Proline Accumulation, Cloning And Transformation Of △~1-Pyrroline-5-Carboxylate Synthetase Gene In Sugarcane (Saccharum Officinarum L.)

Sugarcane (Saccharum officinarum L.) is an important sugar and energy crop in China. The major sugarcane and sugar production in China is dominently located in southern region includes such as Guangxi, Guangdong, Yunnan and Hainan provinces. Drought is the most important abiotic stress limiting sugar productivity in China because about 90% of sugarcane is grown in the upland areas where irrigation is not available. In Guangxi, drought occurs very often in the major sugarcane growing areas, especially in the spring and autumn, which affects cane yield and sugar productivity seriously.Proline accumulation in response to various environmental stresses such as drought, excessive salinity, high or low temperature and heavy metal, has been described in many phylogenetically diverse organisms such as bacterial, mammalian, plants and algae. Efforts toward understanding the proline biosynthetic pathway in sugarcane and to unravel the role of proline in response to osmotic stress may help us to use gene engineering related proline to enhance the resistance of sugarcane under osmotic stress.Three sugarcane varieties, GT21, ROC16 and ROC22, were used as experimental materials. The plants were treated with 25% polyethylene glycol (PEG) 6000 at early and elongation stages, respectively. Effects of PEG stress on proline accumulation and the activities of the key enzymes in leaves of sugarcane were investigated in the present study. At the same time, the isolation and the structure of the sequences of nucleotide acid and deduced amino acid encoding the ScP5CS {Saccharum officinarum L. Δ¹-pyrroline-5-carboxylate synthetase) gene were reported. And the functions of this gene were analyzed by transferring the anti-sense mRNA of ScP5CS gene into tobacco. Expression and resistance under osmotic stress of the sugarcane ScP5CS were analyzed in the transgenic tobacco. The main results as follows:1. At the early growth stage, free proline content in ROC16 increased 1 d after starting of the osmotic stress treatment, that in GT21 also increased 2 d after the starting of the treatment, while there was no difference between the treatment and the control for the variety ROC22 during 12 d. The same results for the activity ofΔ¹-pyrroline-5-carboxylate synthetase (P5CS) were obtained after the stress treatment, the activity in GT21 and ROC16 increased after treatment, but it was contrary in ROC22. For the activities of ornithine-δ-aminotransferase (δ-OAT), there was no remarkable difference between the treatment and control. The proline dehydrogenase (ProDH) activity in GT21 and ROC16 increased at firstly then decreased after 7 d while that in ROC22 increased 7 d after the starting of the treatment.2. At the elongation stage, free proline content in GT21 and ROC16 increased 4 d after the starting of the treatment, while there was no difference between the treatment and the control for the variety ROC22 during 6 d. The same results for the P5CS activity were obtained after the stress treatment, the P5CS activity in ROC22 did not increase until 6 d after the starting of the treatment. For the activities of 5-OAT, there was no remarkable difference between the treatment and the control. The ProDH activity in ROC22 increased after treatment, but it was contrary in the other varieties.3. Effects of water stress on proline content, activities of P5CS,δ-OAT and ProDH in leaves were studied. The results showed that proline is largely accumulated in sugarcane under the stress treatment. Whereas proline can be synthesized from either glutamate or ornithine in plants, the experiments indicate that glutamate, rather than ornithine, is primary precursor for proline biosynthesis in osmotically stressed sugarcane. And the accumulation of proline occurred as the result of both the activation of proline biosynthesis and the inactivation of proline degradation.4. P5CS is the rate-limiting enzyme in proline biosynthesis of glutamate pathway. In the present study, the sugarcane variety ROC22 was used as the experimental material. By homologous based cloning, two specific fragments isolated from sugarcane genome DNA with specific primers, which were confirmed as the sugarcane ScP5CS genes, naming ScPS₁ and ScPS₂. The length of sequence ScPS₁ was 2016 bp, containing 7 introns and 8 extrons, and the GenBank accession number is EF620362. Comparing the extron sequence 977 bp of ScPSi with that of ScP5CS mRNA (EU005373), it showed high identity (99.6%). And the length of ScPS₂ was the same as the transcribed sequence of ScPS₁, showing high identity with the transcribed sequence of ScPS₁ (99.6%) and with ScP5CS mRNA (EU005373) sequence (99.6%). But the introns were absent in the ScPS₂, which is the feature of the pseudogene. So the ScPS₂ was confirmed as the pseudogene.5. With the sugarcane variety ROC22 as experimental material, the cDNA sequence of ScP5CS gene in sugarcane with 2151 bp in length was isolated by homologous cloning, which contained an open reading frame encoding a protein of 716 amino acids, with GenBank accession number EF620362. Comparing the sequence of ScP5CS with that of ScP5CS (EF155655) reported, the nucleotide acid showed high identity (98%), but the deduced amino acid was only 92%. The deduced protein contains putative ATP-binding site, putative leucine domains, Glu-5-kinase domain, putative NADPH-binding domain, conserved GSA-DH domain and feedback inhibition site. Besides, there were more differences in Glu-5-kinase domain from the deduced amino acid of ScP5CS (EF155655), but less for the P5CSs from rice (Oryza sativa) and wheat (Triticum aestivum). So it is confirmed this gene is a new gene of sugarcane P5CS.6. The antisenced expression vectors pBI121/Sc-PCS₁ and pBI121/Sc-PCS₂ were constructed by respectively inserting two different fragments, which were 384 bp (position 1318-1701 bp) and 954 bp (position 1163-2166 bp), of ScP5CS gene, into the plant expression vector pBI121 downstream controlled by CaMV35S promoter. These fragments were contained in gamma-glutamyl phosphate reductase domain. The antisenced expression vectors of ScP5CS gene were used to transformed tobacco plants. The transformed antisence ScP5CS in tobacco inhibited the expression of tobacco P5CS. The transgenic plants grew slowlier, the leaves were easy to get etiolational and the root system development was stunted and the roots were shorter under the NaCl and PEG stress as compared with the control. And the same results were obtained for both the shorter sequence Sc-PCS₁ (384 bp) and the longer sequence SC-PCS2 (954 bp) after the stress treatment, but it was contrary for the plants with the transformed vector pBI121 and the non-transgenic (negative) control.