Construction Of Full-length CDNA Library Of Sesuvium Portulacastrum L. And Cloning Of Salt Tolerance-related Gene

The plant of Sesuvium portulacastrum L. usually lives in tropical and subtropical beaches. Searches have been identidied that Sesuvium portulacastrum L. has a good salt adaptability, and grows well in seawater or freshwater conditions. It is an ideal material to study physiological characteristics and mechanisms of salt-tolerance and clone salt-tolerance gene. In this paper, the full length cDNA library of S. portulacastrum has been constructed; and the analysis of differentially expressed transcriptome of S. portulacastrum between freshwater stress conditions and salt stress conditions has done by Digital Gene Expression-Tag profiling (solexa). The differentially expressed sequenceing tags have been obtained, then, the full-length differentially expressed genes can be cloned from the full length cDNA library.In this paper, the full-length cDNA library of S. portulacastrum was constructed under salt stress:The total RNA was isolated from the salt-stressed plant by using the improved CTAB, and the mRNA was converted into the first single-strand complementary cDNA by SMART method. The double-strand cDNAs (ds-cDNAs) were amplified by long distance polymerase chain reaction (LD-PCR), and then were digested by Proteinase K, and nuclease SfiI. After extraction through CHROMA SPIN+TE-1000 separated columns, the longer than 0.5 kb ds-cDNAs were collected and ligated intoλTripIEx2. The recombinant bacteriophages were packaged by MaxPlaxTM Lambda Packaging Extracts. The titer of the first unamplified library is larger than 4.80x106 pfu/ml, the rate of recombinant clones is 93.75%, and the inserted cDNA fragments are longer than 0.5 kb. The numbers of the dependent clones of the first library is 2.4×106 pfu. The titer of the amplified library was 1.21×109 pfu/mL. These data indicated that the cDNA library construction in the present study was suitable for analysis and cloning of the related genes. It was a high quality of the library.The differentially expressed sequenceing tags have been obtained.763911 sequences of 16 bp freshwater-stress, and 2073628 sequences in salt-stress of S. portulacastrum have been analysed in transcriptome. We have got 450349 tags and 993662 tags from the sequences, respectively. Among the tags, there are 158921 unique tags in salt-stress of Sesuvium portulacastrum L.,3038 unique tags in freshwater-stress of S. portulacastrum. In addition, we have got 226 tags which the quantity of expression is significant difference between the two transcriptomes. Baseing on the differentially expressed sequenceing tags and published salt tolerance gene sequence information in GenBank, the BADH gene was cloned from the full-length cDNA library. The gene has 1503 bp, which codes 500 amino acids. Its theoretical molecular weight of the amino acids is 54.53 kDa, isoelectric point is pI:5.22. The homology is more than 80% of the BADH in Amaranthus hypochondriacus, Kalidium foliatum (Pall.) Moq., Atriplex patens (Litv.), Spinacia oleracea and Triglochin palustre. After preliminary functional identification, the expression of the BADH gene is related to the salt-tolerance in S. portulacastrum.The full-length cDNA library and the differentially expressed sequenceing tags are abundant resources fpr studying the salt-tolerant mechanism of S. portulacastrum. On this basis, we can continue to clone salt-tolerant gene from S. portulacastrum., and the follow-up studying work.Then, we can cultivate transgenic plants to improve the plant soil salinization by genetic engineering technology.