Isolation And Sequence Analysis Of Salt-tolerance Related Gene In Wild Soybean

Salt stress is one of the main factors which affect the growth and output of crops. It isestimated that up to 4×10~8～9×10~8 hectare land was salinized, which is more than three times topresent agricultural land, so how to improve crops salt talerance is becoming more and moreimportant. Cultivated soybean origins from wild soybean,wild soybean has great resistance and isan important germplasm resources for cultivated soybean breeding.Therefore, it is very urgent tostudy salt-tolerance mechanism and isolate excellent genes from wild soybean for cultivatedsoybean improvement.The proline is an important osmolyte. Not only lower plants but also higher plants canaccumulate free proline in response to osmotic stresses. The proline synthesis enzyme (PSCS) is akey enzyme in the proline biosynthesis which limits the synthesis rate of Proline. It has importantsignificance to research on P5CS.Wild soybean ZYD00659 was used in this study. Two pairs of primers were designedaccording to the conserved regions of the P5CS from other plant in Genbank, The gene wasamplified by RT-PCR from the total RNA which was extracted from wild soybean under salt stressin 200mM NaCl. By homologous based cloning, we isolated a cDNA sequence in wild soybeanwith 1569 bp in length. It encodes 523 amino acids. Predicted by software, the molecular weight ofthis peptide is 56,514kDa, its isoelectric point is 6.75. The deduced protein containsNADPH-binding domain, Glu-kinase domain, GSA-dehydrogenase domain and feedbackinhibition site. NCBI database Conserved Domain Search, it has the whole ProA domain, namedGsP5CS1.Use the same method, we also isolated another cDNA sequence in wild soybean with 560bp inlength. It encodes about 186 amino acids. Predicted by software, the molecular weight of thispeptide is 20,186kDa, its isoelectric point is 4.73. NCBI database Conserved Domain Search, it hasthe partial ProA domain, named GsP5CS2.We use GsP5CS2 cDNA sequence designed a pair of primer, amplified a DNA sequence byPCR in genomic DNA. The length of the sequence is 1374bp, contains four introns and fiveextrons. And between introns and extron which have typical GT/AG structure.named gGsP5CS2.We blast the sequence with other plants on NCBI web site, we find they are have highhomologous to other plants.The identity of GsP5CS1 and GsPSCS2 only 71%, but they are all have ProA domain. We suggest that they are different types of P5CS gene in wild soybean.