The Molecular Regulation Mechanism Of Starch Synthesis By ABI4 In Maize Endosperm

Starch is the final product of photosynthesis,and is also the main grain storage substance of crop grains.Crop grouting process is basically dependent on the process of starch accumulation.Many studies have shown that grain weight is closely related to the activities of key enzymes of starch synthesis during the grain filling process.Some Studies indicated that the transcription factor ABI4 regulates the expression of some key enzyme genes of starch synthesis.Thus,in this study,we made the transgenic maize lines with overexpressed ABI4 gene and analysed the function and the molecular regulation mechanism of the transcription factor ABI4 in maize endosperm starch synthesis pathway.The main results are as follows:1.ABI4 was cloned and inserted into the downstream of the ubiquitin promoter of modified pCAMBIA1305.1 vector.The over-expression vector was transformed into Agrobacterium EHA105 and transformed into maize backbone inbred lines 18-599 by infecting the immature embryo.The transgenic maize lines with overexpressed ABI4 gene was obtained by induction of callus induction and seedling differentiation.Positive transformants of T0-T2-generation plants were selected by histochemical staining for GUS,PCR identification,quantitative detection and protein Western Blotting detection.2.We extracted the total RNA of 25 DAP(days after pollination)endosperm of the T2 generation plants,and did the real-time quantitative PCR to analyse the impact of ABI4 on the key enzyme genes of starch synthesis in endosperm.The results showed that,for the ADPG-related genes(ADPG,the direct substrate of starch synthesis),overexpression of ABI4 can improve the expression of AGPL2,AGPLS4 and Sh2 in endosperm;For the starch synthase enzyme gene,overexpression of ABI4 can increase the expression of GBSSI and suppress the expression of SS?a,SS? in endosperm;For starch branching enzyme genes,overexpression of ABI4 can increase the expression of SBE? and SBE?b;For starch debranching enzyme genes,overexpression of ABI4 can promote the expression of ISA1 and ISA2,while suppress the expression of Zpul;For starch phosphorylase genes,overexpression of ABI4 can suppress the expression of PHOH and PHOL.3.We separated the 25 DAP(days after pollination)endosperm of wild-type and T2 generation transgenic plants and measured the starch content.The result showed that the grain starch content of transgenic plants is significantly higher than the wild-type plants.Then we measured the starch chain length distribution of the 25 DAP endosperm by ICS5000+ ion chromatography to test the 25 DAP endosperm starch chain length distribution between wild-type and transgenic T2 generation transgenic plants.The result showed that the grain starch content of transgenic plants is higher than wild-type in chain length of dp6-19,while little difference in chain length of dp20-35.