Research On Proteomics Of Plant Height Trait And ZmMDH Cloning Transformation Of Mutagenic Maize

Maize (Zea Mays L.), also known as Yu shu shu, is an annual Gramineae herb. It is tall, long and wide of leaves; monoecious, with unisexual flowers; male inflorescences grow the top of the plant; pistils develop in the leaf axils (top and middle). It is typical cross-pollination annual crop. Maize has become the important economic food crop for grain and feed all over the world, the average yield is the highest, played an important role in Chinese farm production. To innovate maize germplasm and increase the yield of forage maize,16 superior maize self-bred lines were selected as the example in this study. Pollen of maize was induced by Ethyl Methane Sulfonate (EMS), and M4 generation was studied by two-dimensional polyacrylamide gel electrophoresis (2-DE), combined with biomass spectrometry technology and techniques of differential genes of proteins of Arabidopsis ge-netic transformation and identification, to lay basis for further study of mutation breedin-g, proteomics of maize, differential genes transformation and production of excellent maize self-bred lines. The main results as follows:l.The studies used Pollen of maize induced by Ethyl Methane Sulfonate (EMS) to cre-ate excellent corn germplasm resources. It induced the maize self-bred lines with different genetic background by using EMS, and through observation, selection and identification o-f the successive generations, it indicates from the following three aspects:I.In M1 generat ion. EMS treatment causes more physiological damages and biological variations than nor mal ones after inducing it. The results shows that at seedling stage,it has low rate of surviv al and low emergence rate; at plant stage, it shows that plant rate of survival decreased in M1 generation population and time of male blooming revealed uncertain. II.Observation and evaluation of generation M2 indicated that the seedling stage is mainly of leaf color variation, a large percentage of which is etiolation seedling. While in adult stage, the mutation rate of late maturity、male sterile and plant height variation is high. III. After 16 superior maize self-bred lines was treated with 5 different concentration of EMS,the results showed that with the increase in EMS concentration, the aberration rate of treated subsequent generation plant is also rising. EMS concentration of 0.667×10’3 was chosen as optimal concentration,because it results in high mutation rate and abundant variation. (2) Observation and evaluation of generation M2 indicated that the seedling stage is mainly of leaf color variation, a large percentage of which is etiolation seedling. While in adult stage, the mutation rate of late maturity, male sterile and plant height variation is high. IV. After 16 superior maize self-bred lines was treated with 5 different concentration of EMS, the results showed that with the increase in EMS concentration,the aberration rate of treated subsequent generation plant is also rising. EMS concentration of 0.667×10-3 was chosed as optimal concentration,because it results in high mutation rate and abundant variation.2. In order to solve the interference effect of 2D gel electrophoresis caused by the lower protein contain and great amount of secondary metabolites in corn organization,3 kinds of protei extraction methods were compared on the result of 2D electrophoresis maps, meanwhile, the technical parameters were explored and optimized by protein quantities, isoelectric focusing conditions, SDS polyacrylamide gel concentration, et al. The improved trichloroacetic acid/acetone methold was effective way to extract total proteins form seedling leaves, that was simple operating and had clear 2D map with many more protein dots. The 2D gel electrophoresis suitable for forage com leaf proteomic study was constructed:When the protein sample(protein quantities 800 μg in first IEF, isoelectric focusing condition 20000V.h-1, polyacrylamide gel concentration 12.5%) was running in SDS-PAGE,a good 2D gel electrophoresis map with protein spots distributing equably and syllabify could be obtained protein pattern in 17 cm interpublic strip.3. The samples of in-bred line AMD 16 and hight mutant of maize were collected and seperated by 2D polyacrylamide gel electrophroresis technique. Coupled with MS analysis and other computer-assisted analysis,21 significantly differentially expressed protein spots were recognized,15 of which were identified and confirmed with MALDI-TOF-MS and MASCOT. According to the results, five proteins were specifically expressed in the hight mutant of maize, while two proteins were specifically expressed in the in-bred line AMD16. Seven proteins were up-regulated, while one protein was down-regulated. The function of the mentioned proteins may associated with metastasis, resistance, protein synthesis, chlorophyll synthesis,etc. Furthermore, the malate dehydrogenase which were specifically expressed in the induced mutants, may contributes to the production increase and height change of the maize.4. Malate dehydrogenase plays a key part in resistance and promoting metabolism. The cDNA of ZmMDH6 was cloned from the induced strain of maize and then expressed and verified. The results show that the coding region of ZmMDH6 cDNA, which is 1296bp long, encodes a 432-aa protein (MW:46.80 KDa, p1:5.79).5. The overexpession vector p3300-35S-ZmMDH6 was constructed as follows. First of all, the coding region of ZmMDH6 was cloned and inserted downstream of the 35S promoter on the plant expression vector p3300(pCAMBIA3300). Subsequently, the recombinant vector was transformed into Agrobacterium GV3101 and then introduced into Arabidopsis thaliana. The mutant strain of Arabidopsis thaliana was screened and confirmed by PPT method and sequencing. The acquirement of this mutant will lay the theoretical groundwork for further investigation of how this gene works in production increasing and resistance.