| Waterlogging or hypoxia stress directly affect the growth of maize seedling root system, waterlogging has become to one of the most important abiotic stress in restricting the development of maize production. Micro RNA(mi RNA) have been shown to play critical roles in regulating gene expression at the post-transcriptional level. At present, many experiments have established that mi RNA in plants play an important role in response to a variety of abiotic stress. Previous research has indicated that zma-mi R528 maybe is one of the mi RNA involved in root development and response to waterlogging. However, the mechanisms of zma-mi R528 expression on response to waterlogging stress is not clear.In our research, B73 seedling were used to investigate the influence of abiotic adversity stress on mi R528 expression. And we studied the relationship between natural variation within zma-mi R528 promoter sequences and the expression of zma-mi R528 under waterlogging, furthermore. We performed Yeast one-hybridassay, and we focused on revealing the Cis- and Trans regulatory mechanisms of zma-mi R528 expression on response to waterlogging. The main research results are as follows:1. Stem-loop RT-q PCR analysis indicated that zma-mi R528 in B73 root was significantly induced by ABA, NAA, GA3, KT, Ethylene, Na Cl and 10°C low temperature.2. The promoters of zma-miR528 in maize inbred lines were cloned by PCR. In tatol, we got 130 sequences of the zma-mi R528 a promoter and 122 sequences of the zma-mi R528 b promoter, respectively. B73 genome sequence was used as a reference. The result of the sequences analysis revealed genetic diversity, and particularly in the zma-mi R528 a promoter with 218 bp deletion in-1429 bp to-1647 bp and the zma-mi R528 b promoter with 1077 bp insertion in-637/-636 bp and 248 bp insertion-363/-362 bp respectively. Based on the result of the sequences analysis, the promoter sequences can be divided into 2 class: a set of serial sequence is similar to that of Mo17 and B73, and another group of serial sequence is similar to that of Hz32.3. There were 100 polymorphic sites within zma-mi R528 a promoter in 130 maize inbred lines, including 22 In Dels and 78 SNPs; and there were 60 polymorphic sites within zma-mi R528 b promoter in 122 maize inbred lines, including 12 In Dels and 48 SNPs. Association analysis indicated that 8 sites were significant associated with zma-mi R528 expression under waterlogging, and we detected 6 and 2 sites within zma-mi R528 a and zma-mi R528 b promoter, respectly. These 8 sites were in strong LD(r2>0.9) within zma-mi R528 a and zma-mi R528 b promoter, and formed 4 main haplotypes. The sequnses of 6 sites within zma-mi R528 a promoter formed 2 main haplotypes, haplotype1 and haplotype2, the sequnses of 2 sites within zma-mi R528 b promoter formed 2 main haplotypes, haplotype3 and haplotype4. The expression change of zma-mi R528 in maize inbred lines between haplotype1 and haplotype2 were significant different under waterlogging; and the expression change of zma-mi R528 in maize inbred lines between haplotype3 and haplotype4 were also significant different under waterlogging. The 8 sites were regarded as candidate function sites to regulate zma-mi R528 expression under waterlogging.4. The cis-acting element RHERPATEXPA7 was predicted in haplotype1 sequence. And two motifs which have high homology with the cis-acting elements of OBP and OBF located at the sequence of haplotype1, and the transctiption factors OBP1 and OBF can interact with each other. The cis-acting element LTRECOREATCOR15 was predicted in haplotype2 sequence, and 3 motifs which have high homology with ABRELATERD1 were found within the sequence of haplotype2. No cis-acting element was predicted within the sequence of haplotype3 and haplotype4.5. We have finished to construct the quality of c DNA library. And we constructed 2 p Bait-Ab Ai vector: 528a1-Ab Ai, the-545 bp to-10 bp sequence of zma-mi R528 a promoter in B73 containing the sequence of haplotype1; 528a2-Ab Ai, the-526 bp to-10 bp sequence of zma-mi R528 a promoter in Hz32 containing the sequence of haplotype2. p Bait-Ab Ai vectors have been transformed into yeast strain Y1 H Gold. And. Now, we are screening a one-hybrid c DNA library. |
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