Cloning And Functional Analysis Of Genes Related To Maize Heterosis

Heterosis is a widespread biological phenomenon, having been used to breedingproduction widely. Maize is one of the most important food crop in the world, and isalso one of the most successful crop in cross breeding. Scholars from differentcountries have explored the mechanism of heterosis in theoretical and experimentalstudies for nearly a century. Although with the advance of methods and guidingideology together with many theories, the mechanism of heterosis is still a mystery,there are many issues that need further study.This study highlights the differentially expressed genes between parents and theirreciprocal hybrids on the seedling leaves, roots and the immature embryos afterpollination15days by using cDNA-AFLP. The process is as followed: take thematerials to extract total RNA, using reverse transcriptase to synthesizedouble-stranded cDNA. Double-stranded cDNA was digested by MseI/PstI andMseI/EcoRI, two pairs of restriction enzyme combination. Then the all appropriateadaptors were used in coupled reaction. Finally, after pre-amplified and selectiveamplified with240pairs selective primers, the different expressed genes wereseparated by4.5%polyacrylamide gel electrophoresis.In this study, a total of458differentially expressed fragments were recovered,including180bands detected in roots,170bands detected in leaves, and108bandsdisplayed in immature embryos. After repeat PCR and sequenced,174,156and102fragments were successfully sequenced in three organs respectively. Comparison andanalysis by MaizeGDB database, the result showed that107,107and90homologousgenes were involved in putative functions,and the functional annotation of them werecarried out by using Blast2GO, there were a lot of DEGs make a role in substancemetabolism, energy metabolism, stress physiological activity, plant growth anddevelopment, hormone synthesis, transcription, cell cycle, material transport andsignal transduction pathways, etc. The differentially expressed genes were predictedthat may play a key role in the formation of heterosis. In order to validate the resultsof cDNA-AFLP experiment, quantitative RT-PCR was carried out to study somedifferentially expressed genes. The results showed that is consistent with thecDNA-AFLP, indicate that isolated differentially expression genes by cDNA-AFLP was credible and feasibility. According to the results of qRT-PCR and cDNA-AFLP,we validated the expression patterns of15selected genes based on the expressionlevel of the same gene in different materials and revealed that there are fiveexpression patterns including bands expressed only in two parents, bands expressedonly in one parent, bands expressed only in one parent and two hybrids, bandsexpressed in F1but not in two parents, bands expressed in both parents and F1.Butmost of expression patterns were dominant expression in single parent andhybrid-specific expression patterns in this study.Through functional analysis, the results of qRT-PCR and read a lot of literature,seven of the differentially expressed genes were selected and cloned from theimmature embryos and the roots of seedling. The genes of ZmPUB and MPAO couldpromote growth and development in plants, and could resist to disease and senescence(G-74: polyamine oxidase4, P-3: RING/U-box superfamily protein); the gene ofZmRanGTP1could promote taproot growth and inhibit lateral root growth in plants(G-81: RAN GTPase activating protein1); the gene of ZmSEC-14p could promotegrowth of root hair and resist to cold stress in plants (G-125: Sec14p-likephosphatidylinositol transfer family protein); the genes of ZmSCP and WD40couldcontrol the degree of filling, grain width, embryo development and construction ofplant morphology (P-6: serine carboxypeptidase-like33; P-15: transducin familyprotein/WD-40repeat family protein); the gene of ZmERD15encoding proteins whichhave drought resistance (P-66: dehydration-induced protein-ERD15). It was predictedthat these genes may play an important role in the formation of heterosis, functionalverification by infect Arabidopsis is underway and have got T1generation seeds.In this paper, cDNA-AFLP technique was used to study the differentialexpression genes of the roots, leaves in maize seeding and the immature embryowhich after pollination15days, then the differential erpression genes should validateof functions, that may be helpful to explore the molecular mechanism of heterosis.