Study On Chilling Resistance Gene ZmRLK Cloning, Function Analysis And Genetic Transformation In Maize

Maize(Zea mays L.)is one of the largest acreage crop in the world, the current demand are increasing in agriculture and industry. Therefore, how to improve the maize yield is particularly important event. Maize production is effected by many biotic and abiotic stress. Low temperature stress is very common agro-meteorological disasters in Northeast China. In the maize growing period, in case of low temperature stress, the leaves is lossing water and wilting, photosynthesis and pollen viability are reduced, leading to maize production down. Therefore, chilling resistance maize new varieties and germplasm is very important.In this study, using RACE technology to cloning chilling resistance maize gene Zm RLK and constructing expression vectors and verifing function in Arabidopsis. Maize inbred lines Y423 is used to experimental materialsl, through Agrobacterium-mediated method and pollen tube pathway, we transferred Zm RLK into maize genome for get a new maize veriaty. It is optimized of Maize genetic transformation system of Agrobacterium-mediated key factor. Specific contents and results are as follows:1. Cloned by RACE(Rapid Amplification of c DNA Ends) to obtain a complete chilling resistance genes Zm RLK. The sequencing results showed that: the nucleotide sequence length of the gene is 2028 bp, encoding 675 amino acids, molecular is 166447.1Da, isoelectric point is 4.91. Analysising amino acid sequence and bioinformatics of Zm RLK. The results show that: it is similar more than 80% with the amino acid sequence of sorghum, millet, etc.2. Using p CAMBIA3301 as a based carrier, 35 S and UBI as promoter respectively. To constructed overexpression vector p CAMBIA3301-Zm RLK-35 S, interference vector p CAMBIA3301-UBI-Zm RLK(+)-intron-Zm RLK(-) of maize chilling resistance gene Zm RLK.3. The maize chilling resistance gene Zm RLK is tranferred in Arabidopsis thaliana for functional verification. Results show that Zm RLK can improve plant tolerance to cold stress usually.4. Optimization of Agrobacterium-mediated genetic transformation of maize Transformation System. The results showed that: when the bacteria were resuspended at a concentration of OD600 = 0.55, the infection time was 25 min, AS concentration of 100μmol/L, genetic transformation efficiency is the highest level. Differentiation of NAA concentration of 0.5 mg/L, 6-BA concentration of 0.5 mg/L, rate of seedling regeneration is highest.5. Maize genetic transformation using Agrobacterium-mediated(1) Infecting 12000 maize Y423 immature embryos for transferring maize chilling resistance gene Zm RLK by Agrobacterium-mediated method. Wherein overexpression vector p CAMBIA3301-35S-Zm RLK is infected 6000, Obtain regenerated plants 2207 and there are 89 positive plants, the genetic transformation rate is 4.0%; interference vector p CAMBIA3301-UBI-Zm RLK(+)-intron-Zm RLK(-) is infected 6000, obtained 121 positive plants in regenerated plants 1845, the genetic transformation rate is was 6.6%; empty vector is infected 2000 and obtained 31 positive plants in regenerated plants 693, the genetic transformation rate is 4.5%.(2) Maize inbred line Y423 somatic embryo as explants, infecting 12000 maize Y423 callus by Agrobacterium-mediated method. Wherein overexpression vector p CAMBIA3301-35S-Zm RLK is infected 7200 what obtained 63 positive plants in regenerated plants 946, the genetic transformation rate was 6.7%; interference vector p CAMBIA3301-UBI-Zm RLK(+)-intron-Zm RLK(-) is infected 3800 what obtained 20 positive plants in regenerated plants 343, the genetic transformation rate was 5.8%; empty vector is infected 1000 what obtained 8 positive plants in regenerated plants 103, the genetic transformation rate was 7.8%. Comparing to two ways of immature embryos and somatic embryos, the results show immature embryo as explants of Agrobacterium-mediated genetic transformation method is more higher genetic transformation rates.6. Through pollen tube pathway, chilling expression vector containing the gene Zm RLK is transferred to Y423. 75 ears T0-generation are treated and receive a total of 12530 seeds. wherein 30 ears with overexpression vector, total 5221 maize kernels; wherein 27 ears with interference vector, a total of 4706; empty vector 18, a total of 2603. After selecting by 2‰ Basta and molecular testing, access to 68 positive plants, which overexpression vector 27, interference vector 21, empty vector 20, the genetic transformation rate is 0.52%, 0.45%, 0.77% respectively. The T1 generation seeds are obtained from T0 generation seed cross pollination. Follow the same way adding generation and detection, 2638 T2 generation seeds has received.