Construction Of CDNA Libraries For Sugarcane Leaf And Stem Tissues And Analysis For Expressed Sequence Taq

Sugarcane is not only the most important sugar crop, but also one of the most important energy crops in the world. It can be used in the production of cane sugar, fiber, dextran and green energy such as ethanol, so it occupies important economic position at the international level, all those countries engaging in the production of sugarcane pay great attention to the relevant research, and lots of them aims to study the functional genomics of sugarcane, among them the construction of full-length cDNA library is one of the prerequisites and foundations. In the present study, after sequencing, bioinformatics method, prokaryotic expression technology and Real-time PCR technique were all used to analyze the sequence characteristics and expression profile of the targeted genes and the aim of this study were to obtain some stress-related genes. The main results were as follows:Basing on the conventional Oligo-capping method, we improved some of the steps and our own full-length cDNA construction platform was established. Then, using the improved Oligo-capping method, we have constructed two full-length cDNA libraries, one for sugarcane leaf and the other for sugarcane stem. Libraries check results indicated that the storage capacity of these two libraries were 3.0×106cfu and 2.5×106cfu, respectively; the recombination rates were 89.0% and 91.0%, respectively; the full-length rates were 85.0% and 88.9%, respectively; while the average insert size of both libraries was about 1.0 kb.High-throught sequencing of clones from the sugarcane leaf and stem cDNA library from 5’end resulted in 228 and 283 effective ESTs sequences, respectively; The average length of these sequeces was 464 bp and 459 bp, respectively. 154 and 204 TUTs (tentative unique transcript, TUT) were obtained, respectively, after assembly by SeqmanⅡprogram of DNAStar software. The rates of the redundant sequence of both libraries were 32.4% and 27.9%, respectively. And then, these TUTs sequences were sent to GenBank for homologue search using blastn and blastx program. Out of these, 87 TUTs (including 117 ESTs) from leaf cDNA library and 103 TUTs (including 132 ESTs) from stem cDNA library had significant homology to genes with known function or putative function. These genes were classified according to the functional classification of Arabidopsis genes. Results indicated that genes from leaf cDNA library could be classfied into11 categories, among the largest category was genes related to energy metabolism, the function of the other genes were involved with signal transduction, protein synthesis, transcription, protein destination and storage etc; genes from stem cDNA library could be classfied into12 categories, among the largest category was genes related to protein synthesis, the others were transcription, cell structure, protein destination and storage, metabolism etc.Seven full-length cDNAs of candidate stress-related genes were obtained from the two cDNA libraries through EST sequencing and the corresponding bioinformatics analysis. The analysis of their sequence characteristics and the homology were conducted by bioinformatics software. The coding proteins of the seven stress-related genes were as follows:late embryogenesis abundant protein(LEA), Mn-superoxide dismutase(MnSOD), Zinc finger protein(Zn), S-adenosylmethionine decarboxylase(SAMDC), WRKY transcription factor(WRKY), Glutathione S-transferase(GST) and cyclophilin. And these seven genes were named as Sc-Lea, Sc-MnSOD, Sc-zf, Sc-SAMDC, Sc-WRKY, Sc-GST, Sc-CyP, respectively.The primers designed according to the ORFs sequence of these genes and the restriction enzyme cutting site information of the vector pET29a(+) were used to constructed their prokaryotic expression vectors. Then, the prokaryotic expression vectors containing the corresponding ORFs were successfully constructed and transformed into E.coil BL21 cells and their expression induced by IPTG. The results of SDS-PAGE indicated these genes could express the right targeted protein in E.coil BL21.The sugarcane seedlings were treated by various exogenous chemicals, such as PEG, NaCl, SA, H2O2 and Ustilago scitaminea, and then the expression characters of the seven candidate genes were analyzed by Real-time PCR. The results showed that PEG could induce the expression of Sc-Lea, Sc-SAMDC, Sc-WRKY and Sc-CyP, but inhibit the expression of Sc-zf; NaCl could induce Sc-Lea, Sc-zf, Sc-SAMDC, Sc-WRKY and Sc-CyP; SA could induce the expression of Sc-WRKY and Sc-CyP, but inhibit the expression of Sc-SAMDC; H2O2 could induce the expression of Sc-GST and Sc-CyP,but inhibit the expression of Sc-Lea and Sc-SAMDC; Ustilago scitaminea could induce the expression of Sc-MnSOD, Sc-zf, Sc-WRKY and Sc-GST.