Cloning And Expression Analysis Of Drought And Salt Resbonsive Gene In Alternanthera Philoxeroides

In the wild, Alternanthera philoxeroides can grow abundantly in different habitats ranging from water to semi-aquatic, to even drier terrestrial habitats. Accordingly, adventitious roots of A. philoxeroides seedlings in water condition will grow long thick fleshy roots in drought condition, which are assumed to improve performance of the plant by more efficient reservation and extraction of water from deep soil layers. Besides, A. philoxeroides exhibit osmotic adjustment when grown hydroponically in solutions containing up to 400mM NaCl, a salt concentration greater than that tolerated by most crop plants. For comparing different antioxidative responses to drought and salt stress in Alternanthera philoxeroides and Oriza sativa, content of osmoprotectants and antioxidants were measured. As a result, compared with rice, the content of proline and the activities of SOD, POD and CAT in A. philoxeroides were only a little greater under both control and water stress conditions, while A. philoxeroides was much more resistant to drought than was rice and total antioxidative activity in the former was several-fold higher than that in the latter, which showed that single osmoprotectant or antioxidant has limited contribution to drought resistance. This elucidated why it was unperfect to transfer above a gene. In order to improve crop plants drought-resistance, it may be more advisable to transfer one upstream gene in drought signal pathway than to co-transform downstream genes, the products of which are responsible for the synthesis of single osmoprotectant or antioxidant.In order to clone drought and salt inducible up-regulated gene in A. philoxeroides roots, mRNA differential display technique is used in the present阶段.In order to make sure that all A. philoxeroides seedling have the same genotype,we truncated the singular plant into many portions and planted them in the sand and watered with Hoagland nutrient solution under controlled conditions. Adventitious roots of A. philoxeroides seedlings before and after drought and salt treatment were harvested. Total RNA was extracted from drought-treated and untreated A. philoxeroides roots, treated with RNase-free DNaseI. The RNA was quantified on a spectrophotometer and visualized on 1.2% agarose/formaldehyde gel to ensure high quality. By 78 primer combinations (three anchored primers, twenty-six arbitrary primers ), 126 putative drought and 118 salt up-regulated gene segments were obtained. Two of drought inducible fragments and one of salt inducible fragments were confirmed by reverse northern blot and subsequently cloned, sequenced. Size of two drought inducible and one salt inducible fragments are 612bp ,294 bp and 609bp respectively. We used the BLAST Tool in the NCBI databases to search for sequences homologous to the above fragments and found that 612bp fragments has 36% homology to cryptogein gene and that 294bp fragments has no homology to other gene and that 609bp fragments has some homology to ATP binding / kinase/ protein kinase/ protein serine/threonine kinase/ protein-tyrosine kinase gene.RACE (rapid amplification of cDNA ends) was used to amplify full-length cDNA of the above three fragment and one was get. this gene was named as ApCL and has 738 bp, including a 501 bp open reading frame. The deduced protein is composed of 166 amino acid and predicted MW is 16.7 KD. Semi-quantitative RT-PCR analysis showed that the ApCL gene was up-regulated by drought and salt stress. In yeast, the ApCL conferred increased NaCl and drought tolerance. These suggested that A. philoxeroides ApCL gene may be involved in drought and salt tolerance responses. The possible role of ApCL in resistance mechanism was discussed.