Studies On The Relationship And Regulation Mechanism Of ABI3-like On The Storage Protein Genes During Soybean Seed Development

Abscisic acid insensitive 3 (ABI3) has a regulatory effect on the accumulation of storage proteins, as well as on the dormancy, quality and drought tolerance of Arabidopsis seeds during development. The proteomics research of storage proteins is already well understood; however, the mRNA expression patterns of the ABI3-like genes and their function in soybean remain unknown. There were two ABI3-like transcription factor genes in soybean, GmABI3-8 and GmABI3-18, respectively. Studies on the expression patterns and regulation mechanism between ABI3-like and the storage protein genes, then clarified the role of ABU3-like in promoting the expression of storage protein genes, which was of great significance to improve the protein content of soybean.This study selected high protein soybean varieties NC111-1 which was introduced from the United States as the test material for the study of the expression patterns and interaction mechanism of GmAI3-8, GmABI3-18 and storage protein gene Gyl, Gy2, Gy3, Gy4, Gy5, Gy7,7sa,7s?' and 7s?. The main research contents and results were as follows:(1) The physiological data included seeds and pods size, dry weight, fresh weight, water content and protein content were measured. The results showed that the size variation of pods and seeds were basically the same and changed with development period. The growth rate of pods faster than seeds. There was a significant difference of pods and seeds between the development stage and the dehydration stage. The water content of seeds decreased gradually. The fresh weight was increased gradually in the early stage and then decreased during dehydration stage, while dry weight increased slowly. The protein content was maintained at 42.31%±5.25% level, the maximum appeared in the 20d. The variation curve of a single seed protein quality was "S" type. The development period of seed can be divided into early protein synthesis stage (10d-30d), rapid protein accumulation stage (30d-80d) and slow protein accumulation stage (80d-100d), and the difference between the three was obvious. The rapid protein accumulation stage was a key period of storage protein synthesis.(2) The mRNA expression patterns of GmABI3-8 and GmABI3-18 was detected by RT-qRCR technique. Expression level differences of different tissues in the same period of soybean plants were analyzed. The results showed that GmABI3-8 and GmABI3-18 specific expression in seed and the expression level was significantly higher than that of root, stem, leaf and flower (P<0.05). The expression patterns of GmABI3-8 and GmABI3-18 in the whole development period of seed was analyzed. The results showed that the patterns of GmABI3-8 and GmABI3-18 expression were basically the same and the subtle differences between the two may be caused by genetic evolution. The change of expression level in cotyledon and hypocotyledonary axis during seed germination process were analyzed. The results showed that The mRNA expression level of GmABI3-8 and GmABI3-18 decreased significantly during the seed germination process (P<0.05). The results speculated that their function were related to storage protein accumulation, seed dehydration, dormancy and inhibition of seed germination.(3) The mRNA expression patterns of each storage protein gene was detected by RT-qRCR technique. The relationship between storage protein genes and transcription factor gene GmABI3-8 and GmABI3-18 was analyzed. The results showed that the mRNA expression patterns of storage protein genes (excluding Gy7) exhibited single peak curves and have temporal specificity, with high expression level in 40d-70d. The three highest total expression level of storage protein genes over the whole development period from high to low was 7s?, Gy1 and 7s?, significantly higher than other genes (P<0.05). The time of 7s? started high level expression was the earliest, while the expression level of 7s? was significantly higher than that of other genes at 70d (P<0.05). The expression patterns of Gy7 exhibited double peak curve and its expression level was significantly lower than that of other genes (P<0.05). The expression level of ABI3-like and storage protein genes was significantly positively linearly correlated in 10d-70d (P<0.05, r>0.8). The expression level of GmABI3-8 was more significantly linearly correlated with the expression level of storage protein genes than that of GmABI3-18.(4) By yeast one-hybrid system, the molecular mechanism of Gy2 promoter with GmABI3-8 and removal of RY repeat element Gy2 promoter with GmABI3-8 were analyzed, respectively. The results showed that GmABI3-8 transcription factor could be combined with RY repeat element in yeast one-hybrid system and promoted storage protein genes expression. RY repeat element as a transcriptional activity regulation factor and related with the storage protein genes expression. The promoter activity of the missing RY repeat element was significantly decreased.In summary, this study was the first to investigate the regulation mechanism of ABI3-like on the storage protein genes during the development of soybean seed and confirm that the soybean ABI3-like transcription factor promoting the expression of the storage protein genes by binding RY repeat element presented in the promoter of storage protein genes. This study provided a theoretical basis for high protein content soybean breeding.