Functional Identification And Analysis Of SoSnRK2.1 And SoACLA-1 Genes From Sugarcane

Drought is one of the most stress factors effecting growth, development, productivity and quality of sugarcane. ATP-citrate lyase (ACL) and sucrose non-fermenting 1-related protein kinase 2 (SnRK2) are key regulatory enzymes involved in plant metabolism and stress response. ACL catalyses citrate to acetyl-CoA in the cytoplasm and plays an important role in normal growth and development of plant, while SnRK2 plays a significant role in response to multiple abiotic stresses. In the ABA signal transduction pathways, both of genes were involved in plant development under abiotic stress. In this research, SoACLA-1 and SoSnRK2.1 were used to study the regulatory mechanism in prokaryotic and eukaryotic. The main results are as follows:1. According to the sequences of SoSnRK2.1 and SoACLA-1, both of genes were cloned, the full length of the SoSnRK2.1 and SoACLA-1 genes with the specific primers were amplified and the restriction enzyme cleavage sites were designed for the prokaryotic expression vector pET30a (+), and the plant expression vector pRI101-ON, pRI101-AN and pUBTC. The sequencing analysis was shown that the complete open reading frame of SoACLA-1 and SoSnRK2.1 genes are 1272 and 1002 bp, respectively. The amino acid sequence analysis was showed that both of SoACLA-1 and SoSnRK2.1 from sugarcane have high homology with those of Zea mays and Oryza sativa.2. Both of SoACLA-1 and SoSnRK2.1 were expressed in prokaryote individually. SoACLA-1 and SoSnRK2.1 genes without introns were inserted into the prokaryotic expression vector pET30a (+), which contains the constitutive T7 promoter. The results of prokaryotic expression demonstrated that the sizes of combined proteins were 46 kDa of pET-SoACLA-1 and 38 kDa of pET-SoSnRK2.1, respectively. In addition, recombined proteins of the pET-SoACLA-1 and pET-SoSnRK2.1 existed as inclusion bodies. The purified and concentrated proteins of pET-SoACLA-1 and pET-SoSnRK2.1 were obtained using Ni2+-NTA column affinity chromatography, and dialysis. Furthermore, the function of SoACLA-1 and SoSnRK2.1 were identified through drought (PEG) treatment, which showed the SoACLA-1 and SoSnRK2.1 improved drought tolerance under PEG treatment.3. Identification of SoSnRK2.1 functions was used a model plant tobacco. SoSnRK2.1-GFP-pBI121 under control of a CaMV 35S promoter was successfully transferred to tobacco plants using Agrobacterium-mediated transformation. The results showed that plant genetic transformation rate reached 75%. Growth and morphology of transgenic tobacco demonstrated that over-expression of SoSnRK2.1 enhanced drought tolerance than that wild type tobacco. The transgenic tobacco plants had lower levels of ion leakage (IL), and contents of maleic dialdehyde (MDA) and H2O2, but higher activities of three antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), chlorophyll and relative water content (RWC) as compared with WT tobacco. SoSnRK2.1 could stably transmit to next generation via sexual reproduction. The results indicated that SoSnRK2.1 was involved in the abiotic stress response in sugarcane and may play an important role in regulation plant growth and development.4. The drought resistance of sugarcane SoACLA-1 gene was identified using transgenic tobacco. The transformation harboring SoACLA-1-pRI101-AN expression vector under control of CaMV 35S promoter in Agrobacterium was introduced to WT tobacco. The conversion rate of transgenic tobacco was 56% detected by PCR method. Overexpression of SoACLA-1 enhanced tolerance to drought through observing morphology of the transgenic plants and measuring various physiological and biochemical indexes, which were similar to those in transgenic SoSnRk2.1 gene. The results indicated that SoACLA-1 transgenic plants enhanced drought tolerance than WT plants.5. SoSnRK2.1 gene was successfully transferred in sugarcane. SoSnRK2.1 gene transformation was constructed with pRI101-ON vector, which was transferred into embryogenic callus of sugarcane varieties of ROC22 and YL6 using Agrobacterium-mediated transformation. The conversion rate of ROC22 and Y6 transgenic plants detected target fragment and NPTII marker gene with PCR were 26.5% and 16.7%, respectively. The transgenic sugarcane were potentially enhanced plant drought stress.6. SoACLA-1 gene was successfully transferred in sugarcane. The plants expression vector pUBTC with SoACLA-1 gene was successfully constructed under control of Ubi promoter. The embryogenic callus of ROC22 was used as the acceptors, and genetic transformation system mediated was Agrobacterium-mediated transformation. The phenotype of transgenic plants was observed and compared with the control. The results indicated that the transgene plants improve drought tolerance under drought stress as compared with non-transgenic sugarcane.