Cloning And Expression Of Erianthus Arundinaceus's BADH And Evaluation Of Its Stress Tolerance

Erianthus arundinaceus, the related genara of Saccharum, has good applicability, stress resistance, vigour and ratooning ability. So, it is important gene resource for sugarcane breeding. Based on the comparative analysis for stress resistance of E. arundinaceus and Saccharum, BADH gene from E. arundinaceus was cloned and its expression was analysed in E.coli or under high salinity and drought stress. The results were as follows:The increments of MDA content and the plasma membrane permeability in leaves of E.arundinaceus were less than those in Badila (S. qfficinarum L.) subjected to water deficit or salt stress, and higher activities of SOD and POD in leaves of E.arundinaceus. 10.73 mg/100g FW of glycinbetaine in water-stressed E.arundinaceus was less than that of S. glauca or S. ussuriensis, but higher than those of the other reported plants. No glycine betaine accumulated in water-deficit Badila.The full-length BADHcDNA from E.arundinaceus was isolated by RT-PCR and rapid amplified cDNA ends (RACE). E.arundinaceus BADH was 1718 bp in length, including 35 bp 5'UTR, 165 bp 3'UTR and 1515-base-pair open reading frame (ORF) encoding protein of 54.905 kDa. 75.9% of G and C was found in the 290bp length of its 5'end. The deduced amino acid sequence of E.arundinaceus BADH included a decapeptide sequence "VTLELGGKSP" and a Cysteine residue, which is highly conserved among general aldehyde dehydrogenases (ALDH). It also contained a tripeptide SKL at the C-terminnal, a signal known to target to microbodies. The BLAST analysis in GenBank showed that overall coding region of E. arundinaceus BADH is c. 97% homologous to the Zea mays cDNA sequence ( PCO110216), c. 95% to the partial cDNA of Sorghum bicolor BADH1. However, its 5'end had no homology to O. sativa BADH, H. vulgare BBD2 and T. aestivum BADH.The phylogenetic analysis from 22 kinds of plant BADHs indicated the larger variability in the dicotyledonous plants, one group in the Gramineae plants including BADH genes form E.arundinaceus, S.vulgare, Zea mays, H. vulgare andT. aestivum, which branches out into two groups corresponding to betaine aldehyde dehydrogenase isozymes..Southern and Northern blot analysis were carried out using cDNA fragment of the conserved domain sequence of E.arundinaceus BADH as a probe. Southern blot showed one or two copies of E.arundinaceus BADH in the genome of E. arundinaceus. Northern blot showed the E. arundinaceus BADH was expressed at high levels under drought stress, then decreased after recovering water. However, its expression levels increased with the elongation of salt stress duration, then kept at a certain level. On the tenth day of salt stress, the level of mRNA for Karundinaceus BADH in stem of E. arundinaceus was higher than that in leaves, lower in roots.The expression vector of E.arundinaceus BADH was constructed. E.arundinaceus BADH could be expressed in E.coli BL21.The specific activity of E.arundinaceus BADH induced by IPTG was 9.04 nmolmin-1mg-1protein.Cloning of E. arundinaceus BADH gene and its successful transformation into sugarcane cultivars will be an alternative to improve drought tolerance in sugarcane in future.