Preparation of Polyclonal Antibody of Maize AGPL2 Protein and Analysis of Its Spatiotemporal Expression Analysis

doi:10.7668/hbnxb.2018.06.003

Abstract

Abstract: In order to investigate the functional mechanism of maize AGPL2 in starch accumulation during grain development,this study cloned AGPL2 gene and constructed the GST-tagged prokaryotic expression vector PGEX-6T-1-AGPL2. Using to the inducing expression system of Escherichia coli,GST-AGPL2 fusion protein was able to be obtained expression at high levels by inducing with 0.5 mmol/L was opropyl-β-D-thiogalactoside (IPTG) at 28℃ for 6 h. Then GST (Glutathione S-transferase) tag protein purification medium was used to obtain higher quality GST-AGPL2 fusion protein by affinity chromatography. The AGPL2 polyclonal antibody was prepared by immunizing New Zealand white rabbit with purified GST-AGPL2 recombinant protein,and the antiserum was isolated and purified. Then the GST tag of the antigen GST-AGPL2 fusion protein was removed by rTEV Protease,and Western Blot assayed with the purified AGPL2 polyclonal antibody showed that AGPL2 polyclonal antibody had high specificity and sensitivity and could be used to detect nanogram antigen protein. The distribution and expression patterns of AGPL2 protein in different tissues and pollination stages of maize was studied by Western Blot. The results showed that the expression of AGL2 protein in different tissues of maize was tissue-specific and the highest in endosperm. The expression level of AGPL2 protein increased first and then decreased during the different pollination period of maize endosperm,and reached the maximum in the middle pollination. The results were consistent with the accumulation of starch during the development of maize kernels,and indicated that AGPL2 was mainly present in maize endosperm and might be involved in the synthesis of starch. It also indicated that AGPL2 polyclonal antibody was highly specific and could recognize AGPL2 antigen in corn.

Key words: AGPL2 protein, Gene cloning, Prokaryotic expression, Western Blotting

CLC Number:

Q78
S513.03

LÜ Yanan, YU Guowu, HUANG Yubi. Preparation of Polyclonal Antibody of Maize AGPL2 Protein and Analysis of Its Spatiotemporal Expression Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(6): 17-23. doi: 10.7668/hbnxb.2018.06.003.

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