| Maize (Zea mays L.) is an important crop as food and feed, It plays significant role in agricultural production. Normal maize lack essential amino acid, such as lysine and typtophan, which reduce its the nutritional walue. opaque-2 mutant gene can substantially increase grain lysine content, Therefore, it has become one of the importat ways to improve the quality of existing maize by selecting and breeding high quality protein maize (QPM) which containing the o2 gene and endosperm modifier gene. Backcrss breeding is one of the important ways to breeding QPM, Constructing of o2-NILs by introduce the o2 gene into inbred line can broaden the Germplasm Base of QPM in China. but this method used in o2-NILs construction remain some problems.The methods used in QPM breeding project of our laboratory are introduced in this paper, and the problems of this method are discussed. in addition, most of the previous studies on genes which are regulated by OPAQUE-2 transcription factor are mainly concentrated in the zlcohol soluble protein, amino acid mentalism, plant resistance and so on, rarely involved in starch synthesis related genes. In our laboratory, we used proteomics research found that the expression level of SH1 and SH2 protein was significantly different in o2-NILs, Therefore,02 gene may play an important role in the regulation of the maize starch synthesis process. This paper takes o2-NILs as the study material by the yeast one hybrid, qRT-PCR, Grain Starch Assay and preliminary analysis of 02 transcription factor by affecting SH1 and SH2 expression in maize grain starch synthesis pathway. The main results were as follows:1. Based on our previous studies 44 normal maize o2 near isogenic lines and 3 waxy maize of o2 near isogenic lines were constructed by SSR molecular marker assisted selection. Meanwhile we found the whole process in construction of common maize o2-NILs use phiO57 marker; Construction of o2-NILs waxy maize in backcross generation using phiO57 marker select o2 gene, selfing generation use phiO57 and phiO27 screening o2wx gene.2. Using the yeast single hybridization experiment technology to research whether 02 transcription factor can recognize and bind the promoter of Sh1, Sh2. The results showed that 02 protein can interact with sh1-1, Sh1-3, Shl-5 and Sh1-9 fragments of Sh1 gene promoter and 02 protein can interact with Sh2-7 fragment of Sh2 gene promoter.3. Transcription analysis of different AGPase subunit gene Sh2 in different o2-NILs. We found results as below, in zhong 97-7/o2, Sh2 at 14 DAP,18 DAP,26 DAP expression level were 1.43,1.89,1.44 times in zhong 97-7 and the difference reached significant level (P14DAP= 0.05, P18DAP=0.05, P26DAP= 0.03); in CAL58/o2, Sh2 at 14 DAP,20 DAP,26 DAP expression level were 1.84,1.83,1.79 times in CAL58 and the difference reached significant level (PhDAP=0.02?P22DAP=0.05. P26DAP=0.05). After 14 DAP Sh2 gene transcription level in waxy maize and normal maize o2 introgression lines were higher than the wild type,02 transcription factor plays a negative role in the regulation of Sh2 gene.4. Analysis of starch content of waxy maize inbred lines zhong 97-7,300 male, zhong 501 and their corresponding o2-NILs. We found the crude starch content of 97-7/o2 (72.16%) was higher than that of 97-7 (60.99%), and the difference reached significant level(P=6.6E-5); 300 male/o2 crude starch content(71.31%)higher than 300 male(65.72%) and the difference reached significant level (P=8E-5); The crude starch content of zhong 501/o2 (71.34%) was higher than that in zhong 501 (67.21%), and the difference reached significant level (P=8.4E-4)5. Through the above 2,3,4 experimental results we speculate the 02 transcription factor is a repressor of Sh2 transcription. The wild type 02 gene can encode 02 protein properly,and it can combine with the promoter of Sh2, and restrain transcriptional regulation of Sh2 in wild type, leading to the grain starch content increased eventually. |
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