Expression Profile And Genetic Transformation Of MicroRNA During Embroonic Callus Formation In Maize

As one of important breeding methods, genetic engineering breeding have brought great benefits to agricultural production, directional seed breeding was also realized. The acreage of transgenic crops in creased rapidly since1996, which have made tremendous contributions to global food security. Embryonic callus is an important recepoter material in transgenic, but the induction rate is greatly influenced by the material genotypes. Currently majority of maize inbred lines can not be used directly as a receptor material, because of it’s low induction rate for embryogenic callus. Therefore, it is important to study regulatory mechanisms for maize immature embryos callus in molecular level. miRNAs are endogenous non-coding RNA, which can be involved in various aspects of the process of the plants, by cutting and translation inhibition of target genes, such as cell differentiation, organ morphogenesis, hormone regulation, and so on. In this research, we used18-599R as the research materials that picked from pollination11-13d of immature embryos (1.0-1.5mm), for induction maize embryogenic callus. According to the feature of the immature embryo, the formation progress of embryos callus can be divided into four stages:immature embryo as control, embryo intumesces (1-5d after inoculation), initial callus period (6-10d after inoculation), mature embryonic callus period (11-15d after inoculation). We took total RNA from immature embryo and1-15d induction process of embryogenic callus, then mixed every periods total RNA in the same quality. We choused four sample as deeping sequencing test materials, and detected by real-time fluorescence quantitative. At the same time, the miRNA expression vector were introduced into maize embryonic callus and shoot apical meristems by agrobacterum-mediated transformation, preliminary validation of the role of miRNAs in maize immature embryo induction process. The results obtained are as follows:1. In the embryogenic callus formation process with immature embryo in maize,78differentially expressed miRNA were detected by solexa deeping sequencing, belonged to20miRNA conserved miRNA families, which includes13newly discovered members of the conservative family, belonged to8miRNA families:zma-miR528、zma-miR172、 zma-miR167、zma-miR319、zma-miR393、zma-miR396、zma-miR397、zma-miR408.The sequence between new miRNA and conservative family miRNA found only1-2base difference.2. Seven new miRNAs were discovered. They belonged to four miRNA families, and tentatively named as zma-miR701, zma-miR702, zma-miR703and zma-miR704. At the same time seven new miRNAs are successfully predicted. So we speculated that these seven miRNAs are newly discovered in maize.3.13candidate miRNA expression by deep sequencing were analyzed by RT-qPCR(real-time fluorescence quantitative PCR). The results showed that apart from miR1671and miR827is going downward, the rest of the selected11are expressed in raising trend, several among-st these11are slightly lower after increase. The quantitative results and deep sequencing results are basically identical.4. Targets of candidate miRNA by the RT-qPCR were predicted by online analyzing tool. Some of the miRNA target genes are transcription factor family, including TCP, the F-box-like protein and bZIP transcription factors, and the others are protein kinases and polyphenol oxidase enzymes. The analysis results suggest that they might be mediated by auxin signal way to influence the formation of callus.5. In order to study the miRNA role in the process of embryogenic callus induced immature embryo, this study using the overlap experiment principle, which use the Arabidopsis miR319a precursor as templates to build artificial miRNA expression vector (at the same time research group building miRNA159a express vector), and find the amiRNA precursor sequence at about450bp. This fragment was ligated to PSG105s expression vector, named PSG105s-miRNAs.6. The two expression vectors of PSG105s-miRNA393a and PSG105s-miRNA159a were conducted genetic transformation in maize callus by agrobacterium mediate (research group also carried on the transformation of stem tip). To and T1generation resistance plants were identified to be positive by PCR detection using detection primers, which were designed based on the the promoter and amiRNA sequence. At the same time the T1transgentic positive plant(3-12for miR393intruduced to callus,11-55,12-22for mi159a intruduced to stem tip tissue) were identified the induction rate of embryosbecame callus, the identification result indicated that the induction rate of miR393a (3-12) and miR159a (51,11-12-22transgenic plants were respectively57.89%、40.91%and35.71%, were significantly lower than wild type18-599R and18-599W.