| Maize (Zea mays L.) is one of the most agronomically important crops in theworld. Its products have a key position in the worldwide economy. In the last century,traditional maize breeding has focused primarily on improving the crop yields,through selection and improvement of inbred lines. However, traditional breeding isvery time-consuming and has not always been successful in increasing yield, due tovarious environmental factors such as biotic and abiotic stresses. In recent years,molecular breeding and transgenic techniques, such as tissue culture and genetictransformation, have been used to complement traditional breeding techniques toimprove inbred lines in a relatively short period of time. These techniques will also beof value in enlarging maize germplasm resources. Tissue culture is a key step in themaize transgenic breeding. Studies found that maize is one of the most difficult cropsof in vitro embryogenesis. Therefore, to understanding the molecular mechanism ofmaize embryonic callus formation is an important part of corn transgenic breeding.In this study, a total of10maize inbred lines were used to induce calli thatshowed different induction frequencies, and successfully verified that embryonic calliinduction rate of the inbred line H99on N6basal media is higher than other inbredlines. At the same time, the paraffin section observations and scanning electronmicroscopy results showed that there are significant differences in internal structureand external structure between the embryonic calli and non-embryonic calli of inbredline H99. Above results laid the theoretical basis for future experiments.To identify candidate genes associated with the embryonic calli, a cDNA-AFLPapproach was used to evaluate gene expression profiles from non-embryonic calli andembryonic calli which were sub-cultured four times. A total of192selective-amplification primer combinations were used in this study. According to thecDNA-AFLP analysis, approximately2,430differentially expressed TDFs were identified, including1,350bands detected in non-embryonic calli and1,080bandsdisplayed in embryonic calli. To validate the differentially expressed TDFs, a total of101sequences with insertions longer than100bp were used for further analysis. TheTDFs could be classified into several categories according to their putative functions,including energy production and conversion (3.06%), cell growth and division(4.08%), transporters (3.06%), signal transduction (8.16%), metabolism (6.12%),defense responses (3.06%), transcription (6.12%), protein synthesis and degradation(12.24%) and cell structure (6.12%). These results showed that the formation processof maize embryogenic callus is a complex regulatory mechanism, which needs allkinds of genes work together to complete.After analysis the expression profile of embryonic calli and non-embryonic calli,we obtained five candidate genes that may play a role in the formation of embryoniccalli. According to the TDFs sequences, three candidate genes cDNA full-lengthsequences were assembled from the GenBank database and the maize genomesequence database to generate1,419bp,2,016bp and5,388bp cDNA sequencedesignated ZmKHCP, ZmTypA and ZmARF-GEP, and the GenBankit no. is JN107649,JN107650and JN107651, respectively. Bioinformatics analysis showed that thesecandidate genes had high similarity with the function known genes. At the same time,the prokaryotic expression vector pET-28a-ZmTypA has been successfully constructed,and the ZmTypA purified protein has been obtained. Enzyme activity analysis showedthat the protein has GTPase activity.In addition, the total protein of embryonic calli and non-enbryonic calli has beensuccessfully extracted using trichloroacetic acid/acetone method. After measured theprotein concentrations found that embryonic calli is3.7μg/μl, while non-embryoniccalli is1.7μg/μl. The result of SDS-PAGE showed that the protein content ofembryonic calli increased in the50kDa and20kDa. Using2-DE technique to analysisdifferential expression protein between the two types calli showed that about1,200spots were detected on each silver-stained gel,42protein spots were noticeablydifferent (P <0.05). Among them,33proteins were differentially expressed at leastthreefold, with15up-regulated and18down-regulated in embryonic calli versus non-enbryonic calli, and nine proteins had emerged (four in embryonic calli and fivein non-enbryonic calli).According to the differential abundance of the spots,38were excised from thegels, digested with trypsin, and analyzed using mass spectrometry (MS/MS), theresults showed that29spots have good matches, identified with an identificationsuccess rate of78.95%. According to their homologies, these proteins were classifiedinto six functional groups, including cell proliferation (10.34%), transcription andprotein processing (17.24%), stress response (10.34%), signal transduction (3.45%),metabolism and energy (48.28%) and hypothetical function (10.34%). In order tovalidate the proteomic results, qRT-PCR was performed to assess the transcriptionlevel of10genes. The results showed that the expression levels of five genes haveclear correlation with the profiles detected in the2-DE gels. Similar mRNA levels fortwo genes were detected in two samples. However, the other genes were in contrast tothe results of the proteomic analysis, presumably due to the proteomic approachfocusing on protein isoforms, whereas the gene expression levels analyzed by PCRmay verify the quantification of the abundance of related transcripts. Therefore, thebest explanation for these discrepancies could be because of post-translationalmodification or protein degradation. Comprehensive of these results, thesedifferentially expression proteins related to plant growth and development, furtherverified that the formation of two types calli needs of various proteins regulation.In this study, a large number of experiments showed that maize embryonic callusformation is a complex regulatory process. This study broaden our understanding ofthe molecular mechanism of maize embryonic callus formation on the transcriptionallevel and protein translation level, and provided an important theoretical basis forestablishing good maize genetical receptor system.
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