of living standards, people pay more attention to its nutritional quality. But corn has low content of amino acids, among which lysine necessary to people has limited effect on the promotion of its nutritional quality. So only by increasing the content of lysine can we improve corn’s nutritional value. Although the varieties carrying the homozygous allele o2 have a high level in lysine content, the mutant gene brings some adverse traits.In order to study the function of Opaque2, our laboratory constructed a few pairs of near isogenic lines(NILs). The 18 days after pollination(DAP) grain endosperm of Liao2345 and Liao2345/o2, among which genetic coefficient reached 91%, are used for two-dimensional electrophoresis(2-DE). Based on the results of 2-DE, we studied the composition and microstructure of the NILs. The main results are as follows: 1. The nutrients, including crude protein, crude starch, crude fat, total amino acid content and each content of 17 amino acids, were compared between the NILs Liao2345 and Liao2345/o2. Compared with Liao2345, the content of fat, protein and total amino acid of Liao2345/o2 decreased significantly; 14 kinds of amino acid were significant difference, especially lysine, which improved by 76.5%. 2. Mature grains and grains of 22 DAP were used for scanning electron microscopy(SEM) and transmission electron microscopy(TEM), respectively. Observation found that the starch grains of Liao2345 were rectangular or polygonal, packed closely, and sheath proteins were distributed in clearance of starch granule; starch grains of Liao2345/o2 were arranged round or oval loosely, and matrix proteins were decentralized in clearance of starch granule. 3. 12 different expressed protein points in 2-DE were selected for q RT-PCR, for detecting gene expressions of 10 DAP, 14 DAP, 18 DAP, 22 DAP and 26 DAP of Liao2345 and Liao2345/o2. We found results as below. Compared with Liao2345, gamma zein precursor gene(LOC541920), serine hydroxymethyl transferase gene(Shmt), Pyruvate orthophosphate dikinase(Ppdk), ubiquitin conjugating enzyme gene(Ubc), copper zinc superoxide dismutase gene(Sod4ap), lactoylglutathione lyase gene(Lgl), sucrose synthase gene(Sh1) and S-homocysteine methyltransferase 3 homologue gene(Hmt3) of Liao2345/o2 were expressed in lower level; Bifunctional Hageman Factor Amylase Inhibitor gene(Chfi), legumin 1 gene(Leg), sorbitol dehydrogenase gene(Sdh) and glucose-1-phosphate adenylyltransferase large subunit gene(Sh2) were expressed in higher level. We concluded that OPAQUE2(O2) could activate or promote the expression of LOC541920, Shmt, Ppdk, Ppdk, Ubc, Sod4 ap, Lgl, Sh1, and could inhibit the expression of Chfi, Leg, Sdh and Sh2. The specific mechanisms need further investigation. 4. As key enzymes in starch synthesis pathway, Sucrose Synthase(Su Sy) and ADP-Glucose Pyrophosphorylase(AGPase) were selected for enzyme activity analysis. Comparing with Liao2345, the results showed that Su Sy had a higher activity in 10 DAP and 14DAP; AGPase had a higher activity in 14 DAP and 26 DAP. Furthermore, we detected the enzymes’ activity of other five NILs. The results showed that the wild types had higher Su Sy activity, except for D huang 212 and lower AGPase activity, and the activity of the two enzymes in Zheng 58 and Zheng58/o2 were of no significant difference. Those were consistent with the results of two-dimensional electrophoresis. So, we concluded that O2 had a promoting effect on Su Sy and an inhibiting effect on AGPase. 5. Yeast two hybrid(Y2H) system was used to verify whether O2 could interact with the coding products of Sh1 and Sh2. Recombinant carriers p GBKT7-X(X=Sh1, Sh2) were co-transformed with p GADT7-O2 and p GADT7 into yeast strains AH109, respectively. We found that SH1, the product of Sh1, had the ability of self-activation and could interact with O2; SH2, the product of Sh2, could interact with O2 strongly. 6. Yeast one hybrid(Y1H) system was applied to detect if O2 can control transcription of Sh1 and Sh2. Recombinant carriers p Lac Zi-Y(Y=Sh1-1,Sh1-2,Sh1-3,Sh1-4,Sh1-5,Sh2-1,Sh2-2,Sh2-3,Sh2-4)were co-transformed with p B42AD-O2 and p B42 AD into yeast strains EGY48, respectively. We found that Sh1-1, Sh1-2 and Sh2-4 had self-activate ability, for their strains turned blue, and O2 could combine with Sh1-1, Sh1-2 and Sh2-4 in a deeper blue color. 7. From what has been discussed above, we thought that O2 could promote the expression of Sh1 and interact with SH1, which induced high level of RNA, SH1 and Su Sy. Besides, O2 could inhibit the expression of Sh2 and interact with SH2, resulting in low level of RNA, SH2 and AGPase.