Cloning,characterization,and Transformation Of Cold Resistant Gene ZmGDH2 In Maize (Zea Mays L.)

Maize is one of the most important cereal crops in the world.The northeast of China is the main maize producing zone.However,maize production often affects by low temperature in this zone,because of the geographical and climatic factors,that will directly cause physiological damage,metabolic inhibition,oxidation,hypertonic and other reaction in maize.Low temperature could reduce maize production seriously.Therefore,it is important to improve the low temperature resistance of maize by genetic methods.In this study,a maize cold resistant gene ZmGDH2(maize glutamate dehydrogenase gene)were researched,the results as follows:1.A 1236 bp ORF of ZmGDH2 was cloned in the cold resistance maize inbred line w9816,which encodes 411 amino acids and was located on chromosome 10.The full length of ZmGDH2 is 4453 bp,which contains 9 exons.By using Thermotoga maritima GDH(PDB:1B3B)as template,we constructed the three-dimensional structure of maize GDH2.The results showed that the maize GDH2 had poor thermal stability and high flexibility,which could make it more useful in the low temperature environment.The maize GDH2 amino acid sequence was compared with other plants by DNAMAN.The results indicated that GDH2 proteins might be conserved.The similarity was more than 88% between and other plants GDH2.2.The maize inbred lines W9816 were treated at 4? for 0h,6h,12 h,24h and48 h,respectively,.and then the ZmGDH2 expression in root,stem and leaf tissue were detected by qRT-PCR.The results showed that ZmGDH2 expressed differently in different treatment time points or tissueses,the most obvious response was detected in leaves.3.Overexpression vector p CHF3300-35S-ZmGDH2 was constructedsuccessfully.And,over expression ZmGDH2 transgenic Arabidopsis materials were obtained.We meatured the physiological indexes MDA,PRO and POD in transgenic Arabidopsis,wild type Arabidopsis,and ZmGDH2 homozygous mutant.Comparing the results,we found that over-expressing ZmGDH2 could improve cold tolerance in Arabidopsis4.ZmGDH2 was transformed into immature embryos and callus of maize inbred line Y423 by Agrobacterium tumefaciens mediated method.607 regenerated plantlets were obtained from immature embryos.and 29 plants were identified transformed successfully by PCR,the positive ratio was 4.78%.944 regenerated plants were obtained from callus.34 plants were identified transformed successfully by PCR,and the positive ratio was 3.6%.ZmGDH2 expression was detected by qRT-PCR in the T0 generation positive plants,and the result showed that the expression of ZmGDH2 in positive transgenic plants were significantly higher than in control.5.Agrobacterium mediated transformation system was optimized in maize.The results showed that the conversion efficiency was increased from 4.32% to 5.06% by adding 10 grams glucose into 1 liter transformation medium(Co-culture medium and suspension),and by heat shock treating at 45? for 3min before infection,transformation rate of immature embryo was increased from 3.70% to 6.25%;and transformation rate of callus was increased from 2.85% to 3.93%.6.By pollen tube pathway,we transformed ZmGDH2 into maize inbred lines10Z27.Twelve T0 generation corn ears and total 6956 seeds were obtained.After0.15% Basta solution screening and PCR identifying,76 positive plants were reserved and the conversion rate was 1.1%.Now,we have harvested.2798 T1 generation grains.