Cloning Of Salt Tolerance-Related CDNAs From Mangrove Plants Sesuvium Portulacastrum L.

Salinization of soils is an increasingly threat to agriculture, forest and environment. There are 0.96 billion hectares of salted soil worldwide amounting to one third of the total terrestrial area on earth. There are about 100 million hectares of salted soil in our country that severely limits agricultural production. How to use and develop the salted soil in order to increase crop production and improve environment is one of the top projects all over the world. Many scientists have done their best to solve this problem and have made a lot of encouraging advancements in plant's salt tolerance mechanism, salt tolerance gene cloning and salt stress signal conduction. Based on the practical experience in the past two decades it is clear that the breeding of salt, drought and cold tolerant crop varieties by cloning the controlling genes is the ultimate, most economical and most reliable measure for utilizing salted soil. Mangrove plants which grow in seawater environments are truly salt-tolerant plants. They may have acquired specific genes essential for salt tolerance during the course of their evolution. Finding such genes may enhance agricultural productivity and contribute significantly to the study on salt tolerance mechanism.In this study Sesuvium portulacastrum L. (Family Aizoaceae), a mangrove plant was used as the experimental material. In order to screen for salt-sensitive plants by abduction the offsprings reproduced asexually by one Sesuvium portulacastrum plant were placed in freshwater and then transferred into seawater. Whereas the roots of the salt-sensitive plants would die and their leaves would wilt, the salt-tolerant plants remained growing well. Suppressive subtractive hybridization (SSH) was conducted by using Poly(A+) RNA of salt-tolerant plants as Tester and Poly(A+) RNA of salt-sensitive plants as Driver. The differential cDNA fragments were cloned into pDrive cloning vector. The false positive clones were eliminated by colony PCR and the real differential cDNA fragments were screened out by Reverse Northern Dot-Blot and Northern Blot. Finally, the full length cDNA of real differential fragments were cloned by performing both 5'- and 3'- rapid amplification of cDNA ends (RACE) and then the functional screening was performed by transferring the full length cDNA into Arabidopsis thaliana or Nicotiana tabacum plant.The salt-sensitive plant population of No.6 salt-tolerant plant of Sesuviumportulacastrum (SP6) was obtained by freshwater abduction screening. As the RNA extraction technique for mangrove total RNA extraction was developed the total RNA of SP6 was extracted in large quantity. Reverse northern dot-blots were performed for 241 clones selected from SSH. 61 of these clones have hybridization signals on the X-photos of the reverse northern blot. Then northern blots were performed for 23 of these 61 clones: Nos. 4, 17, 18, 23, 28, 66-1, 69, 84, 89-1, 94, 97, 101, 108, 152, 156-1, 157-1, 165, 172, 175, 183, 185, 191 and 233. Northern blot results show that Nos. 66-1, 84, 89-1, 97, 108, 152, 175 and 233 have stronger signal in SP6-Tester than in SP6-Driver; and No. 23 has weak signal only in SP6-Tester, Nos. 94, 165, 172, 185 and 191 have similar hybridization signals in both SP6-Tester and SP6-Driver; Nos. 4, 17, 18, 28, 6 9, 101, 156-1, 157-1 and 183 do not reveal hybridization signals in both SP6-Tester and SP6-Driver; The results of sequencing and BLASTN and BLASTX on NCBI indicate that No. 23 cDNA (846bp) has significant alignments with Nicotiana tabacum mRNA for elicitor inducible beta-1-glucanase Nt-SubE76, and Arabidopsis thaliana clone 7119 for glycosyl hydrolase family 17 (protein id: At5g55180.1, supported by cDNA: 7119, supported by cDNA:gi_l 87001 54) and Arabidopsis thaliana beta-1-glucanase-like protein (gi₂ 1594590); No.84 cDNA(560bp) has significant alignment with Lotus corniculatus aspartate aminotransferase mRNA (complete cds Length = 1685, gi|2605931|gb|AF029898.1|AF029898) for aspartate aminotransferase; No.89-1 cDNA has significant alignment with Arabidop...