Study On Cloning Of MsCAMTA1 Gene And Alfalfa Transfomation

CAMTA1 gene encodes a calmodulin-binding transcription factor 1 protein. It plays an important role in plant development and resistance to abiotic stress. In the present study, we cloned a novel CAMTA1 gene from Medicago sativl L. and designated it as MsCAMTA1. Full length ORF of MsCAMTA1 were separately cloned into two plant expression vector pCBM and pYLCRISPR/Cas9-DH for gene over-expression and knockout. The summary of the results are as follows:1. Cloning and sequence analysis of MsCAMTA1 geneMs CAMTA1 was obtained by RT-PCR. Sequence analysis of MsCAMTA1 showed that it is 3123 nucleotides long, and encodes a 1040 amino acid protein. The MsCAMTA1 protein contains CG-1 domain, TIG domain, ANK repeat and IQ motif. Compared to Medicago truncatula CAMTA1, there are 32 mutations in the amino acid sequence of MsCAMTA1, including deletion, insertion and single base substitution.2. Construction of plant expression vector pCBM-MsCMATA1 and pYLCRISPR/Cas9- MsCMATA1The ORF of MsCMATA1 was inserted into plant expression vecto pCBM under the drive of 35 S promoter of Cauliflower mosaic virus(CaMV). The vector carries the bar gene;Plant expression vector pYLCRISPR/Cas9-MsCMATA1 was constructed. It carrys three pieces of target sequence which combines MsCAMTA1 by complementary base pairing and hygromycin resistance gene.3. Study of MsCAMTA1 gene transformation in alfalfa via Agrobacterium-mediated methods(1)The study of alfalfa section cotyledon and hypocotyl resestance to glufosinate and hygromycin respectively.Alfalfa explants section cotyledon and hypocotyl were put into inductive medium with diferent concentration of glufosinate or hygromycin for testing resistance pressure. The results showed that 0.5 mg/L glufosinate is suitable for screening cotyledon, and 4mg/L glufosinate is suitable for hypocotyl; the concentration of hygromycin selection is 20mg/L for cotyledon, and 5mg/L for hypocotyl.(2)Transformation of alfalfa by using explants section cotyledon and hypocotyl.Section of cotyledon was selected as explants, and inserted into MS medium containing 1 mg/L 6-BA. By cultivating 3 days for pre-incubate, the explants were infected 20 min with Agrobacterium tumefaciens with an OD600 in the range of 0.4 to 0.6, then co-cultured for another 3-5 days. Then the explants were washed with liquid medium containing 500mg/L Cef, and inserted them into induction medium. Elongation of adventitious bud happened in MS medium containing 0.5 mg/L 6-BA, Cef and screening reagent; Taking root hanppened in 1/2 MS. The adventitious buds were cut down after elongation, and inserted into 1/2 MS medium to take root. About 500 cotyledons were infected in total, and 13 plants transformed pCBM-MsCMATA1 with resistance of glufosinate and 8 plants transformed pYLCRISPR/Cas9- MsCMATA1 with resistance of hygromycin were obtained by this way.The explants hypocotyls were cut down to infect immediately, Agrobacterium tumefaciens with OD600 around 0.6~0.8 should be diluted 5 times, then put them on sterile filter paper above MS containing 2,4-D 2mg/L, KT 0.25 mg/L and acid hydrolysis of casein 2g/L. After 2 days’ co-culture, the explants were taken out, washed to remove bacteria and put on inductive medium containing Cef and screening reagent. These swollen callus were transferred to tissue differentiation medium including 6-BA 0.5 mg/L and NAA 0.05mg/L, Small bud from differentiation of callus should take root in 1/2 MS. About 300 hypocotyls were infected in total, 7 plants transformed pCBM-MsCMATA1 with resistance of glufosinate and 6 plants transformed pYLCRISPR/Cas9- MsCMATA1 with resistance of hygromycin were obtained.