The Establishing Of The Rapid Regeneration System And The Preliminary Functional Studies Of NAC1 Gene In Cerasus Humilis

Cerasus humilis belongs to Rosaceae cherry fruit trees, perennial deciduous dwarf shrubs. It resistant to cold, drought, barren and salt stress, with solid soil and water conservation, preventing dust, reducing surface runoff, strong adaptability. Therefore it is an excellent ecological tree. In the study, we successfully established in vitro regeneration system of Cerasus humilis, and can provide pure, homogenization material for subsequent test. In order to lay the foundation for building up the genetic transformation system, an efficient protocol for the rapid micropropagation of Cerasus humilis has been standardized using leaf and stem explants in our study. Finally, we established shoot culture and in vitro regenerated plants of Cerasus humilis. The results were as follows:1.We established a regeneration system of Cerasus humilis through the induction of callus(1) We found that the best disinfection on explant is 75% ethanol for 1.0min and 1% NaCIO for 4min.(2)The optimal medium for embryogenic callus induction is MS+30g/Lsucrose+8g/Laga r+1.0mg/L6-BA+0.6mg/LNAA using leaves for explants; but MS+30g/L sucrose+8g/L agar+0.5mg/L 6-BA+0.8mg/LNAA using stem for explants.(3)Our results showed that the callus were amplified on MS medium supplemented with 0.5mg/L6-BA and 0.6mg/L2.4-D.(4)Shoots were produced from callus on Murashige and Skoog (MS) medium supplemented with different concentrations of 5.0mg/L6-BA+0.9mg/LNAA.(5)Shoots were produced from axillary buds on Murashige and Skoog (MS) medium supplemented with different concentrations of 2.0mg/L6-BA+0.9mg/L NAA.(6)Rooted plantlets obtained on half-strengh MS medium supplemented with plant growth regulator (PGR) 0.5 mg/LNAA and were successfully acclimatized to greenhouse conditions.2.Through the previous transcriptome analysis of the Cerasus humilis, we isolated one NAC transcription factor sequence. As a reference, a full-length cDNA sequence of NAC1 gene was obtained from Cerasus humilis leaves, named ChNAC1 (K.P314213.1). The full length of ChNACl gene was 997 bp, which contained a 894 bp open reading frame(ORF), and encoded a protein with 297 amino acids. Bioinformatics analysis suggested that this protein was a stable protein with a calculated molecular mass of 33.94kD and a theoretical pI of 8.09, located in the nucleus.3.The ChNAC1 was respectively cloned into the Plant expression vector under the control of CaMV35S promoter. Using freeze thawing method, transferred vector pROK2-ChNAC1 into Agrobacterium tumefaciens receptor EHA105, then mediated Arabidopsis transformation by Floral-dip, which caused a high-efficiency expression on ChNACl transgenic Arabidopsis. Use selective mediums to obtain transgenic lines named linel and line4. Our research discovered positive bu dual PCR with characteristic primer ChNAC1. The transgenic plants were subjected to drought stress for 20d, followed by re-watering. Chlorophyll content of each plant were measured. The results showed that the transgenic plants had better resistance to drought tolerance.